Final Report Summary - DEVELAGE (Pathways common to brain development and ageing: defining strategies for preventive therapy and diagnostics)
Executive Summary:
The European health care system is facing a crisis due to a steadily increasing elderly population and a concomitant increase in the prevalence of age-related diseases. Common age-related neurodegenerative disorders, including Alzheimer’s disease (AD), have dramatic repercussions on our society and health systems due to the involved nature of patient care and absence of a treatment. Understanding the link between brain development and aging is critical for advancing our knowledge of pathological ageing and defining new therapeutic and diagnostic strategies.
DEVELAGE focuses on characterising shared molecular pathways between early developmental processes in the brain and brain ageing in order to identify early pathological processes contributing to AD. DEVELAGE is based on the hypothesis that disorders of neural development contribute to age-related neurodegeneration. On the one hand, this means that factors essential for the development of the brain may have a role in neurodegeneration, and conversely those neurodegeneration-related proteins and genes might be important during the development of the brain.
The DEVELAGE project takes a unique brain-tissue-based approach using detailed molecular analysis of the spectrum of developmental and ageing changes in neural cell populations in the very same brain samples. The project as a whole has placed special emphasis on the evaluation of protein modification, altered cellular functions, gene expression and genetic and epigenetic alterations that take place during the development and ageing process. It further compares these results with transgenic mice and a non-human primate model, Microcebus murinus (MIM). Using these approaches, DEVELAGE contributed to: 1) the definition of distinguishing factors between normal and Down syndrome developing brains; 2) the understanding of the hippocampus and related structures, which are early affected in Alzheimer’s disease; 3) the understanding of early metabolomic and transcriptomic alterations in brains, which have not yet fully developed Alzheimer’s disease; 4) the understanding of the epigenetic alterations of genes and factors related to Alzheimer’s disease; 5) the understanding of the spectrum of brain disease affecting the brains of the elderly; 6) the understanding of protective genetic constellations associated with healthy brain ageing; 7) the characterisation of the non-human primate model as a competitive tool for research on neurodegenerative disorders.
Professional project management and dissemination of results significantly contributed to the successful collaboration of the DEVELAGE beneficiaries, whose active dissemination resulted in 53 peer-reviewed publications, with a number of additional publications expected. DEVELAGE work was also presented 73 times at international scientific conferences. Public awareness of DEVELAGE has been addressed with a website and press releases. DEVELAGE was very successful in attracting the attention of two major media outlets, including a documentary film by the EC-funded ASAPS project and ‘EuroNews’ award winning science documentary Futuris, where DEVELAGE was featured in their video ‘Deep inside the brain’, estimated to have been viewed by approximately 1 million viewers.
In summary, DEVELAGE was highly successful and met essentially all objectives on schedule. DEVELAGE has brought together internationally renowned experts in closely interacting work packages to achieve world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s Disease. DEVELAGE has improved the competitiveness of European Universities by collaboration on methodological and thematic aspects that will bring forth added value to all participants. The results of DEVELAGE have the potential to lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Project Context and Objectives:
The DEVELAGE context
The increasing number of elderly people resulting from an ever increasing life-expectancy will have a major impact on the prevalence of age-related diseases in the near future leading to enormous costs and challenges for health systems in Europe. Age-related common degenerative disorders (Alzheimer’s disease, AD; mild cognitive impairment; Parkinson’s disease, PD) affect ~20 million European citizens today, with women particularly affected especially with AD. If no progress is achieved in the understanding of how preventive therapy can be initiated, it could result in dramatic consequences or the collapse of European and world-wide health-systems. It is therefore of the upmost importance that scientists and physicians work together to translate the latest findings from basic research into new treatments and prevention schemes.
Age-associated cognitive decline and normal or non-pathological (normative) cognitive ageing is a human experience, which differs in extent between individuals. Such age-related cognitive changes have been ascribed to alterations in synapses and neuronal loss in the brain that are multiplied in pathological brain ageing. Experimental evidence suggests a complex scenario, including mitochondrial dysfunction, compromised stress responses, synaptic rearrangements and altered protein (e.g. amyloid beta, tau, or α-synuclein) expression, to cause age-related brain changes. Cellular genes that are mutated in neurodegenerative diseases code for proteins that are expressed throughout neural development. These proteins interact with numerous other cellular proteins that are components of signalling pathways involved in patterning of the neural tube and in regional specification of neuronal subtypes. These observations support the notion that neurodegeneration-related proteins are important during the normal brain development.
Many elderly individuals experience no major, or only limited, functional impairment emphasising the ability of the human brain to compensate for potential cognitive decline. Many fascinating studies have shown that new neurons are actively generated in the adult brain, mainly in the subventricular zone but also in the subgranular zone of the dentate gyrus in the hippocampus. Failure of adult neurogenesis may lead to defective memory, a characteristic feature of age-associated cognitive decline. However, it is not clear which factors influence adult hippocampal neurogenesis and its failure. Since neurogenesis and complex interaction of glial cells and neurons (“guidance”) are basic requirements of physiological brain development, comparison of these pathways in the most exclusive human-specific model (i.e. the developing brain) with normal and pathological ageing may serve as a rationale for defining targets for the maintenance of the physiological milieu for neurogenesis. Research on pathological ageing has for a long time focused on defining the causes of neuronal dysfunction and death during adult life. However, increasing evidence supports the concept that neurodegenerative disorders could represent a disorder of neural development.
Efficient therapeutic strategies for age-related neurodegenerative disease (i.e. AD) require an improved understanding of early stages of the disease, when pathology is still reversible. Known clinical features of neurodegenerative disease (NDD) occur at relatively advanced stages, when the degenerative process has overcome thresholds that render the process irreversible. However, practically nothing is known about changes at earliest stages of the disease, as the human brain remains one of the most inaccessible areas for biomedical research. Previous studies have shown that NDD are not the mere result of accumulation of altered proteins, as for example Aβ and tau in AD, but a deleterious interaction of different pathways results in impaired neuronal function. A complex scenario has led to the concept of neuronal exhaustion, which precedes neuronal death in neurodegeneration. Further identification of altered molecular pathways will serve to delineate a comprehensive scenario of first alterations in the entorhinal cortex and hippocampus in cases with AD-related pathology.
The factors contributing to the variability of age-related cognitive change comprise demographic, social, educational, medical, nutritional, and biological (genetic and epigenetic) influences. Epigenetic modifications, such as DNA methylation and histone acetylation regulate replication, transcription and translation activity contribute to chromatin modelling, epigenetic modifications and DNA imprinting. Methylation status is essential for normal brain biology, and low methylation is associated with markers related to neurodegeneration. Epigenetic mechanisms play a pivotal role in brain development and may contribute to the course and development of AD. It is conceivable that brain-specific transcription is regulated by methylation on an epigenetic level much more frequently than tissue-specific expression in other organs. Low methylation status is strongly associated with neurological and cognitive deficits. However, it is still not fully clear whether epigenetic changes actually represent a cause or a consequence of the disease. This is mainly due to the fact that epigenetic studies involved AD patients in an advanced stage of the disease. Thus, further research is needed to decide the rationale for therapeutic interventions improving the methylation status. In addition, it needs to be clarified whether and how epigenetic alterations of pathways crucial for the developing hippocampus happen, in particular which may have consequences later in life, mediated by aberrant epigenetic patterns. Alteration of SAM (S-adenosyl-methionine)/HCY (homocysteine) metabolism is known to occur in AD and neurodegeneration. It has the potential to be exploited as a target for therapeutic approaches to improve synaptic function and/or neuronal replacement in normal ageing and neurodegenerative diseases.
In addition to environmental factors, genetic influences also account for the variance in adult cognitive abilities to varying degrees depending on the age of the study group. The search for genetic contributions to cognitive ageing faces a lot of aspects, including the problem of non-reproducibility due to varying definitions. In large autopsy series, the oldest-old individuals (>85 y) are recognised with relatively well-preserved brain tissue and with mild or no deposition of proteins linked to neurodegenerative diseases. Strict neuropathology-based stratification of study groups can help to define protective genetic constellations contributing to healthy ageing. Pathways related to these genes open new avenues for the definition of targets for neuroprotective therapies.
Models as close as possible to humans are needed for a better understanding of age-related changes. So far, the vast majority of studies have been performed in rodents, but experimental findings and conclusions are highly dependent on animal and strain models. Moreover, the complexity of the primate brain with its behaviour and cognitive abilities are dramatically greater than in rodents, so results of neurological decline in rodents cannot be easily extrapolated to humans. Preliminary data shows that ageing MIM-brains may show some AD-like pathology. Defining a relevant animal model of age-related brain disorders is an important research strategy in ongoing trials to understand the pathology and to test new treatments.
In spite of efforts to clarify the causes and pathogenesis of AD, our current knowledge is insufficient to recognise the transition of normal brain ageing into AD-like brain disease when the process is still reversible. This is partly due to the fact that research focuses on clinically manifest or terminal stages of brain diseases or uses experimental conditions that are not easily translated to, or never validated for, human disease. Moreover, due to the plasticity of the brain, patients admitted to medical care after first symptoms have already severe loss of neurons together with modifications of proteins, which is a major restriction of effective therapy. At this stage only palliative but no preventive or curative therapy can be initiated. Thus, elucidation of complex pathogenetic pathways characterising the earliest stage of the detrimental road to AD in human brain is needed, since it represents the most promising opportunity for therapeutic intervention to reverse pathological processes and prevent onset of the disease.
The DEVELAGE project took a unique brain-tissue-based approach, with detailed molecular analysis of the spectrum of developmental and ageing changes in neural cell populations in the very same brain samples used for a wide array of investigations. We addressed age-related common neurodegenerative disorders, including Alzheimer’s disease (AD), that together affect ~20 million European citizens today, especially women. In addition, the project increased our understanding of normal as well as pathological brain development. This knowledge will contribute to our understanding of early pathological events in neurodegenerative disorders and prove instrumental in developing concepts for therapeutic intervention when a condition is still reversible
Thus, the OVERALL AIM of DEVELAGE has been to characterise shared molecular pathways between early developmental processes in the brain and brain ageing, in order to identify early pathological processes contributing to AD. This overall DEVELAGE aim encompassed the following objectives:
1) Evaluation of proteins characterising age-related neurodegenerative disorders and their modifications throughout brain development and ageing.
2) Characterisation of key mechanisms involved in neurogenesis including the role of neurotrophic factors.
3) Mapping the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology.
4) Characterisation of methylation sensitive genes in normal ageing and early phases of AD-like neurodegeneration.
5) To use precise morphological definitions and perform whole genome sequencing to evaluate genetic constellations on the background of a healthy state of the brain in oldest-old individuals.
6) To develop and evaluate a non-human primate model (MIM), based on the results obtained from our evaluation of human brains.
To this end, DEVELAGE implemented a collaborative research project using a wide variety of methods including the evaluation of protein modifications, altered cellular functions and gene expression, genetic and epigenetic alterations, and comparison with transgenic mice and a non-human primate model, Microcebus murinus (MIM). To complement our descriptive observations, we identified target molecules and pathways, which could serve as entry points to exploit our knowledge for the development of therapeutic and diagnostic solutions. In addition, we evaluated the effects of SAM (S-adenosyl-methionine), a compound acting on the epigenetic level in experimental models. A focus was placed on the evaluation of the temporal and entorhinal cortex and hippocampus, due to their importance in age-related changes, learning, memory and neurogenesis.
For Objective 1 (Evaluation of proteins characterising age-related neurodegenerative disorders and their modifications throughout brain development and ageing), DEVELAGE addressed the understanding of the expression pattern and cellular distribution of genes and proteins related to neurodegeneration, in normal brain development as well as in brain tissue from patients with Down syndrome, and in focal malformations of cortical development.
For Objective 2 (Characterisation of key mechanisms involved in neurogenesis including the role of neurotrophic factors), DEVELAGE sought to explore and characterise the mechanisms that are involved in the sequential formation of different layers in the cortex and in the hippocampus, at various stages of development and ageing in human; indispensable knowledge for designing therapeutic strategies towards neurodegenerative diseases.
For Objective 3 (Mapping the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology), DEVELAGE used convergent methods to map the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology, categorised as stages I-II of Braak & Braak (from the six stages of neurofibrillary degeneration), compared with non-affected age-matched controls.
For Objective 4 (Characterisation of methylation sensitive genes in normal ageing and early phases of AD-like neurodegeneration), DEVELAGE addressed the important role played by the methylation of CpG dinucleotides in regulation of gene expression in the brain.
For Objective 5 (To use precise morphological definitions and perform whole genome sequencing to evaluate genetic constellations on the background of a healthy state of the brain in oldest-old individuals), DEVELAGE addressed the difficulties of separating non-pathological from pathological brain ageing through the whole genome sequencing of strictly defined cases of elderly individuals.
For Objective 6 (To develop and evaluate a non-human primate model (MIM), based on the results obtained from our evaluation of human brains), DEVELAGE addressed the importance of developing and evaluating a non-human primate model to facilitate the extrapolation of therapeutic approaches to humans.
In order to address these objectives DEVELAGE has brought together internationally renowned experts in closely interacting work packages to achieve world-class collaborative research (see Figure 1). DEVELAGE successfully organised the complementary expertise of partners in relevant fields of basic and clinical research and facilitated the sharing of methodologies between a critical mass of partners, thereby increasing their scientific impact and bringing added value to all participants. DEVELAGE emphasised active dissemination to increase the public awareness of the DEVELAGE-project and age-related degenerative disorders as an important individual and public health issue. DEVELAGE has produced knowledge that will significantly contribute to the development of pharmacologic strategies to treat age-related common degenerative disorders, including Alzheimer’s disease. Further research will show to what extent the results of DEVELAGE could lead to the development of individualised pharmacological strategies, in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Project Results:
DEVELAGE has essentially achieved all of the overall scientific objectives and those within the six scientific work packages (WP1-WP6).
WP1 Insight into the expression pattern and cellular distribution of genes and proteins related to neurodegeneration in human brain development and pathological processes during ageing.
When ageing, the human brain shows morphological alterations that are reminiscent of so called neurodegenerative diseases (NDDs). Current molecular pathological classification of NDDs is based on the evaluation of pathologically altered proteins and in particular the evaluation of where these proteins are deposited in the human brain. Increasing evidence supports the concept that aberrant neural development can contribute to the process of neurodegeneration in the ageing brain. Accordingly, several genes that are mutated in NDDs (such as amyloid precursor protein, APP, and presenilin, PSEN) encode for proteins that participate in fundamental neurodevelopmental processes, including the precisely orchestrated cascade of events which underlies the development of the cerebral cortex. Additionally, major pathways essential to normal cortical development (including CDK5 and the mTOR signalling pathways) have been shown to be potentially involved in the mechanisms underlying cellular ageing and neurodegeneration.
Data obtained in this WP provides valuable insight into pathways common to brain development and pathological processes during ageing. This information is necessary to improve our understanding about the abnormal (re-)activation of pathways essential to neurodevelopment, and how this might contribute to early events in neurodegeneration. This knowledge provides the potential for novel avenues of investigation into suitable therapeutic approaches to target critical pathways at a stage when pathological progress can still be reverted.
Our Consortium had unique access to clinically and morphologically characterised normal human brain at different development ages, on which we could perform comparative analysis of both neurodegeneration- and neurogenesis-linked proteins, in developing versus aged hippocampus and temporal cortex. In the first 12 months we selected and carried out a neuropathological evaluation of autopsy brains at different developmental ages from control, and Down syndrome patients. Additionally, adult brain tissue was obtained from controls, patients with post-traumatic brain injury, individuals with Down syndrome (DS) and patients with Alzheimer’s disease (AD). This cohort of tissue samples was used for the further studies of this WP.
We performed a systematic evaluation of the hippocampus and temporal cortex, at different developmental ages, for various neurodegeneration related proteins using immunohistochemistry and western blot analysis.
We have studied the developmental expression of amyloid precursor protein (APP) and of the death receptor-6 (DR6) in both control and Down syndrome brains. Our study provided the first description of the expression pattern and cellular localisation of DR6 in human hippocampus and neocortex during the pre- and early postnatal development. Our findings demonstrated a developmental regulation of DR6 in human hippocampus and highlighted the potential role of DR6 in specific types of disease-associated neuronal degeneration. Preliminary observations from MIM (Microcebus murinus; non-human primate model used in our project) brain at different ages support the idea of a developmental regulation of DR6, reactivated during ageing.
We also analysed the developmental expression of several other neurodegeneration-related proteins (TDP-43, p62 and ubiquitin) and demonstrated a developmental regulation of TDP-43, p62 and ubiquitin in human hippocampus, supporting a role for these proteins during early development in the hippocampus, as well their contribution to the pathogenesis AD-associated pathology.
We demonstrated that phosphorylation of tau protein may have important physiological effects during development. Remarkably, this physiological phosphorylation seems to be critically reduced in Down syndrome and therefore can be used as a distinguishing marker of Down syndrome.
We demonstrated significant differences in the number and density of non-neuronal cells between DS patients and controls. These alterations may contribute to or reflect a compensatory response to defective neurogenesis, reduced neuron number, and delayed myelination during the development of foetal DS brain. These alterations might contribute to the development of mental retardation in DS patients.
We evaluated the developmental changes occurring in human healthy foetal and postnatal hippocampus by combining high-resolution 7T Magnetic Resonance Imaging (MRI) and histological and immunohistochemical analysis. Our study showed the potential utility of high-field MRI for detection of developmental changes in human foetal and postnatal hippocampus. These data will be a promising tool in the understanding of hippocampal abnormalities occurring in developmental disorders when in the future high resolution MRI might be used in clinical practice.
A major task was the investigation of the relationship between neurodegeneration related proteins and specific neurodevelopmental pathways. We studied the activation of mTOR signalling using, as marker of mTOR, the phospho-S6 (pS6) protein in DS specimens at different developmental ages followed by the evaluation of other components of the mTOR signalling pathway (i.e. phospho-p70S6 kinase1, phospho-4EBP1). Evaluation of pS6 during hippocampal development showed increased immunoreactivity in CA1 neurons prior to establishment of AD pathology.
Further characterisation of pS6 positive cells, as well as evaluation of other components of the mTOR signalling pathway was performed. The expression pattern and cellular distribution of components of the mTORC1 signalling (phosphorylated (p)S6, p70S6K, p4E-BP1 and pmTOR) were investigated immunohistochemically in the developing hippocampus from controls and patients with Down's syndrome, as well as in adults with DS and Alzheimer’s disease-associated pathology. Our study provides evidence of mTOR pathway activation in DS hippocampus. Since DS is a model of AD-related pathology, the alterations detected early during brain development support the notion that mTOR is an important pathway in AD, and also that already during development neurodegeneration related changes are initiated in the brain practice.
Specimens from tuberous sclerosis complex (TSC) and focal cortical dysplasias (FCD) patients represent a unique opportunity to study the relationship between neurodegeneration related protein expression and the persistent activation of specific developmental pathways. We examined FCD and TSC cases for the expression of several proteins involved in major neurodegenerative diseases. We compared the abnormal cortex with adjacent normal cortex and cases of FCD (type I) without evidence of upregulation of CDK5 or activation of the mTOR pathway. Sections were processed for TUNEL labelling and immunohistochemistry using markers for the evaluation of apoptosis signalling pathways and neurogeneration -related proteins/pathways. We provided evidence of complex, but similar mechanisms of cell injury in focal malformations of cortical development associated with mTOR pathway hyperactivation, with prominent induction of apoptosis signalling pathways a premature activation of mechanisms of neurodegeneration. In addition, mTOR signalling was evaluated immunohistologically in foetal brains of TSC patients, with the results indicating that brain malformations in TSC are likely a consequence of increased mTOR activation during embryonic brain development.
We also examined the expression of several proteins involved in major neurodegenerative disease in developmental brain tumours (glioneuronal tumors, GNT) characterised by mTOR pathway hyperactivation. Sections were processed for immunohistochemistry using markers for the evaluation of apoptosis signalling pathways and neurodegeneration -related proteins/pathways. Our data provide evidence for the premature activation of mechanisms of neurodegeneration in glioneuronal tumours associated with mTOR pathway activation.
We characterised the cultures of neural stem (NS) cells prepared from foetal human brain and the protocol to induce them to differentiate into neurons or astrocytes. We demonstrated that a pure population of NS cells can be isolated from the adult human sub-ventricular zone (SVZ), which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis and studying the relationship between neurodegeneration-related proteins and specific developmental pathway.
We also provided evidence supporting the regulation of GFAP expression by epigenetic mechanisms. Our data demonstrate that histone acetylation controls transcription, splicing, and the assembly of GFAP in astrocytes. These data also imply that a tight regulation of histone acetylation in astrocytes is essential; since dysregulation of its gene expression causes pathological aggregation of GFAP.
We studied selected mRNAs, microRNA (miRNA) and proteins identified in WP3. First, we investigated the role of miR-146a, (upregulated in Alzheimer’s disease) in the modulation of astrocyte-mediated inflammation. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miRNA may represent a novel target for therapeutic strategies.
The amyloid hypothesis of Alzheimer’s disease suggests that soluble amyloid β is an initiator of a cascade of events eventually leading to neurodegeneration. Recently, it has been reported that amyloid beta deranged Ca2+ homeostasis specifically in hippocampal astrocytes by targeting key elements of Ca2+ signalling, such as mGluR5 and IP3R1. We further dissected a cascade of signalling events and suggest that nanomolar concentrations of amyloid beta deregulates Ca2+ homeostasis via calcineurin and its downstream target NF-kB, possibly via the cross-talk of Bcl10 in hippocampal astrocytes.
We demonstrated developmental regulation of mGluR5 in human hippocampus and suggest a role for this receptor in astrocytes during early development in DS hippocampus, as well as a potential contribution of mGluR5 to the pathogenesis AD-associated pathology.
We also addressed the adenosine hypothesis of neurodegeneration and comorbidities, which implies that astrogliosis, via overexpression of adenosine kinase (ADK), induces a deficiency in purine ribonucleoside adenosine, which is a homeostatic network regulator of the brain. We found increased expression of ADK in astrocytes surrounding amyloid deposits and tangle-containing neurons, in the hippocampus of AD patients, and in adult DS patients with widespread AD-associated neurodegeneration. Interestingly, strong expression of ADK has been detected in human foetal brain (in both controls and DS), possibly reflecting the enzyme’s expression in the deep compartments of the cortical wall (VZ/SVZ; ventricular/subventricular zone) at early stages of corticogenesis.
WP2 Neurogenesis during brain development and in adults
Neurogenesis in adult brain contributes to the plasticity of the hippocampus and plays a role in learning and memory. Research on pathological ageing has for a long time focused on defining the causes of neuronal dysfunction and death during adult life. However, recent findings point towards alterations in neurogenesis in neurodegenerative diseases. Experimental studies suggest that neurogenesis is influenced by a number of intrinsic and extrinsic factors. Additionally, numerous signalling pathways and trophic factors implicated in development, contribute to the regulation of adult neurogenesis.
The molecular mechanisms regulating cell-cycle progression during neurogenesis and the tightly regulated occurrence of proliferative versus differentiative divisions are not completely understood.
This WP was designed to explore and characterise the mechanisms involved in the neurogenesis and sequential formation of the different layers in the cortex and in the hippocampus, at various stages of development in humans, and to characterise the molecular mechanisms regulating cell-cycle progression during neurogenesis. We also characterised the stem cell niches that persist during adult life in ageing people with or without Alzheimer’s disease.
We aimed to provide insights into the signalling pathways, which control the cell cycle of the progenitors of the temporal cortex and the hippocampus in developing brain, healthy and AD adults. Using frozen and formalin fixed brains we examined the expression of markers of the cell cycle, mitosis and cell cycle exit, in order to recognise various types of progenitors and the modalities of cell division. The fate of ventricular cells and sub-granular germinal zone cells has been examined using multiple labelling with a combination of markers, such as SOX2, Nestin, GFAP, TBR2, PAX6, Ki67 and layer markers. We have evaluated three major anatomical regions as follows:
A-Proliferating areas and generation of neurons in the dorsal telencephalon
We have characterised for the first time the setting of different compartments of progenitor cells proliferation and neurogenesis in the human foetuses at all stages.
B-Proliferating areas and generation of multiple neuronal subtypes in the hippocampal formation during development
The molecular mechanisms underlying the formation of the hippocampus are unknown in humans. To increase our knowledge of potential regulatory molecules, we investigated the expression of progenitor markers and cell fate molecules. We described for the first time the development of the hippocampus from the early foetal age (GW9) to the end of the gestation (see Figure 2). In particular, we characterised the evolution of proliferating zones and the formation of pyramidal and dentate granular layers.
Our findings demonstrate for the first time the presence of a germinal subventricular zone (SVZ) in the hippocampus of human foetuses. In addition, we provide molecular evidence of the heterogeneity of the pyramidal neurons in the hippocampal formation. The connectivity of the hippocampal formation has been established in animals and emphasises that differences between species exist. Our data provide new information to further investigate the connectivity of the hippocampal formation and to better understand the susceptibility of specific neuronal subtypes during neurodegenerative diseases.
C-Proliferating areas and generation of neuronal subtypes in the dentate gyrus of the hippocampal formation during development and ageing
The molecular mechanisms that orchestrate the development of the human dentate gyrus are unknown. We characterised the formation of human dentate, fimbrial progenitors and post-mitotic neurons from GW9 to GW25, to further our understanding of this region of the hippocampal formation.
We established how some signalling pathways interfere with neurogenesis in different neuronal populations, cell cycle regulation in the hippocampus and temporal cortex at different developmental ages, including healthy and AD adults. We determined which types of neuronal populations are dependent on these pathways and the regional expression of their downstream transduction factors. We also established the expression pattern of trophic factors involved in neurogenesis in the hippocampus and temporal cortex in human at different developmental ages, including healthy adults.
In addition, we showed that during congenital cytomegalovirus infection (CMV) the virus preferentially targets these neural progenitors in the brain. CMV infection is the most prevalent cause of congenital neurological handicap and is a major public health concern. The pathogenesis of cerebral lesions remains unclear. We investigated the neuropathologic substrates, the immune response, and the cellular targets of CMV infected human foetal brains. Our results indicate that the 2 main factors influencing the neuropathologic outcome at this stage are: the density of CMV-positive cells and the tropism of CMV for stem/progenitor cells. This suggests that a large spectrum of CMV-induced brain abnormalities are caused not only by tissue destruction, but also by the particular vulnerability of stem cells during early brain development. Florid infection of the hippocampus and the olfactory bulb may expose these patients to the risk of neurocognitive and sensorineural handicap, even in cases of infection at late stages of gestation.
We also established the phenotypic and genotypic characteristics of foetuses diagnosed with L1 syndrome, a disease responsible for abnormal axonal development and pathfinding. L1 syndrome results from mutations in the L1CAM gene located at Xq28 (X chromosome marker). L1 syndrome encompasses a wide spectrum of disease phenotypes, with X-linked hydrocephalus being the most severe phenotype, detected in utero, and whose pathophysiology is incompletely understood. The aim of this study was to report detailed neuropathological data from patients with mutations. Additionally, to delineate the neuropathological criteria (required for L1CAM gene screening in foetuses) by characterising the sensitivity, specificity and positive predictive value of the cardinal signs, as well as to discuss the main differential diagnoses in non-mutated foetuses, in order to delineate closely related conditions without L1CAM mutations. These data underline the existence of closely related clinical entities, whose molecular bases are currently unknown. The identification of the causative genes would greatly improve our knowledge of the defective pathways involved in these cerebral malformations.
We were also able for the first time to successfully culture neuronal precursors, immature and mature neurons, as well as cells from the glial lineage (precursors and differentiated astrocytes), from our non-human primate model, and maintain these cell types in culture over time (see Figure 3). This allowed us to describe the characteristics of the various cell types, and analyse the MIM neural precursor cells for stem cell potency.
WP3 Complex molecular pathways in AD as a model for age-related neurodegeneration
Very little is known about changes occurring at the earliest stages of AD, Braak & Braak stages I-II, in which neurofibrillary pathology (one of the hallmarks of AD) is restricted to the entorhinal and transentorhinal cortices. The fact that we do not know whether a particular individual would have developed full symptoms of AD has for years prevented a profound study of the brain at these very early stages of the disease. Yet these first stages are crucial as the majority of people over 65 years have neurofibrillary tangle pathology in the entorhinal and transentorhinal cortices, and 100% of AD cases have entorhinal and transentorhinal pathology. Previous studies have shown that in addition to the accumulation of altered proteins, neurodegenerative diseases are characterised by the loss of the ability of neurons to cope with energy requirements. This scenario has led to the concept of neuronal exhaustion, which precedes neuronal death in neurodegeneration.
Several studies have analysed gene expression in AD brains. However, there is a paucity of of mRNA expression studies focused on the entorhinal cortex and hippocampus, in cases with AD-related pathology at Braak & Braak stage I-II. MicroRNAs (miRNAs) belong to the family of small silencing non-coding RNAs that are involved in the guidance of diverse formats of gene regulation, typically resulting in reduced expression of target genes. The miRNA pathways alter and modulate the expression of thousands of genes and contribute to a variety of physiological processes, providing an intricate network of regulation for the fine-tuning of the transcriptome, proteome and metabolome.
The aim of this WP was to map the transcriptome and metabolome of the entorhinal cortex and hippocampus, in the very same individuals affected by first stages of AD-related pathology (Braak & Braak stages I-II) compared with non-affected age-matched controls. To achieve this, convergent methods were used including: mRNA expression, microRNA expression profiling, protein expression, oxidation damaged proteins, impaired enzymatic function; altered metabolic pathways identification (metabolomics) focused on energy metabolism pathways and bioinformatic processing. In addition, lipidomics has been added as valuable complementary source of information. Animal studies using the APP/PS1 transgenic mice have also been employed as a model of familial AD or of familial amyloid beta deposition in brain.
The objective of our study of mRNA expression was to identify deregulated genes, and most importantly, to identify clusters of altered genes that recognise particular metabolic pathways. Once these clusters were obtained, the data was validated and further combined with proteomic, metabolomic and lipidomic studies in order to obtain functionally altered interactomes in ageing and disease processes. Moreover, the data was shared with other members of the consortium so that they could apply these results to their studies of development and animal models, and to analyse mechanisms related to altered regulation (for example DNA methylation studies of specific genes).
The four main altered clusters correspond to: olfactory and taste receptors, cytokines and mediators of the immune response, purine metabolism, and protein synthesis. The studies were expanded to other pathologies including Parkinson’s disease, tauopathies and prion diseases, in addition to ageing and Alzheimer’s disease, in order to understand commonalities and differences among these distinct neurodegenerative processes. The responses have been shown to be disease-specific and not mere reflections of accelerated ageing.
To complement our studies in humans, we produced a hierarchical clustering heatmap of the expression intensities of the significantly regulated transcripts versus the APP-PS1 and wild-type (WT) arrays of an animal model of AD. A clear separation was seen between APP/PS1 and WT mice at 6 and 12 months of age. We found that neuroinflammation associated with ageing, and neuroinflammation at early stages of sporadic Alzheimer’s disease (but not in APP/PS1 transgenic mice), does not depend on amyloid beta deposition, soluble amyloid beta or abnormal tau deposition.
One of the altered clusters uncovered by our expression analysis corresponded to purine metabolism. This could reflect functional alterations in energy metabolism, which is a feature of both ageing and Alzheimer’s disease, and occurs through altered mitochondria and oxidative stress damage. Purines form the core of DNA, RNA, nucleosides and nucleotides. Nucleotides participate in a wide variety of crucial metabolic pathways including energy metabolism and cell signalling. In addition to intracellular signalling, purines and their products may function as extracellular signals acting upon other cells, either between neurons, or between neurons and glial cells equipped with appropriate receptors. In this WP we validated at the middle and later stages region-dependent deregulation of several enzymes of the purine metabolism. No modifications were observed at earlier stages, in clear contrast with the early appearance of neuroinflammation. This study has been complemented with targeted metabolomics, showing that not only is there a deregulation of the enzymes but also an abnormal expression of several metabolites, thus indicating that purine deregulation has metabolic implications. Purine metabolism has also been examined in the substantia nigra and frontal cortex in Parkinson’s disease at different stages of disease progression. The pattern of altered profiles differed in the substantia nigra, where purine metabolism is largely compromised, when compared with the cerebral cortex where it is relatively unaffected
In this WP, we identified altered microRNAs that can regulate RNA expression, and therefore modulate protein synthesis in different settings. Additionally, we identified altered proteomic profiles and clusters of altered proteins from specific metabolic pathways. Results were also shared with other members of the consortium, to analyse these altered clusters during development and in animal models. Moreover, we evaluated post-translational modifications of proteins that may alter protein function. Changes resulting from oxidative damage are particularly important, as even though the protein expression level is unchanged, the function (for example the enzymatic activity) of a particular protein is altered by oxidative and nitrosative damage.
We combined our metabolomics data with the human metabolic model (Recon 2), in order to generate a tissue specific model of the metabolic fluxes occurring during neurodegeneration in the ageing brain. Using a constraint-based modelling (CBM) approach, our data will lead to network-based predictions that will be used to determine changes in metabolite levels revealing potential nodes of interest or biomarkers.
We processed the large amount of data obtained from transcriptomics, proteomics and metabolomics. Subsequently we employed a systems biology approach in order to identify altered interactomes, and understand the interaction of altered interactomes and metabolomes in disease processes, with a focus on the transition between ageing and the early stages of AD-related pathology. Finally, we created brain-ageing interactomes containing genes associated with our epigenetic, gene expression, non-coding RNAs, proteome and metabolome results, as well as proximal interactors. We created a protein network of all known human genes and/or proteins from three different network resources: iREFINDEX, ConsensusPath DB, and the HI Yeast-Two-Hybrid prepublication dataset downloaded from Human Interactome Database website. We used this network to examine the similarity of the brain interactome to common neurological disorders. Thus, Bioinformatics has been the tool that has permitted the identification of altered clusters of mRNAs, proteins, metabolites and interactomes.
To complement the data obtained by using the other “omics” approaches we focused on lipidomics, as cell membranes in the nervous system are very rich in lipids and alterations of lipid composition impair protein processing. Increasing age is accompanied by alterations in the lipid matrix of brain cortex lipid rafts from both WT and APP/PS1 transgenic mice, which we coined the “lipid-raft ageing” concept. However, in AD mice the process is accelerated and animals exhibit the oldest phenotype prematurely, at the age of 9 months. Progression of lipid changes in brain cortex lipid rafts as a function of age in WT and APP/PS1 mice.
WP4 Epigenetic alterations in brain development and ageing
Methylation of CpG dinucleotides plays an important role in regulation of gene expression in the brain. It was discovered that brain specific promoter-related sequences are surprisingly enriched in CpG sites. This leads to the conclusion that it is likely that brain-specific transcription is regulated by methylation at an epigenetic level much more frequently than tissue specific expression in other organs. Low methylation status is strongly associated with neurological and cognitive deficits.
Many epidemiological studies have shown that factors connected to low methylation status such as elevated total homocysteine, low folate or low cobalamin levels are associated with increased risk of cognitive decline, dementia and brain atrophy. In recent years, hyperhomocysteinemia has begun to be widely considered a risk factor in AD and this may be ascribed to alteration of the S-adenosylmethionine/homocysteine (SAM/HCY) metabolism. SAM is known to be the primary methyl-donor present in eukaryotes and it is involved in methylation of target molecules as DNA, RNA, proteins, lipids, and polyamines synthesis. SAM appears to be altered in some neurological disorders, including AD. About 95% of SAM is engaged in methylation reactions. It is then transformed in S-adenosylhomocysteine (SAH) and further hydrolysed into homocysteine (HCY) and adenosine. The reaction is strongly reversible and HCY, if not rapidly transformed into methionine or cystathionine, forms SAH, which is a potent inhibitor of methyl-transferases. These metabolic alterations may be responsible for the generalised reduction of DNA methylations observed in ageing, which can lead to the overactivation of methylation-controlled genes.
With this WP we aimed to define the DNA methylation pattern of neurodevelopment and AD relevant genes during both development and ageing, and to verify the efficacy of epigenetic drugs (SAM) in modulating DNA methylation. Thus, we studied gene regulation mediated by CpG methylation in developing and ageing TgCRND8 mice, and in healthy and AD individuals, focusing on genes related to neurodevelopment and neurodegeneration. Within this framework, we investigated if/how methylation patterns typical of early stages of neurodevelopment vary during normal and pathological ageing. The rationale for selecting specific genes is: (i) they play essential roles both in developing and adult brain, being involved in neuronal migration, synaptic connectivity formation and plasticity (Reelin and BDNF), and in neurodegeneration (PSEN1); (ii) their expression is subjected to an epigenetic control.
The three genes studied, Psen1, Reelin and BDNF, showed a complex expression pattern, depending on the age, genotype and gender. A clear correlation between promoter methylation and expression patterns (transcripts/proteins) was observed only for Psen1. DNA methylation analyses by bisulfite modification and DNA sequencing showed that Reelin and BDNF promoters display a very low level of CpG methylation. No age/gender/genotype-dependent differences were observed, leading to the conclusion that Reelin and BDNF expression are not modulated by DNA methylation in TgCRND8 and WT mice. We found a clear correlation between promoter methylation and expression patterns (transcripts/proteins) for PSEN1. In particular, female mice showed PSEN1 promoter hypomethylation at PN90. We demonstrated sexually dimorphic expression of Reelin and its downstream signalling pathway.
We analysed the methylation status of PSEN1 in cortices and hippocampi from healthy and demented human individuals to evidence differential methylation patterns between different brain areas and stage of disease progression. DNA methylation of this gene was assessed through bisulfite modification techniques. This data were correlated to the conditions of normal ageing or dementia (and related stage). Methylation data indicated no difference in methylation of CpG moieties studied by bisulfite assay during early neurodevelopment (from 14 GW (gestation week) to 40 GW). During late neurodevelopment (2 months and 17 years), PSEN1 CpG methylation is significantly higher than it is in embryos, with no differences between the two conditions. CpGs in the PSEN1 promoter are hypomethylated in aged subjects; methylation further decreases in AD patients. Non-CpG methylation was detected at very low level during early and late neurodevelopment (14 GW to 17 years), although it slightly increases from 17 years to elderly. In AD subjects, non-CpG methylation decreases significantly compared to healthy controls. Both CpG and non-CpG methylation are decreased in AD subjects versus controls. PSEN1 promoter hypomethylation, in the cortex of AD subjects, correlates with the gene expression.
We assessed the DNA methylation of selected up- and down-regulated genes involved in AD-related pathology, as identified in WP3, to reveal whether modulation of these genes corresponds to the methylation of CpG sites. The three genes selected (based on up-regulation of their expression during the early AD stages and supporting literature) belong to the family of activated pro-inflammatory cytokines (IL-1B, Interleukin 1B; IL-6, Interleukin 6) and C3AR1 (Complement Component 3A Receptor). The expression of IL-1B is markedly up-regulated in the early phase of the pathology. It is then down-regulated (although still higher than in control samples) in the late phase, which is correlated with markedly reduced non-CpG methylation in AD I-II subjects compared to control and AD V-VI samples.
An aspect of therapeutic intervention is the application of SAM, which has already been shown to partially silence overexpressed genes. However, with this WP we had the opportunity to evaluate the restorative effects of treatment with SAM in mouse models of AD.
We evaluated the efficacy of perinatal and postnatal SAM supplementation on DNA methylation modulation in TgCRND8 mice. The aim was to analyse the effect of epigenetic drugs (SAM) on DNA methylation patterns of neurodevelopment-related genes by means of SAM supplementation during the perinatal period and as adults. The effect of SAM effect on at least 5 neurodevelopment- and AD-related genes in mice was determined. SAM supplementation to pregnant mice (3 pregnant females per strain, corresponding to about 10-15 embryos) allowed the evaluation of the effect on gene methylation and expression during brain development in embryos through the collection of embryonic brain areas. 3 additional pregnant females per strain were treated in parallel, but were not sacrificed, in order that perinatally-treated pups could be analysed as adults (3 months), when amyloid plaques normally appear. Analyses were repeated on these mice versus untreated mice. Moreover, DNA methylation, gene and protein expression were evaluated in these perinatally-treated mice at the adult stage for those AD-related genes, already known to have altered DNA methylation and gene expression in these mice (PSEN1).
WP5 Genetic influences on normal and pathological brain ageing
Age-related cognitive decline and dementia is one of the major threats to the quality of life of the elderly. Definition of factors in the background of dementia in the elderly is hindered by the fact that humans differ in their cognitive abilities. It is far from clear how to distinguish non-pathological from pathological brain ageing. Age-related cognitive decline forms a continuum and people’s cognitive level in old age depends also on their intelligence and cognitive abilities. Thus, studies performing cross-sectional analysis must be interpreted with caution, either in the sense that non-pathological cognitive ageing is not invariably non-pathological, or vice versa. One problem in distinguishing normal and abnormal ageing is that AD-like changes are also seen in the elderly without cognitive decline or dementia. Thus, the most critical issue for progress in genomic studies of cognitive ageing is the identification with strict criteria and selection of appropriate phenotypes.
This WP was intended to provide whole genome sequencing of elderly individuals selected according to morphological criteria of healthy ageing in the brain and compare these with AD type brain ageing. The major advantage of our approach was that we evaluated very strictly defined cases that lacked morphological/biochemical evidence of neurodegeneration-related alterations.
To fulfil these criteria we identified brains from individuals above the age of 80-85 years without or with very mild AD-related neuropathological alterations, and brains from individuals with end-stage AD (Braak & Braak stage V-VI). Cases were selected from the Vienna Trans-Danube Ageing (VITA) study. Since the year 2000, the VITA study has followed longitudinally a community-based cohort of every inhabitant of the Vienna area on the left shore of the river Danube (districts 21 and 22) born between May 1925 and June 1926. Neuropathological evaluation included a systematic evaluation of 2x2.5 cm sized paraffin embedded tissue block sections from at least 15 anatomical regions. In sum we examined approximately 4,600 immunostained sections. The neuropathological examination demonstrated the high variability of pathological alterations in the ageing brain, which supported our concept of using very strict criteria to select cases for genome analysis.
During the evaluation of this cohort we noticed that there is a difference in the subregional tau pathology of the hippocampus in tauopathies when compared to Alzheimer’s disease. Thus, we performed an additional study in which we investigated the spatial and temporal distribution of hyperphosphorylated tau protein in a cohort of 105 patients stratified according the neuropathological diagnosis. Some diseases revealed unique combination of deposits within different cell types and neuropil in certain subregions. This may be the morphological background for clinical symptoms. In addition, some other diseases showed very similar patterns of the protein distribution. This explains similar clinical symptoms and difficult clinical diagnosis. Our data suggests that stratification of the patients with dementia, according to spatial and temporal distribution of pathologic proteins, is a key feature for correct diagnosis and possibly therapy development. We developed a mathematical model to compare tau constellations in the hippocampus. Our data suggest that some diseases are ‘closer’ to each other, whereas others are more distant.
Whole genome sequencing from neuropathologically characterised individuals without or with very mild AD-related pathological alterations (28 cases) and with end-stage AD-related pathology (12 cases) was performed using the Illumina HiSeq 2000 sequencing system platform. Bioinformatic analysis was then performed by Thrace University (Prof. Peristera Paschou, Thessaloniki, Greece). In order to enrich for functional variants, we focused on non-synonymous, splicing, stoploss and stopgain variants, which affect the transcript. We identified 41,493 functional variants before filtering for unique variants in cases and control individuals.
Our initial analysis focused on genes that are known to be involved in longevity phenotypes.
1. ApoE Haplotype and variants that have been previously implicated in longevity
The Apolipoprotein E (ApoE) haplotype has been widely studied and found to be one of the most consistent associations in human longevity studies. We were able to replicate published findings of the significant association with SNPs on TOMM40, ApoE and APOC1 genes, all found on chromosome 19.
2. Association tests of the sirtuin genes
In the next step we focused on the sirtuins. We evaluated sirtuins due to their association with longevity. Sirtuins have been implicated in influencing a wide range of cellular processes like ageing, transcription, apoptosis, inflammation and stress resistance, as well as energy efficiency and alertness during low-calorie situations. We were able to recover indicative association with markers on SIRT1, SIRT2, SIRT5 and SIRT6.
3. Association tests with SNPs that have been previously implicated in AD
We investigated the association of previously implicated Alzheimer’s disease related SNPs in our own sample of AD patients and normal brain ageing controls. Besides the APOE4 signal there was an indicative signal at SLC24A4.
Our further analysis revealed genes implicated in healthy ageing, genes involved with the nervous system like cell adhesion molecules, and genes that had functional roles in neurodegenerative diseases.
In addition to the whole genome analysis to define protective genes, we had the opportunity to study a family with neurodegenerative disease to define novel genes implicated in dementia. The family examined comprised of two affected siblings and one control aunt without symptoms. One sibling was clinically diagnosed with Alzheimer-type dementia and had a neuropathologically proven neurodegenerative disease with tauopathy, but not amyloid beta deposition. The other sibling was a clinically demented patient, who clinically resembled Alzheimer’s dementia. Neuropathology and biochemical examinations were performed. Our combined ranked method highlighted a number of interesting genes and their interactions in the background of the complex phenotype.
We examined the expression of sirtuins in healthy and diseased elderly, as well as in developing brain (hippocampus). Sirtuins were selected according to literature reports on their association with longevity and healthy ageing. Our whole genome analysis also confirmed that sirtuins do indeed play a role in healthy ageing. We microdissected and evaluated the hippocampus white matter and the entorhinal cortex using western blotting and immunohistochemistry for Sirt1, Sirt3, and Sirt5. We found that the protein level (western blot) show that with the progression of Alzheimer-related changes (Braak & Braak stages), the expression of Sirt1 and Sirt3 significantly decreases. Interestingly, the protein expression of Sirt5 showed positive correlation with the progression of Alzheimer’s disease pathology and showed an inverse correlation with the expression of Sirt1 and Sirt3. Based on these findings, we interpreted that Sirt5 could be upregulated as a compensatory response to the loss of Sirt1. Using a combination of immunohistochemical observations and immunoblotting on subcellular fractions of two selected cases (Braak & Braak stage II and stage VI) we were able to characterise the subcellular localisation of Sirt1, Sirt3, and Sirt5. With our unique approach we were able to define the precise subcellular localisation of sirtuins and their consequences on the development of brain pathology. These revelations can be used as rationale for developing therapies targeting the sirtuin system.
Furthermore, we focused on proteins related to age-related neurodegenerative diseases and we were able to perform additional experiments and studies. First, we focused on dementia with Lewy bodies (DLB) and Parkinson's disease (PD), which are frequent age-related neurodegenerative conditions characterised by the deposition of disease-associated α-synuclein. Our findings suggested a prion-like cell-to-cell spread of α-synuclein in neurons by uptake from surrounding structures. Second, we performed a study to evaluate whether epilepsy-related changes can be detected in Alzheimer’s diseased brains. In order to achieve this we evaluated the expression of Calbindin (Cb), one of the major Ca2+ binding proteins, that exhibits neuromodulatory functions such as long-term potentiation (LTP), synaptic plasticity, and memory functions. Cb is expressed in hippocampal interneurons, pyramidal cells and granule cells of the dentate gyrus (DGCs). A recent study suggested a link between Amyloid beta-induced Alzheimer's disease-related cognitive deficits and neuronal depletion of Cb. To evaluate whether this is specific for Alzheimer's disease, we performed a comparative study of Cb immunoreactivity of DGCs in cases with Alzheimer's disease-related neuropathologic change, grouped according to the stages of Braak & Braak, Creutzfeldt-Jakob-disease, FTLD (frontotemporal lobar degeneration)-tau Pick's disease type, argyrophilic grain disease, and FTLD-TDP types A and B. We concluded, that late stage Alzheimer's disease-neuropathologic change (Braak & Braak stages V and VI) associates with significantly higher ratios of Cb negative DGCs and this correlates with advanced Braak & Braak stage. This might suggest an accumulative effect of an epilepsy-like pathway on the Cb expression or the direct influence of local pathological protein deposits on the DGCs.
Finally, we analysed genome-wide sequencing data for one healthy old mouse lemur (MIM) and one animal with an Alzheimer phenotype. The initial part of this project focused on quality control and pre-processing of the raw sequence data obtained from the high throughput-sequencing instrument. We are now focusing on the comparison of the sequence with those of humans.
In summary, we achieved the goals and tasks for WP5. We were able to perform whole genome analysis using very strict neuropathology-based criteria and a longitudinal ageing cohort. We were able to select a gene product (sirtuins) that plays a role in healthy ageing, which was confirmed by literature data and our own whole genome studies. We were also able to evaluate and characterise their expression in healthy and pathological ageing in normal and Down syndrome developing brains. In addition, we were able to expand the literature data on the pathogenic role of neurodegeneration-related proteins, in particular of -synuclein and we were able to show morphological data on the pathogenesis of Alzheimer’s disease (epilepsy-like alterations). Finally, we were able to perform the first comparative genomic analysis of humans and the non-human primate model.
WP6 Animal models for ageing
Adequate animal models are crucial to study human brain ageing. Most of the previously used models have been genetically modified rodents and have proved highly important for modelling specific aspects of neuropathological disorders including AD. Unfortunately, results obtained in these models cannot necessarily be extrapolated to humans. Thus, the use of non-human primates has been recommended for the testing of drugs before developing a therapeutic approach in humans. An interesting alternative non-human primate model for human ageing and ageing related neuropathologies is one of the 18 species of mouse lemurs, the gray mouse lemur, Microcebus murinus (MIM). It is small, nocturnal, non-human primate, genetically much closer to humans than rodents. Its lifespan is no more than 8 years under natural conditions, but up to 15 years in captivity. It has been maintained and bred successfully in captivity since the 1960s. Comparable to humans during ageing, some, but not all captive mouse lemurs naturally display some of the neuropathological signs of AD with the presence of amyloid beta plaques and/or neurofibrillary tangles, the development of cerebral atrophy and sensory, motoric and cognitive deficits.
DEVELAGE has unique access to two of the world’s largest colonies of the MIM model with a total of more than 350 individuals, representing all different age cohorts as well as matrilines with AD-like brain disorders. This allowed us to use the colonies in France and Germany to pursue the establishment of a standardised and integrative emotional, hormonal, cognitive, neuro-imaging and genomic approach: to disentangle for the first time healthy ageing from AD-like phenotypes at an early stage and to compare molecular pathways linked to the development of AD in the human and non-human primate brain.
This WP was designed to promote interdisciplinary collaboration to increase our understanding of the causes of AD and translate this knowledge into innovative approaches for preventive therapeutics of neurodegenerative disease. This was achieved through the development of genetic and genomic aspects, brain imaging and a behavioural and cognitive phenotyping platform. The integrative approach applied to this new animal model has led to novel possibilities for modelling human ageing and exploring new avenues for therapeutic intervention of related ageing disorders such as Alzheimer's disease.
We performed a genomic analysis to establish groups for phenotyping and to identify candidate genes for AD. We sequenced the whole genome of one AD-like carrier (showing numerous amyloid beta deposits in the brain) and one wild-type MIM matched for age. The phenotypic selection of animals by in vivo diagnosis of cerebral atrophies (using MRI) and by ex vivo diagnosis of amyloid deposits (by immunohistochemistry) was performed to increase the number of AD-like animals. We established in vivo Magnetic Resonance Imaging (MRI) protocols for MIM to discriminate between healthy ageing and AD.
We aimed to establish and validate behavioural and hormonal tools to phenotype subjects in both colonies, to apply them to discriminate in the long run between wild-type phenotypes and AD-like carriers and thereby to validate the mouse lemur as a primate brain ageing model.
Previous research on AD in humans and animal AD models had discussed if the prevalence of AD is linked to an emotional phenotype reflected in the personality traits of anxiousness and novelty-seeking and to chronic stress hormones. To measure phenotypic markers of emotionality in mouse lemurs and to validate them for repeated usage in the same individuals, we established a behavioural test battery, commonly used in biomedical research, the ‘sleeping box emergence’ test, the ‘open-field’ test and the ‘novel object’ test. With the behavioural traits measured, it was then possible to characterise the emotional phenotype of an animal by the two personality traits ‘anxiety’ and ‘novelty-seeking’. We performed cognitive phenotyping of animals in order to explore how this linked to potential predictors of diseases. Furthermore, we collected stool samples from MIM, fed with a standardised diet, and phenotyped emotionally and/or cognitively. Additionally, a MIM validated ELISA test, in collaboration with the German Primate Center in Göttingen, to determine the glucocorticoid metabolite levels in the stool samples, as an indicator for chronic stress. For cognitive evaluation we aimed to translate cognitive tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) to the model MIM. The CANTAB battery, developed 25 years ago by the Cambridge University, is a well renowned set of tasks, which allows the assessment of the cognitive abilities of humans. By testing subjects with these rapid, computerised and standardised tasks, disorders in cognition may be detected at an early stage. Performance failure in a specific test may help to characterise which brain region is affected and may help in diagnosing age-related disorders like AD.
We programmed, explored and optimised training and testing protocols of three cognitive tasks of the CANTAB battery for the model MIM.
Two learning and memory tasks, named the visual pairwise discrimination task (PD) and its reverse (PDR), explored appetitive, visual associate learning as well as cognitive flexibility. They are claimed to be sensitive to alterations of the medial temporal lobe (visual discrimination learning) and the prefrontal cortex and interconnected subcortical areas (cognitive flexibility). Tasks were performed in an experimental chamber, specifically adapted to MIM and based on an operant conditioning paradigm with positive reinforcement. We trained 48 mouse lemurs in the PD pretask training. 34 subjects entered the actual PD task with 32 of them reaching the desired 80% criterion in both PD acquisition and the reversal phase. Among the 14 animals, which failed to be trained in the pretask training, most of the animals (N=9) could not reach the criterion in the ‘Must Touch’ training phase. This was probably due to a lack of motivation to touch the screen.
The performance in the PD acquisition and PDR task of one group of 20 young adults (10 males, 10 females; mean age = 2.6 years; range 1.1 to 4.1) and one group of 10 aged adults (4 males, 6 females; mean age = 7.9 years; range 6.9 to 9.5 years) was compared. In the PD acquisition we found a significant difference in the number of sessions and trials required to reach the criterion between young and aged adults. Young mouse lemurs needed fewer trials than aged ones to reach the criterion. Great inter-individual variability in the number of trials to reach the criterion was found: the fastest animals (2 young: 1.1 and 2.4 years old) reach the criterion within only 120 trials (4 sessions), the slowest (the oldest: 9.5 years old) needed 780 trials (26 sessions). Lastly, we checked whether there was a difference in the attention to the task and in the motivation between the age groups. We found that young and aged mouse lemurs did not significantly differ in their latency to respond after the stimuli were displayed, or in their latency to collect the reward. Within each age group, we did not find any significant differences in these variables between the sexes.
In the PDR task, we found a statistical trend (p < 0.1) towards a difference in the number of sessions and a significant difference in the number of trials required to reach the 80% learning criterion between young and aged adults. Young mouse lemurs needed fewer trials than aged ones to reach the criterion. Again, great inter-individual variability in the number of trials to reach the 80% criterion was found: the fastest animal (1 young: 3.1 years old) reached the criterion within only 208 trials (7 sessions), the slowest (the oldest: 9.5 years old) needed 1800 trials (60 sessions) which was a far outlier (young range: 208-960; aged range: 300-1020). Among young adults, we found that females tended to require fewer trials to reach the criterion than males. We did not find this difference among aged adults, but the sample size was smaller though. Young and aged mouse lemurs did not significantly differ in their latency to respond after the stimuli display or in their latency to collect the reward. Within each age group, we did not find any differences in these variables between sexes.
We explored in a subgroup of animals, for which data on the hormonal, emotional and cognitive level were available by linear mixed models, whether different factors such as emotional phenotype and hormones predict cognitive performance, so that AD-like cognitive decline may be identified at an early stage. Findings showed that faecal glucocorticoids were more strongly related to cognitive performance than to personality traits.
All in all, our findings provide the first evidence in MIM of age-related effects on learning and memory, comparable to humans. Our integrative approach shows that MIM mimics the complex phenomena of human brain ageing and AD-like dysfunctions much closer than other known primate models. The DEVELAGE approach thus presents promising avenues to identify cognitive decline at an early stage in MIM, and to relate them to potential AD-predictors such as dysfunctions in brain biochemical signalling pathways, candidate genes, brain atrophies, chronic stress hormones or personality traits. DEVELAGE has made important steps towards further characterisation of this novel animal model for ageing and AD-research on the cognitive level.
Potential Impact:
Potential impact (including socio-economic impact and wider societal implications)
The increasing number of elderly people, resulting from an ever increasing life-expectancy, will have a major impact on the prevalence of age-related diseases in the near future leading to enormous costs and challenges for health systems in Europe. These diseases have dramatic repercussions on our society and health systems resulting from the involved nature of patient care and the fact that there is currently no treatment to halt their progress. DEVELAGE has contributed to all of the expected impacts as listed in the work programme and the technical annex. DEVELAGE has used a multidisciplinary basic biomedical approach to perform world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s disease (AD). DEVELAGE has addressed issues starting from understanding brain development and the molecular pathways underlying age-related neurodegenerative disorders, through the use of methylation sensitive genes to understand the influence of gender on Alzheimer’s disease and characterising the mouse lemur as a new experimental model of ageing. DEVELAGE ultimately aimed to produce basic research with the potential to inform new treatments and prevention schemes by uncovering drug targets or regulatory mechanisms to interfere with. In this sense, DEVELAGE has contributed a wealth of new findings to address the urgent need for novel strategies to reduce the burden of age-related common degenerative disorders on society.
Specific impact of DEVELAGE actions and results
In the DEVELAGE project we evaluated proteins characterising age-related neurodegenerative disorders and their modifications. We confirmed that brain development and ageing are linked, and provided evidence that pathways, which are important for the developing brain, are altered in epilepsy or Down syndrome (DS) and could also contribute to the pathogenesis of Alzheimer’s disease. In particular we provide the first description of the expression pattern and cellular distribution of components of mTORC1 in human hippocampus during the pre- and early postnatal development in DS patients. These findings suggest a critical role for mTOR signalling pathway in the DS hippocampus. They support the idea that a dysregulated mTOR pathway may contribute to the early disease pathogenesis and that hippocampal functional impairment, involving this pathway, may develop before neurodegeneration. Future investigation targeting mTOR signalling in DS experimental models might be worthwhile to further develop our current understanding of the role of this pathway, as a link between neurodevelopmental disorders and neurodegeneration.
We investigated the role of miR-146a, (which is upregulated in Alzheimer’s disease) in the modulation of astrocyte-mediated inflammation. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. With this result we defined an important role for miR-146a in human neurological disorders associated with chronic inflammation, which may represent a novel target for therapeutic strategies. Implementation of anti-inflammatory miRNAs at brain levels by synthetic mimics may represent a strategy for preventing the pathologic consequences of neuroinflammation associated with neurodegeneration, which is worth pursuing in experimental models.
We characterised key mechanisms involved in neurogenesis including the role of neurotrophic factors. We have made significant steps towards an improved understanding of the development and ageing of the hippocampus and the entorhinal cortex, which are crucial structures for human memory, where Alzheimer’s disease originates. The mechanisms of hippocampal neurogenesis are mainly investigated in adult animal models, providing preclinical models for therapeutic strategies. However, it is well known that differences between species exist. Our studies provide a gold standard for the knowledge of various progenitor subtypes and the mechanisms involved in neurogenesis at various stages of life, in both healthy people and during neurodegenerative diseases, and will help in improving the design of potential therapeutic strategies when translated from experimental studies.
DEVELAGE mapped the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology. We established what happens in the brains of aged individuals before clinical symptoms related to Alzheimer’s disease arise. We identified a major shift in brain energy metabolism and increased oxidative stress damage. In addition an altered composition of lipids and an altered neuroinflammatory response precede the outbreak of symptoms. The study provided a wealth of data, enabling further study, and providing novel targets for therapeutic strategies. Olfactory and taste receptors have been implicated for the first time as new chemoreceptors in the brain, which are deregulated in neurodegenerative diseases. We recognised early neuroinflammatory responses in the aged brain, which are linked to the presence of soluble oligomers and precede the appearance of tau deposits and amyloid plaques. Together these serve as a rationale for developing treatments geared to the prevention of age-related molecular changes that fuel neurodegenerative diseases in old age.
We characterised methylation sensitive genes in normal ageing and the early phases of AD-like neurodegeneration We explored the pattern of DNA methylation in many neurological disorders, such as Alzheimer’s disease. Based on our DNA methylation maps of mouse and human brain we identified new target genes in Alzheimer’s disease. Our study of the methylation and expression patterns of our selected candidate genes (BDNF, Reelin and PSEN1) showed that Reelin and BDNF mRNA and protein levels are independent on DNA methylation of their gene promoters. The higher PSEN1 expression in females seems to be dependent on DNA hypomethylation at specific CpG sites
The finding that Reelin, BDNF and PSEN1 expression, in addition to age and genotype, are also influenced by gender represents a major breakthrough. Our findings highlight gender as a novel and important variable to be considered for further studies aimed at characterising molecular mechanisms underlying AD pathogenesis. In fact, it offers a novel framework for a better understanding of the higher AD incidence observed in the females of our species.
We used precise morphological definitions and whole genome sequencing to evaluate genetic constellations of healthy state brain in oldest-old individuals. In the DEVELAGE project we have compared a group of individuals who demonstrated normal brain ageing to individuals with Alzheimer’s disease in search of a protective factor. This work also has the potential to serve as basis for the development of personalised therapy. Indeed, we were able to define a target molecule for the family of sirtuins that we evaluated in brain tissue to assess their role in the progression of disease. We observed complex patterns of sirtuin expression in correlation with tissue damage, which has the potential to inform the development of therapies interacting with sirtuins. In addition, we demonstrated that human brain cells internalise disease-associated α-synuclein reminiscent of that of prion protein seen in prion diseases. This observation in humans supported the notion of prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures based on experimental studies. Demonstration of this mechanism in the human brain is crucial for the development of therapies against Parkinson’s disease or dementia with Lewy bodies since it suggests that therapies, which block the uptake of pathological α-synuclein, may prevent the damage of neurons affected in these disorders. In this study we used a novel antibody that selectively binds to the pathological but not to the physiological normal form of α-synuclein. We used a complex approach based on electron microscopic examination, which enabled us to visualise how the pathological molecules are transported in the cells.
We characterised and evaluated the gray mouse lemur, a non-human primate model of ageing, using an integrative approach. Firstly, we sequenced the whole genome of one AD-like carrier of numerous amyloid beta deposits. Our analysis of the genetic variations of the APP gene, which is involved in Alzheimer’s disease, seems to show that this gene is also associated with the AD-like trait in the mouse lemur. This result supports the case for this species being a very good primate model in which to study Alzheimer’s disease.
We have succeeded in establishing for the first time a validated behavioural test battery for the model mouse lemur with which boldness, neophobia and general activity can be measured reliably. These are phenotypic markers of emotionality implicated as indicative of the presence of AD. We successfully implemented a CANTAB comparable cognitive test battery and translated three tasks with which we could compare cognitive performance of cohorts of young and aged mouse lemurs.
All in all, the establishment of these methods is the first step towards the further validation of brain imaging, behavioural and cognitive phenotyping tools, in this new primate brain ageing model across laboratories, an important issue for the usage of this primate model for therapeutic interventions.
There are numerous direct beneficiaries from the knowledge generated by this research project. These include the investigators working on the project and their staff and students, who have benefited from training and experience and are likely to continue investigating age-related common degenerative disorders under additional peer-reviewed external funding. Additional beneficiaries are the DEVELAGE partners, whose awareness of the role of specific issues involved in age-related common degenerative disorders has been substantially enhanced by regular discussion in DEVELAGE project meetings, and the global scientific community, who will benefit from access to data when these are published in peer-reviewed journals.
Impact on health care issues
Population ageing is a highly generalised process. It is most advanced in the most highly developed countries. With the ageing of population, diseases such as AD type dementia will affect more and more people. AD affects 3 to 5% of the population aged between 65 and 74, with this figure increasing to around 50% in those over 85 years old. Society problems linked to dementia are numerous and affect not only patients, but also those in their surroundings, as most patients require long-term care. Dementia is a progressive brain dysfunction, leading to a restriction of daily activities. People with dementia suffer mainly from impaired memory and orientation, limitations of concentration, planning and judgement, personality changes and later also perceptual, speech and walking disorders. During the course of dementia, patients often lose their independence in managing everyday life. There are effects on perception and social relationships; people become more and more dependent on care. The average annual financial cost of dementia in the UK has recently been estimated by the Alzheimer’s Society (UK) to be £32,250 (approximately €44,400) per person (including costs of healthcare and social care at home or hosted in long stay establishments). If no progress is achieved in the understanding of how preventive therapy could be initiated, the consequences could be dramatic; even the collapse of European and world-wide health-systems.
DEVELAGE results could lead to improvements in the health of European citizens by having addressed a major health problem with a particularly effective approach. The results of the DEVELAGE project will enable researchers to define the first alterations in the human brain preceding the development clinical symptoms. These observations could be translated either into biomarkers that can predict cognitive decline or help to define targets for neuroprotection. This is crucial, since currently the effectiveness of the therapies used is reduced due to their relatively late application, i.e. when the symptoms are already present. This concept is also supported by genomic studies defining protective and risk constellations. In summary, the definition of ‘protective’ and ‘risk’ genomic and transcriptomic constellations can be used for predicting when preventive therapy should be initiated and the characterisation of proteins that may be used as biomarkers with predictive and prognostic value for age-related disorders. This could lead to a reduction in the costs of nursing of patients and reduce the time lost from active work, since therapy can be initiated at an early phase of disease. Elongation of a patient’s active life period by even 3-5 years can dramatically decrease the workload and emotional distress of family members and society. DEVELAGE has presented further findings, which represent the first important steps towards developing effective therapies. The demonstration of the internalisation process of α-synuclein in neurons provides support for the development of vaccination strategies to block the uptake of pathological α -synuclein. The description of the role of sirtuins could help to develop better targeted therapies acting on sirtuins, increasing the likelihood of therapeutical trial success by providing the exact knowledge of what happens in the human brain during the progression of Alzheimer’s disease. The discovery of microRNA disturbances opens up an innovative novel approach to modify harmful aspects of brain disease. In addition, the studies on the developing brain clarify many open questions as to how some brain structures are formed. This is important for individuals with mental retardation and in particular for individuals with Down syndrome. Several observations in the DEVELAGE project defined differences which are already present during the brain development of normal and Down syndrome individuals. These aspects can be used to define strategies leading to the improved mental capability of individuals with Down syndrome and could also help to retard the development of Alzheimer’s disease in these individuals. Our findings on the methylation of genes in Alzheimer’s disease supports the notion that therapies or even food supplements (like SAM) could be effective in hindering harmful pathways in Alzheimer’s disease. Finally, for the development of any therapies a reliable animal model is needed. DEVELAGE has been able to make the first steps towards the validation of a highly promising non human primate animal model of brain ageing.
Cross-thematic impact
Currently, researchers either work in the field of developmental neuroscience, the field of age-related neurodegeneration or in behavioural neuroscience, and the cross-talk between these disciplines is rare. Our project is well positioned to have an impact on all of these fields and opens the pathways for collaborations. DEVELAGE provided several results on the neurogenesis in brain structures important for cognition and memory. These are important for researchers in the field of Alzheimer’s disease, since they allow us to better understand which cell populations might be disease-sensitive and need support during age related cognitive decline. The issue of neurogenesis in the ageing brain is still an open question; accordingly our observations contribute to this field by showing how the cell layers develop in the hippocampus and related structures, and which cell populations may have the capacity to provide new cells, even in the ageing brain. Conversely, our strategy to evaluate neurodegeneration-related protein in the developing brain also revealed important results, in particular the demonstration of different patterns of tau phosphorylation in the developing brain as compared to the developing Down syndrome brain. It is important that we evaluated factors important for the brain development in the ageing brain, which was an important link between the two disciplines. Findings of ageing-protective genetic constellations also provide information for researchers in the field of development; in fact we found several genes which are important in brain development and show up as protective in brain ageing. These concepts were also implemented in a non-human primate model where we provided the first rationale to evaluate the animal model from developmental aspects and not only from the ageing aspect. These results render the DEVELAGE project as a successful model to bring researchers working on seemingly different fields together in highly collaborative research.
Competitiveness and European added value
DEVELAGE has improved the competitiveness of European Universities and research-based SMEs by collaboration on methodological and thematic aspects that will bring forth added value to all participants. DEVELAGE has hence increased the innovative capacity of European health-related industry and business by facilitating tight interactions with world-class European researchers that will probably last beyond the end of the project. DEVELAGE collaborations have thereby strengthened the international competitiveness of European academies and SMEs that will generate added European value for SMEs and academic research.
By bringing together internationally renowned experts in closely interacting work packages, DEVELAGE has achieved world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s disease. The complementary expertise of partners in project relevant fields of basic and clinical research has been successfully organised. DEVELAGE facilitated sharing of methodologies between a critical mass of partners thereby potentiating their scientific impact. Moreover, DEVELAGE has made technically sophisticated methodologies available to its partners, such as: the exchange of protocols for brain imaging; behavioural phenotyping and cognitive phenotyping of the primate brain aging model mouse lemur (UMP2/TIHO, TIHO/UMP2) as a prerequisite to standardise methods; the exchange of protocols for the characterisation of dysfunctions of brain molecular pathways in mouse lemur and human brains (MUV/UMP2); and the characterisation of mouse lemur brains phenotyped by UMP2/TIHO with comparable immunhistochemical methods as used in humans (MUW/TIHO/UMP2). Overall, DEVELAGE has brought about breakthroughs in research on age-related common degenerative disorders. DEVELAGE has produced knowledge that will significantly contribute to the development of pharmacologic strategies to treat age-related common degenerative disorders, including Alzheimer’s disease. The results of DEVELAGE could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
The strategic importance of this research project lies in the fact that there is an important potential demand and that the European Union must be at the edge of technology in the field. Indeed all European countries are concerned by age-related neurodegenerative diseases and therefore the efforts have to be considered at the European level. Indeed, the goals of the Joint Programming in Neurodegenerative Disease project (JPND) were defined as to find cures for neurodegenerative diseases and to enable early diagnosis for early targeted treatments (http://www.neurodegenerationresearch.eu/). DEVELAGE has impact on these goals, since our aims comprise also the characterisation of the earliest stages of AD. DEVELAGE also fits well with the Innovation Union strategy, to enhance European competitiveness while tackling societal challenges (http://ec.europa.eu/research/innovation-union/index_en.cfm?section=activehealthy-ageing). With the implementation of this project the European investment into basic research of high potential for the health industry is strengthened at a time when the US is cutting investments into research. This investment will therefore create career opportunities for young researchers in Europe and strengthen an important area of research in Europe.
Main dissemination activities and exploitation of results
Professional project management and dissemination of results has also significantly contributed to the successful collaboration of the DEVELAGE beneficiaries. The Coordinator has encouraged all participants to actively disseminate their project aims and results to colleagues, stakeholders and the wider public
At the beginning of the project, a logo and an overall project design was created (see Figure 4) in order to identify the project. It is used together with the Grant Agreement number HEALTH-F2-2011-278486 and the FP7 logo on any printed and electronic issues for the public and for any other official contact.
The DEVELAGE project was very successful in attracting the attention of two major media outlets, including being featured by the EuroNews award winning science documentary Futuris in their video ‘Deep inside the brain’, which was estimated to have been viewed by approximately 1 million viewers and coincided with the European Month of the Brain initiative in May 2013. Additionally, the DEVELAGE project was selected as one of the most interesting research projects currently being funded by the EC on cognitive ageing for a documentary filming by ASAPS www.asaps-sharingage.eu (see Figure 5).
On the web, public awareness of DEVELAGE has been addressed by the project website www.develage.eu. The website content was regularly updated and includes key background information about the project and its beneficiaries. Additionally, fact sheets are available in 5 languages for external stakeholder to be able to obtain information at a glance. The news and media section provides visitors with the latest publications and dissemination activities of DEVELAGE, as well as a series of DEVELAGE video clips, which were released over a period of months during the 2nd year of the project, in order to generate significantly greater exposure for the project and increased impact.
A major step for the dissemination of the project, both in this reporting period and as a lasting tribute to the achievements and impact created by the DEVELAGE project, was achieved through the creation and launch of the DEVELAGE ‘gateway’ website. The concept for the new ‘gateway’ website was to provide a project overview with a focus on: partners, results, collaboration and impact. This would require neither updates nor maintenance in the future and would represent a digital final results summary, thus providing an effective substitute to the planned print version, but with the considerable advantage of increased visibility and the possibility to update if required. The focus of the ‘gateway’ website is on large images and graphic elements with the text kept deliberately short to provide a quick summary overview to the whole project. The website was designed in a modular fashion to allow easy implementation of possible future updates, such as announcements of symposia, workshops and other public events. The new ‘gateway’ website also provided the opportunity to accommodate changing technology trends by providing full compatibility for viewing on mobile devices, including mobile phones and tablets.
The original project website was updated throughout the reporting period and is now archived but still accessible by means of a prominent link available on the new ‘gateway’ website, with a prominent reciprocal link also featured on every page of the original website redirecting back to the summary page. In this way interested parties still have access to the information available on the original DEVELAGE website.
The Consortium also took measures to provide DEVELAGE exposure to the public through conventional PR media work and new media, which complemented the public access website, particularly for those that do not have internet access.
The scientific community has a vested interest in keeping up with DEVELAGE progress and results to build on the body of knowledge that currently exists. This will be achieved through conventional scientific dissemination means, i.e. peer reviewed publications and presentations at international conferences. 53 peer-reviewed publications were published during the project funding period as part of the project and partners have presented DEVELAGE work world-wide 73 times at international scientific conferences. These publications also included a significant number with contributions from multiple partners, emphasising the impressive level of cross-consortium cooperation, which the DEVELAGE project has demonstrated. A number of additional publications are at the preparation or review stage and are expected to be published in the near future.
Major dissemination highlights during the last year of the project included the DEVELAGE satellite symposium being accepted by, and organised as part of, the XVIIIth International Congress of Neuropathy, which was held in Rio de Janeiro, Brazil in September 2014. This symposium was attended by 50 congress participants and received extremely positive feedback with the conclusion of the attendees being that the concept is very novel and highly interesting. Additionally, a further DEVELAGE symposium under the title ‘Linking Pathways in the Developing and Ageing Brain with Neurodegeneration’ was organised by the project management team, and held at the Medical University of Vienna, Austria on 27th November 2014, in month 35 of the project. The DEVELAGE Vienna symposium was held in cooperation with the Clinical Neurosciences (CLINS) Doctoral Programme, and consisted of talks presented by DEVELAGE partners and also young researchers from DEVELAGE and CLINS. In total the symposium was attended by 62 people. Three training events in Barcelona, Montpellier and Vienna were organised during the project, to provide an opportunity for young researchers from across Europe to benefit from the expertise and insights of the DEVELAGE project, these also included a ‘hands-on’ brain training course, restricted to 18 people.
List of Websites:
Website:
http://www.develage.eu/
Contact details:
Project Coordinator
Medizinische Universität Wien
Prof. Gabor Kovacs
Clin. Inst. Neurology
AKH Wien 4J
Währinger Gürtel 18-20, 1090 Vienna, Austria
E-mail: gabor.kovacs@meduniwien.ac.at
Phone: +43-1-40 400 55 07
Fax:
biolution - DEVELAGE office
Helmut-Qualtinger-Gasse 2/2
1030 Vienna, Austria
E-mail: office@develage.eu
Phone: +43-1-786 95 95
Fax: +43-1-786 95 95 20
List of DEVELAGE partners
Beneficiaries
Medical University of Vienna (Coordinator) – Gabor Kovacs - Austria
The Bellvitge Institute of Biomedical Research - Isidro Ferrer - Spain
Amsterdam Medical Center – Eleonora Aronica - Netherlands
Sapienza University of Rome – Sigfrido Scarpa - Italy
Tierärztliche Hochschule Hannover – Elke Zimmermann - Germany
University of Montpellier 2 - Jean-Michel Verdier - France
Universite Paris Diderot – Homa Adle-Biasette - France
biolution GmbH (SME) - Iris Grünert - Austria
The European health care system is facing a crisis due to a steadily increasing elderly population and a concomitant increase in the prevalence of age-related diseases. Common age-related neurodegenerative disorders, including Alzheimer’s disease (AD), have dramatic repercussions on our society and health systems due to the involved nature of patient care and absence of a treatment. Understanding the link between brain development and aging is critical for advancing our knowledge of pathological ageing and defining new therapeutic and diagnostic strategies.
DEVELAGE focuses on characterising shared molecular pathways between early developmental processes in the brain and brain ageing in order to identify early pathological processes contributing to AD. DEVELAGE is based on the hypothesis that disorders of neural development contribute to age-related neurodegeneration. On the one hand, this means that factors essential for the development of the brain may have a role in neurodegeneration, and conversely those neurodegeneration-related proteins and genes might be important during the development of the brain.
The DEVELAGE project takes a unique brain-tissue-based approach using detailed molecular analysis of the spectrum of developmental and ageing changes in neural cell populations in the very same brain samples. The project as a whole has placed special emphasis on the evaluation of protein modification, altered cellular functions, gene expression and genetic and epigenetic alterations that take place during the development and ageing process. It further compares these results with transgenic mice and a non-human primate model, Microcebus murinus (MIM). Using these approaches, DEVELAGE contributed to: 1) the definition of distinguishing factors between normal and Down syndrome developing brains; 2) the understanding of the hippocampus and related structures, which are early affected in Alzheimer’s disease; 3) the understanding of early metabolomic and transcriptomic alterations in brains, which have not yet fully developed Alzheimer’s disease; 4) the understanding of the epigenetic alterations of genes and factors related to Alzheimer’s disease; 5) the understanding of the spectrum of brain disease affecting the brains of the elderly; 6) the understanding of protective genetic constellations associated with healthy brain ageing; 7) the characterisation of the non-human primate model as a competitive tool for research on neurodegenerative disorders.
Professional project management and dissemination of results significantly contributed to the successful collaboration of the DEVELAGE beneficiaries, whose active dissemination resulted in 53 peer-reviewed publications, with a number of additional publications expected. DEVELAGE work was also presented 73 times at international scientific conferences. Public awareness of DEVELAGE has been addressed with a website and press releases. DEVELAGE was very successful in attracting the attention of two major media outlets, including a documentary film by the EC-funded ASAPS project and ‘EuroNews’ award winning science documentary Futuris, where DEVELAGE was featured in their video ‘Deep inside the brain’, estimated to have been viewed by approximately 1 million viewers.
In summary, DEVELAGE was highly successful and met essentially all objectives on schedule. DEVELAGE has brought together internationally renowned experts in closely interacting work packages to achieve world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s Disease. DEVELAGE has improved the competitiveness of European Universities by collaboration on methodological and thematic aspects that will bring forth added value to all participants. The results of DEVELAGE have the potential to lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Project Context and Objectives:
The DEVELAGE context
The increasing number of elderly people resulting from an ever increasing life-expectancy will have a major impact on the prevalence of age-related diseases in the near future leading to enormous costs and challenges for health systems in Europe. Age-related common degenerative disorders (Alzheimer’s disease, AD; mild cognitive impairment; Parkinson’s disease, PD) affect ~20 million European citizens today, with women particularly affected especially with AD. If no progress is achieved in the understanding of how preventive therapy can be initiated, it could result in dramatic consequences or the collapse of European and world-wide health-systems. It is therefore of the upmost importance that scientists and physicians work together to translate the latest findings from basic research into new treatments and prevention schemes.
Age-associated cognitive decline and normal or non-pathological (normative) cognitive ageing is a human experience, which differs in extent between individuals. Such age-related cognitive changes have been ascribed to alterations in synapses and neuronal loss in the brain that are multiplied in pathological brain ageing. Experimental evidence suggests a complex scenario, including mitochondrial dysfunction, compromised stress responses, synaptic rearrangements and altered protein (e.g. amyloid beta, tau, or α-synuclein) expression, to cause age-related brain changes. Cellular genes that are mutated in neurodegenerative diseases code for proteins that are expressed throughout neural development. These proteins interact with numerous other cellular proteins that are components of signalling pathways involved in patterning of the neural tube and in regional specification of neuronal subtypes. These observations support the notion that neurodegeneration-related proteins are important during the normal brain development.
Many elderly individuals experience no major, or only limited, functional impairment emphasising the ability of the human brain to compensate for potential cognitive decline. Many fascinating studies have shown that new neurons are actively generated in the adult brain, mainly in the subventricular zone but also in the subgranular zone of the dentate gyrus in the hippocampus. Failure of adult neurogenesis may lead to defective memory, a characteristic feature of age-associated cognitive decline. However, it is not clear which factors influence adult hippocampal neurogenesis and its failure. Since neurogenesis and complex interaction of glial cells and neurons (“guidance”) are basic requirements of physiological brain development, comparison of these pathways in the most exclusive human-specific model (i.e. the developing brain) with normal and pathological ageing may serve as a rationale for defining targets for the maintenance of the physiological milieu for neurogenesis. Research on pathological ageing has for a long time focused on defining the causes of neuronal dysfunction and death during adult life. However, increasing evidence supports the concept that neurodegenerative disorders could represent a disorder of neural development.
Efficient therapeutic strategies for age-related neurodegenerative disease (i.e. AD) require an improved understanding of early stages of the disease, when pathology is still reversible. Known clinical features of neurodegenerative disease (NDD) occur at relatively advanced stages, when the degenerative process has overcome thresholds that render the process irreversible. However, practically nothing is known about changes at earliest stages of the disease, as the human brain remains one of the most inaccessible areas for biomedical research. Previous studies have shown that NDD are not the mere result of accumulation of altered proteins, as for example Aβ and tau in AD, but a deleterious interaction of different pathways results in impaired neuronal function. A complex scenario has led to the concept of neuronal exhaustion, which precedes neuronal death in neurodegeneration. Further identification of altered molecular pathways will serve to delineate a comprehensive scenario of first alterations in the entorhinal cortex and hippocampus in cases with AD-related pathology.
The factors contributing to the variability of age-related cognitive change comprise demographic, social, educational, medical, nutritional, and biological (genetic and epigenetic) influences. Epigenetic modifications, such as DNA methylation and histone acetylation regulate replication, transcription and translation activity contribute to chromatin modelling, epigenetic modifications and DNA imprinting. Methylation status is essential for normal brain biology, and low methylation is associated with markers related to neurodegeneration. Epigenetic mechanisms play a pivotal role in brain development and may contribute to the course and development of AD. It is conceivable that brain-specific transcription is regulated by methylation on an epigenetic level much more frequently than tissue-specific expression in other organs. Low methylation status is strongly associated with neurological and cognitive deficits. However, it is still not fully clear whether epigenetic changes actually represent a cause or a consequence of the disease. This is mainly due to the fact that epigenetic studies involved AD patients in an advanced stage of the disease. Thus, further research is needed to decide the rationale for therapeutic interventions improving the methylation status. In addition, it needs to be clarified whether and how epigenetic alterations of pathways crucial for the developing hippocampus happen, in particular which may have consequences later in life, mediated by aberrant epigenetic patterns. Alteration of SAM (S-adenosyl-methionine)/HCY (homocysteine) metabolism is known to occur in AD and neurodegeneration. It has the potential to be exploited as a target for therapeutic approaches to improve synaptic function and/or neuronal replacement in normal ageing and neurodegenerative diseases.
In addition to environmental factors, genetic influences also account for the variance in adult cognitive abilities to varying degrees depending on the age of the study group. The search for genetic contributions to cognitive ageing faces a lot of aspects, including the problem of non-reproducibility due to varying definitions. In large autopsy series, the oldest-old individuals (>85 y) are recognised with relatively well-preserved brain tissue and with mild or no deposition of proteins linked to neurodegenerative diseases. Strict neuropathology-based stratification of study groups can help to define protective genetic constellations contributing to healthy ageing. Pathways related to these genes open new avenues for the definition of targets for neuroprotective therapies.
Models as close as possible to humans are needed for a better understanding of age-related changes. So far, the vast majority of studies have been performed in rodents, but experimental findings and conclusions are highly dependent on animal and strain models. Moreover, the complexity of the primate brain with its behaviour and cognitive abilities are dramatically greater than in rodents, so results of neurological decline in rodents cannot be easily extrapolated to humans. Preliminary data shows that ageing MIM-brains may show some AD-like pathology. Defining a relevant animal model of age-related brain disorders is an important research strategy in ongoing trials to understand the pathology and to test new treatments.
In spite of efforts to clarify the causes and pathogenesis of AD, our current knowledge is insufficient to recognise the transition of normal brain ageing into AD-like brain disease when the process is still reversible. This is partly due to the fact that research focuses on clinically manifest or terminal stages of brain diseases or uses experimental conditions that are not easily translated to, or never validated for, human disease. Moreover, due to the plasticity of the brain, patients admitted to medical care after first symptoms have already severe loss of neurons together with modifications of proteins, which is a major restriction of effective therapy. At this stage only palliative but no preventive or curative therapy can be initiated. Thus, elucidation of complex pathogenetic pathways characterising the earliest stage of the detrimental road to AD in human brain is needed, since it represents the most promising opportunity for therapeutic intervention to reverse pathological processes and prevent onset of the disease.
The DEVELAGE project took a unique brain-tissue-based approach, with detailed molecular analysis of the spectrum of developmental and ageing changes in neural cell populations in the very same brain samples used for a wide array of investigations. We addressed age-related common neurodegenerative disorders, including Alzheimer’s disease (AD), that together affect ~20 million European citizens today, especially women. In addition, the project increased our understanding of normal as well as pathological brain development. This knowledge will contribute to our understanding of early pathological events in neurodegenerative disorders and prove instrumental in developing concepts for therapeutic intervention when a condition is still reversible
Thus, the OVERALL AIM of DEVELAGE has been to characterise shared molecular pathways between early developmental processes in the brain and brain ageing, in order to identify early pathological processes contributing to AD. This overall DEVELAGE aim encompassed the following objectives:
1) Evaluation of proteins characterising age-related neurodegenerative disorders and their modifications throughout brain development and ageing.
2) Characterisation of key mechanisms involved in neurogenesis including the role of neurotrophic factors.
3) Mapping the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology.
4) Characterisation of methylation sensitive genes in normal ageing and early phases of AD-like neurodegeneration.
5) To use precise morphological definitions and perform whole genome sequencing to evaluate genetic constellations on the background of a healthy state of the brain in oldest-old individuals.
6) To develop and evaluate a non-human primate model (MIM), based on the results obtained from our evaluation of human brains.
To this end, DEVELAGE implemented a collaborative research project using a wide variety of methods including the evaluation of protein modifications, altered cellular functions and gene expression, genetic and epigenetic alterations, and comparison with transgenic mice and a non-human primate model, Microcebus murinus (MIM). To complement our descriptive observations, we identified target molecules and pathways, which could serve as entry points to exploit our knowledge for the development of therapeutic and diagnostic solutions. In addition, we evaluated the effects of SAM (S-adenosyl-methionine), a compound acting on the epigenetic level in experimental models. A focus was placed on the evaluation of the temporal and entorhinal cortex and hippocampus, due to their importance in age-related changes, learning, memory and neurogenesis.
For Objective 1 (Evaluation of proteins characterising age-related neurodegenerative disorders and their modifications throughout brain development and ageing), DEVELAGE addressed the understanding of the expression pattern and cellular distribution of genes and proteins related to neurodegeneration, in normal brain development as well as in brain tissue from patients with Down syndrome, and in focal malformations of cortical development.
For Objective 2 (Characterisation of key mechanisms involved in neurogenesis including the role of neurotrophic factors), DEVELAGE sought to explore and characterise the mechanisms that are involved in the sequential formation of different layers in the cortex and in the hippocampus, at various stages of development and ageing in human; indispensable knowledge for designing therapeutic strategies towards neurodegenerative diseases.
For Objective 3 (Mapping the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology), DEVELAGE used convergent methods to map the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology, categorised as stages I-II of Braak & Braak (from the six stages of neurofibrillary degeneration), compared with non-affected age-matched controls.
For Objective 4 (Characterisation of methylation sensitive genes in normal ageing and early phases of AD-like neurodegeneration), DEVELAGE addressed the important role played by the methylation of CpG dinucleotides in regulation of gene expression in the brain.
For Objective 5 (To use precise morphological definitions and perform whole genome sequencing to evaluate genetic constellations on the background of a healthy state of the brain in oldest-old individuals), DEVELAGE addressed the difficulties of separating non-pathological from pathological brain ageing through the whole genome sequencing of strictly defined cases of elderly individuals.
For Objective 6 (To develop and evaluate a non-human primate model (MIM), based on the results obtained from our evaluation of human brains), DEVELAGE addressed the importance of developing and evaluating a non-human primate model to facilitate the extrapolation of therapeutic approaches to humans.
In order to address these objectives DEVELAGE has brought together internationally renowned experts in closely interacting work packages to achieve world-class collaborative research (see Figure 1). DEVELAGE successfully organised the complementary expertise of partners in relevant fields of basic and clinical research and facilitated the sharing of methodologies between a critical mass of partners, thereby increasing their scientific impact and bringing added value to all participants. DEVELAGE emphasised active dissemination to increase the public awareness of the DEVELAGE-project and age-related degenerative disorders as an important individual and public health issue. DEVELAGE has produced knowledge that will significantly contribute to the development of pharmacologic strategies to treat age-related common degenerative disorders, including Alzheimer’s disease. Further research will show to what extent the results of DEVELAGE could lead to the development of individualised pharmacological strategies, in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Project Results:
DEVELAGE has essentially achieved all of the overall scientific objectives and those within the six scientific work packages (WP1-WP6).
WP1 Insight into the expression pattern and cellular distribution of genes and proteins related to neurodegeneration in human brain development and pathological processes during ageing.
When ageing, the human brain shows morphological alterations that are reminiscent of so called neurodegenerative diseases (NDDs). Current molecular pathological classification of NDDs is based on the evaluation of pathologically altered proteins and in particular the evaluation of where these proteins are deposited in the human brain. Increasing evidence supports the concept that aberrant neural development can contribute to the process of neurodegeneration in the ageing brain. Accordingly, several genes that are mutated in NDDs (such as amyloid precursor protein, APP, and presenilin, PSEN) encode for proteins that participate in fundamental neurodevelopmental processes, including the precisely orchestrated cascade of events which underlies the development of the cerebral cortex. Additionally, major pathways essential to normal cortical development (including CDK5 and the mTOR signalling pathways) have been shown to be potentially involved in the mechanisms underlying cellular ageing and neurodegeneration.
Data obtained in this WP provides valuable insight into pathways common to brain development and pathological processes during ageing. This information is necessary to improve our understanding about the abnormal (re-)activation of pathways essential to neurodevelopment, and how this might contribute to early events in neurodegeneration. This knowledge provides the potential for novel avenues of investigation into suitable therapeutic approaches to target critical pathways at a stage when pathological progress can still be reverted.
Our Consortium had unique access to clinically and morphologically characterised normal human brain at different development ages, on which we could perform comparative analysis of both neurodegeneration- and neurogenesis-linked proteins, in developing versus aged hippocampus and temporal cortex. In the first 12 months we selected and carried out a neuropathological evaluation of autopsy brains at different developmental ages from control, and Down syndrome patients. Additionally, adult brain tissue was obtained from controls, patients with post-traumatic brain injury, individuals with Down syndrome (DS) and patients with Alzheimer’s disease (AD). This cohort of tissue samples was used for the further studies of this WP.
We performed a systematic evaluation of the hippocampus and temporal cortex, at different developmental ages, for various neurodegeneration related proteins using immunohistochemistry and western blot analysis.
We have studied the developmental expression of amyloid precursor protein (APP) and of the death receptor-6 (DR6) in both control and Down syndrome brains. Our study provided the first description of the expression pattern and cellular localisation of DR6 in human hippocampus and neocortex during the pre- and early postnatal development. Our findings demonstrated a developmental regulation of DR6 in human hippocampus and highlighted the potential role of DR6 in specific types of disease-associated neuronal degeneration. Preliminary observations from MIM (Microcebus murinus; non-human primate model used in our project) brain at different ages support the idea of a developmental regulation of DR6, reactivated during ageing.
We also analysed the developmental expression of several other neurodegeneration-related proteins (TDP-43, p62 and ubiquitin) and demonstrated a developmental regulation of TDP-43, p62 and ubiquitin in human hippocampus, supporting a role for these proteins during early development in the hippocampus, as well their contribution to the pathogenesis AD-associated pathology.
We demonstrated that phosphorylation of tau protein may have important physiological effects during development. Remarkably, this physiological phosphorylation seems to be critically reduced in Down syndrome and therefore can be used as a distinguishing marker of Down syndrome.
We demonstrated significant differences in the number and density of non-neuronal cells between DS patients and controls. These alterations may contribute to or reflect a compensatory response to defective neurogenesis, reduced neuron number, and delayed myelination during the development of foetal DS brain. These alterations might contribute to the development of mental retardation in DS patients.
We evaluated the developmental changes occurring in human healthy foetal and postnatal hippocampus by combining high-resolution 7T Magnetic Resonance Imaging (MRI) and histological and immunohistochemical analysis. Our study showed the potential utility of high-field MRI for detection of developmental changes in human foetal and postnatal hippocampus. These data will be a promising tool in the understanding of hippocampal abnormalities occurring in developmental disorders when in the future high resolution MRI might be used in clinical practice.
A major task was the investigation of the relationship between neurodegeneration related proteins and specific neurodevelopmental pathways. We studied the activation of mTOR signalling using, as marker of mTOR, the phospho-S6 (pS6) protein in DS specimens at different developmental ages followed by the evaluation of other components of the mTOR signalling pathway (i.e. phospho-p70S6 kinase1, phospho-4EBP1). Evaluation of pS6 during hippocampal development showed increased immunoreactivity in CA1 neurons prior to establishment of AD pathology.
Further characterisation of pS6 positive cells, as well as evaluation of other components of the mTOR signalling pathway was performed. The expression pattern and cellular distribution of components of the mTORC1 signalling (phosphorylated (p)S6, p70S6K, p4E-BP1 and pmTOR) were investigated immunohistochemically in the developing hippocampus from controls and patients with Down's syndrome, as well as in adults with DS and Alzheimer’s disease-associated pathology. Our study provides evidence of mTOR pathway activation in DS hippocampus. Since DS is a model of AD-related pathology, the alterations detected early during brain development support the notion that mTOR is an important pathway in AD, and also that already during development neurodegeneration related changes are initiated in the brain practice.
Specimens from tuberous sclerosis complex (TSC) and focal cortical dysplasias (FCD) patients represent a unique opportunity to study the relationship between neurodegeneration related protein expression and the persistent activation of specific developmental pathways. We examined FCD and TSC cases for the expression of several proteins involved in major neurodegenerative diseases. We compared the abnormal cortex with adjacent normal cortex and cases of FCD (type I) without evidence of upregulation of CDK5 or activation of the mTOR pathway. Sections were processed for TUNEL labelling and immunohistochemistry using markers for the evaluation of apoptosis signalling pathways and neurogeneration -related proteins/pathways. We provided evidence of complex, but similar mechanisms of cell injury in focal malformations of cortical development associated with mTOR pathway hyperactivation, with prominent induction of apoptosis signalling pathways a premature activation of mechanisms of neurodegeneration. In addition, mTOR signalling was evaluated immunohistologically in foetal brains of TSC patients, with the results indicating that brain malformations in TSC are likely a consequence of increased mTOR activation during embryonic brain development.
We also examined the expression of several proteins involved in major neurodegenerative disease in developmental brain tumours (glioneuronal tumors, GNT) characterised by mTOR pathway hyperactivation. Sections were processed for immunohistochemistry using markers for the evaluation of apoptosis signalling pathways and neurodegeneration -related proteins/pathways. Our data provide evidence for the premature activation of mechanisms of neurodegeneration in glioneuronal tumours associated with mTOR pathway activation.
We characterised the cultures of neural stem (NS) cells prepared from foetal human brain and the protocol to induce them to differentiate into neurons or astrocytes. We demonstrated that a pure population of NS cells can be isolated from the adult human sub-ventricular zone (SVZ), which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis and studying the relationship between neurodegeneration-related proteins and specific developmental pathway.
We also provided evidence supporting the regulation of GFAP expression by epigenetic mechanisms. Our data demonstrate that histone acetylation controls transcription, splicing, and the assembly of GFAP in astrocytes. These data also imply that a tight regulation of histone acetylation in astrocytes is essential; since dysregulation of its gene expression causes pathological aggregation of GFAP.
We studied selected mRNAs, microRNA (miRNA) and proteins identified in WP3. First, we investigated the role of miR-146a, (upregulated in Alzheimer’s disease) in the modulation of astrocyte-mediated inflammation. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miRNA may represent a novel target for therapeutic strategies.
The amyloid hypothesis of Alzheimer’s disease suggests that soluble amyloid β is an initiator of a cascade of events eventually leading to neurodegeneration. Recently, it has been reported that amyloid beta deranged Ca2+ homeostasis specifically in hippocampal astrocytes by targeting key elements of Ca2+ signalling, such as mGluR5 and IP3R1. We further dissected a cascade of signalling events and suggest that nanomolar concentrations of amyloid beta deregulates Ca2+ homeostasis via calcineurin and its downstream target NF-kB, possibly via the cross-talk of Bcl10 in hippocampal astrocytes.
We demonstrated developmental regulation of mGluR5 in human hippocampus and suggest a role for this receptor in astrocytes during early development in DS hippocampus, as well as a potential contribution of mGluR5 to the pathogenesis AD-associated pathology.
We also addressed the adenosine hypothesis of neurodegeneration and comorbidities, which implies that astrogliosis, via overexpression of adenosine kinase (ADK), induces a deficiency in purine ribonucleoside adenosine, which is a homeostatic network regulator of the brain. We found increased expression of ADK in astrocytes surrounding amyloid deposits and tangle-containing neurons, in the hippocampus of AD patients, and in adult DS patients with widespread AD-associated neurodegeneration. Interestingly, strong expression of ADK has been detected in human foetal brain (in both controls and DS), possibly reflecting the enzyme’s expression in the deep compartments of the cortical wall (VZ/SVZ; ventricular/subventricular zone) at early stages of corticogenesis.
WP2 Neurogenesis during brain development and in adults
Neurogenesis in adult brain contributes to the plasticity of the hippocampus and plays a role in learning and memory. Research on pathological ageing has for a long time focused on defining the causes of neuronal dysfunction and death during adult life. However, recent findings point towards alterations in neurogenesis in neurodegenerative diseases. Experimental studies suggest that neurogenesis is influenced by a number of intrinsic and extrinsic factors. Additionally, numerous signalling pathways and trophic factors implicated in development, contribute to the regulation of adult neurogenesis.
The molecular mechanisms regulating cell-cycle progression during neurogenesis and the tightly regulated occurrence of proliferative versus differentiative divisions are not completely understood.
This WP was designed to explore and characterise the mechanisms involved in the neurogenesis and sequential formation of the different layers in the cortex and in the hippocampus, at various stages of development in humans, and to characterise the molecular mechanisms regulating cell-cycle progression during neurogenesis. We also characterised the stem cell niches that persist during adult life in ageing people with or without Alzheimer’s disease.
We aimed to provide insights into the signalling pathways, which control the cell cycle of the progenitors of the temporal cortex and the hippocampus in developing brain, healthy and AD adults. Using frozen and formalin fixed brains we examined the expression of markers of the cell cycle, mitosis and cell cycle exit, in order to recognise various types of progenitors and the modalities of cell division. The fate of ventricular cells and sub-granular germinal zone cells has been examined using multiple labelling with a combination of markers, such as SOX2, Nestin, GFAP, TBR2, PAX6, Ki67 and layer markers. We have evaluated three major anatomical regions as follows:
A-Proliferating areas and generation of neurons in the dorsal telencephalon
We have characterised for the first time the setting of different compartments of progenitor cells proliferation and neurogenesis in the human foetuses at all stages.
B-Proliferating areas and generation of multiple neuronal subtypes in the hippocampal formation during development
The molecular mechanisms underlying the formation of the hippocampus are unknown in humans. To increase our knowledge of potential regulatory molecules, we investigated the expression of progenitor markers and cell fate molecules. We described for the first time the development of the hippocampus from the early foetal age (GW9) to the end of the gestation (see Figure 2). In particular, we characterised the evolution of proliferating zones and the formation of pyramidal and dentate granular layers.
Our findings demonstrate for the first time the presence of a germinal subventricular zone (SVZ) in the hippocampus of human foetuses. In addition, we provide molecular evidence of the heterogeneity of the pyramidal neurons in the hippocampal formation. The connectivity of the hippocampal formation has been established in animals and emphasises that differences between species exist. Our data provide new information to further investigate the connectivity of the hippocampal formation and to better understand the susceptibility of specific neuronal subtypes during neurodegenerative diseases.
C-Proliferating areas and generation of neuronal subtypes in the dentate gyrus of the hippocampal formation during development and ageing
The molecular mechanisms that orchestrate the development of the human dentate gyrus are unknown. We characterised the formation of human dentate, fimbrial progenitors and post-mitotic neurons from GW9 to GW25, to further our understanding of this region of the hippocampal formation.
We established how some signalling pathways interfere with neurogenesis in different neuronal populations, cell cycle regulation in the hippocampus and temporal cortex at different developmental ages, including healthy and AD adults. We determined which types of neuronal populations are dependent on these pathways and the regional expression of their downstream transduction factors. We also established the expression pattern of trophic factors involved in neurogenesis in the hippocampus and temporal cortex in human at different developmental ages, including healthy adults.
In addition, we showed that during congenital cytomegalovirus infection (CMV) the virus preferentially targets these neural progenitors in the brain. CMV infection is the most prevalent cause of congenital neurological handicap and is a major public health concern. The pathogenesis of cerebral lesions remains unclear. We investigated the neuropathologic substrates, the immune response, and the cellular targets of CMV infected human foetal brains. Our results indicate that the 2 main factors influencing the neuropathologic outcome at this stage are: the density of CMV-positive cells and the tropism of CMV for stem/progenitor cells. This suggests that a large spectrum of CMV-induced brain abnormalities are caused not only by tissue destruction, but also by the particular vulnerability of stem cells during early brain development. Florid infection of the hippocampus and the olfactory bulb may expose these patients to the risk of neurocognitive and sensorineural handicap, even in cases of infection at late stages of gestation.
We also established the phenotypic and genotypic characteristics of foetuses diagnosed with L1 syndrome, a disease responsible for abnormal axonal development and pathfinding. L1 syndrome results from mutations in the L1CAM gene located at Xq28 (X chromosome marker). L1 syndrome encompasses a wide spectrum of disease phenotypes, with X-linked hydrocephalus being the most severe phenotype, detected in utero, and whose pathophysiology is incompletely understood. The aim of this study was to report detailed neuropathological data from patients with mutations. Additionally, to delineate the neuropathological criteria (required for L1CAM gene screening in foetuses) by characterising the sensitivity, specificity and positive predictive value of the cardinal signs, as well as to discuss the main differential diagnoses in non-mutated foetuses, in order to delineate closely related conditions without L1CAM mutations. These data underline the existence of closely related clinical entities, whose molecular bases are currently unknown. The identification of the causative genes would greatly improve our knowledge of the defective pathways involved in these cerebral malformations.
We were also able for the first time to successfully culture neuronal precursors, immature and mature neurons, as well as cells from the glial lineage (precursors and differentiated astrocytes), from our non-human primate model, and maintain these cell types in culture over time (see Figure 3). This allowed us to describe the characteristics of the various cell types, and analyse the MIM neural precursor cells for stem cell potency.
WP3 Complex molecular pathways in AD as a model for age-related neurodegeneration
Very little is known about changes occurring at the earliest stages of AD, Braak & Braak stages I-II, in which neurofibrillary pathology (one of the hallmarks of AD) is restricted to the entorhinal and transentorhinal cortices. The fact that we do not know whether a particular individual would have developed full symptoms of AD has for years prevented a profound study of the brain at these very early stages of the disease. Yet these first stages are crucial as the majority of people over 65 years have neurofibrillary tangle pathology in the entorhinal and transentorhinal cortices, and 100% of AD cases have entorhinal and transentorhinal pathology. Previous studies have shown that in addition to the accumulation of altered proteins, neurodegenerative diseases are characterised by the loss of the ability of neurons to cope with energy requirements. This scenario has led to the concept of neuronal exhaustion, which precedes neuronal death in neurodegeneration.
Several studies have analysed gene expression in AD brains. However, there is a paucity of of mRNA expression studies focused on the entorhinal cortex and hippocampus, in cases with AD-related pathology at Braak & Braak stage I-II. MicroRNAs (miRNAs) belong to the family of small silencing non-coding RNAs that are involved in the guidance of diverse formats of gene regulation, typically resulting in reduced expression of target genes. The miRNA pathways alter and modulate the expression of thousands of genes and contribute to a variety of physiological processes, providing an intricate network of regulation for the fine-tuning of the transcriptome, proteome and metabolome.
The aim of this WP was to map the transcriptome and metabolome of the entorhinal cortex and hippocampus, in the very same individuals affected by first stages of AD-related pathology (Braak & Braak stages I-II) compared with non-affected age-matched controls. To achieve this, convergent methods were used including: mRNA expression, microRNA expression profiling, protein expression, oxidation damaged proteins, impaired enzymatic function; altered metabolic pathways identification (metabolomics) focused on energy metabolism pathways and bioinformatic processing. In addition, lipidomics has been added as valuable complementary source of information. Animal studies using the APP/PS1 transgenic mice have also been employed as a model of familial AD or of familial amyloid beta deposition in brain.
The objective of our study of mRNA expression was to identify deregulated genes, and most importantly, to identify clusters of altered genes that recognise particular metabolic pathways. Once these clusters were obtained, the data was validated and further combined with proteomic, metabolomic and lipidomic studies in order to obtain functionally altered interactomes in ageing and disease processes. Moreover, the data was shared with other members of the consortium so that they could apply these results to their studies of development and animal models, and to analyse mechanisms related to altered regulation (for example DNA methylation studies of specific genes).
The four main altered clusters correspond to: olfactory and taste receptors, cytokines and mediators of the immune response, purine metabolism, and protein synthesis. The studies were expanded to other pathologies including Parkinson’s disease, tauopathies and prion diseases, in addition to ageing and Alzheimer’s disease, in order to understand commonalities and differences among these distinct neurodegenerative processes. The responses have been shown to be disease-specific and not mere reflections of accelerated ageing.
To complement our studies in humans, we produced a hierarchical clustering heatmap of the expression intensities of the significantly regulated transcripts versus the APP-PS1 and wild-type (WT) arrays of an animal model of AD. A clear separation was seen between APP/PS1 and WT mice at 6 and 12 months of age. We found that neuroinflammation associated with ageing, and neuroinflammation at early stages of sporadic Alzheimer’s disease (but not in APP/PS1 transgenic mice), does not depend on amyloid beta deposition, soluble amyloid beta or abnormal tau deposition.
One of the altered clusters uncovered by our expression analysis corresponded to purine metabolism. This could reflect functional alterations in energy metabolism, which is a feature of both ageing and Alzheimer’s disease, and occurs through altered mitochondria and oxidative stress damage. Purines form the core of DNA, RNA, nucleosides and nucleotides. Nucleotides participate in a wide variety of crucial metabolic pathways including energy metabolism and cell signalling. In addition to intracellular signalling, purines and their products may function as extracellular signals acting upon other cells, either between neurons, or between neurons and glial cells equipped with appropriate receptors. In this WP we validated at the middle and later stages region-dependent deregulation of several enzymes of the purine metabolism. No modifications were observed at earlier stages, in clear contrast with the early appearance of neuroinflammation. This study has been complemented with targeted metabolomics, showing that not only is there a deregulation of the enzymes but also an abnormal expression of several metabolites, thus indicating that purine deregulation has metabolic implications. Purine metabolism has also been examined in the substantia nigra and frontal cortex in Parkinson’s disease at different stages of disease progression. The pattern of altered profiles differed in the substantia nigra, where purine metabolism is largely compromised, when compared with the cerebral cortex where it is relatively unaffected
In this WP, we identified altered microRNAs that can regulate RNA expression, and therefore modulate protein synthesis in different settings. Additionally, we identified altered proteomic profiles and clusters of altered proteins from specific metabolic pathways. Results were also shared with other members of the consortium, to analyse these altered clusters during development and in animal models. Moreover, we evaluated post-translational modifications of proteins that may alter protein function. Changes resulting from oxidative damage are particularly important, as even though the protein expression level is unchanged, the function (for example the enzymatic activity) of a particular protein is altered by oxidative and nitrosative damage.
We combined our metabolomics data with the human metabolic model (Recon 2), in order to generate a tissue specific model of the metabolic fluxes occurring during neurodegeneration in the ageing brain. Using a constraint-based modelling (CBM) approach, our data will lead to network-based predictions that will be used to determine changes in metabolite levels revealing potential nodes of interest or biomarkers.
We processed the large amount of data obtained from transcriptomics, proteomics and metabolomics. Subsequently we employed a systems biology approach in order to identify altered interactomes, and understand the interaction of altered interactomes and metabolomes in disease processes, with a focus on the transition between ageing and the early stages of AD-related pathology. Finally, we created brain-ageing interactomes containing genes associated with our epigenetic, gene expression, non-coding RNAs, proteome and metabolome results, as well as proximal interactors. We created a protein network of all known human genes and/or proteins from three different network resources: iREFINDEX, ConsensusPath DB, and the HI Yeast-Two-Hybrid prepublication dataset downloaded from Human Interactome Database website. We used this network to examine the similarity of the brain interactome to common neurological disorders. Thus, Bioinformatics has been the tool that has permitted the identification of altered clusters of mRNAs, proteins, metabolites and interactomes.
To complement the data obtained by using the other “omics” approaches we focused on lipidomics, as cell membranes in the nervous system are very rich in lipids and alterations of lipid composition impair protein processing. Increasing age is accompanied by alterations in the lipid matrix of brain cortex lipid rafts from both WT and APP/PS1 transgenic mice, which we coined the “lipid-raft ageing” concept. However, in AD mice the process is accelerated and animals exhibit the oldest phenotype prematurely, at the age of 9 months. Progression of lipid changes in brain cortex lipid rafts as a function of age in WT and APP/PS1 mice.
WP4 Epigenetic alterations in brain development and ageing
Methylation of CpG dinucleotides plays an important role in regulation of gene expression in the brain. It was discovered that brain specific promoter-related sequences are surprisingly enriched in CpG sites. This leads to the conclusion that it is likely that brain-specific transcription is regulated by methylation at an epigenetic level much more frequently than tissue specific expression in other organs. Low methylation status is strongly associated with neurological and cognitive deficits.
Many epidemiological studies have shown that factors connected to low methylation status such as elevated total homocysteine, low folate or low cobalamin levels are associated with increased risk of cognitive decline, dementia and brain atrophy. In recent years, hyperhomocysteinemia has begun to be widely considered a risk factor in AD and this may be ascribed to alteration of the S-adenosylmethionine/homocysteine (SAM/HCY) metabolism. SAM is known to be the primary methyl-donor present in eukaryotes and it is involved in methylation of target molecules as DNA, RNA, proteins, lipids, and polyamines synthesis. SAM appears to be altered in some neurological disorders, including AD. About 95% of SAM is engaged in methylation reactions. It is then transformed in S-adenosylhomocysteine (SAH) and further hydrolysed into homocysteine (HCY) and adenosine. The reaction is strongly reversible and HCY, if not rapidly transformed into methionine or cystathionine, forms SAH, which is a potent inhibitor of methyl-transferases. These metabolic alterations may be responsible for the generalised reduction of DNA methylations observed in ageing, which can lead to the overactivation of methylation-controlled genes.
With this WP we aimed to define the DNA methylation pattern of neurodevelopment and AD relevant genes during both development and ageing, and to verify the efficacy of epigenetic drugs (SAM) in modulating DNA methylation. Thus, we studied gene regulation mediated by CpG methylation in developing and ageing TgCRND8 mice, and in healthy and AD individuals, focusing on genes related to neurodevelopment and neurodegeneration. Within this framework, we investigated if/how methylation patterns typical of early stages of neurodevelopment vary during normal and pathological ageing. The rationale for selecting specific genes is: (i) they play essential roles both in developing and adult brain, being involved in neuronal migration, synaptic connectivity formation and plasticity (Reelin and BDNF), and in neurodegeneration (PSEN1); (ii) their expression is subjected to an epigenetic control.
The three genes studied, Psen1, Reelin and BDNF, showed a complex expression pattern, depending on the age, genotype and gender. A clear correlation between promoter methylation and expression patterns (transcripts/proteins) was observed only for Psen1. DNA methylation analyses by bisulfite modification and DNA sequencing showed that Reelin and BDNF promoters display a very low level of CpG methylation. No age/gender/genotype-dependent differences were observed, leading to the conclusion that Reelin and BDNF expression are not modulated by DNA methylation in TgCRND8 and WT mice. We found a clear correlation between promoter methylation and expression patterns (transcripts/proteins) for PSEN1. In particular, female mice showed PSEN1 promoter hypomethylation at PN90. We demonstrated sexually dimorphic expression of Reelin and its downstream signalling pathway.
We analysed the methylation status of PSEN1 in cortices and hippocampi from healthy and demented human individuals to evidence differential methylation patterns between different brain areas and stage of disease progression. DNA methylation of this gene was assessed through bisulfite modification techniques. This data were correlated to the conditions of normal ageing or dementia (and related stage). Methylation data indicated no difference in methylation of CpG moieties studied by bisulfite assay during early neurodevelopment (from 14 GW (gestation week) to 40 GW). During late neurodevelopment (2 months and 17 years), PSEN1 CpG methylation is significantly higher than it is in embryos, with no differences between the two conditions. CpGs in the PSEN1 promoter are hypomethylated in aged subjects; methylation further decreases in AD patients. Non-CpG methylation was detected at very low level during early and late neurodevelopment (14 GW to 17 years), although it slightly increases from 17 years to elderly. In AD subjects, non-CpG methylation decreases significantly compared to healthy controls. Both CpG and non-CpG methylation are decreased in AD subjects versus controls. PSEN1 promoter hypomethylation, in the cortex of AD subjects, correlates with the gene expression.
We assessed the DNA methylation of selected up- and down-regulated genes involved in AD-related pathology, as identified in WP3, to reveal whether modulation of these genes corresponds to the methylation of CpG sites. The three genes selected (based on up-regulation of their expression during the early AD stages and supporting literature) belong to the family of activated pro-inflammatory cytokines (IL-1B, Interleukin 1B; IL-6, Interleukin 6) and C3AR1 (Complement Component 3A Receptor). The expression of IL-1B is markedly up-regulated in the early phase of the pathology. It is then down-regulated (although still higher than in control samples) in the late phase, which is correlated with markedly reduced non-CpG methylation in AD I-II subjects compared to control and AD V-VI samples.
An aspect of therapeutic intervention is the application of SAM, which has already been shown to partially silence overexpressed genes. However, with this WP we had the opportunity to evaluate the restorative effects of treatment with SAM in mouse models of AD.
We evaluated the efficacy of perinatal and postnatal SAM supplementation on DNA methylation modulation in TgCRND8 mice. The aim was to analyse the effect of epigenetic drugs (SAM) on DNA methylation patterns of neurodevelopment-related genes by means of SAM supplementation during the perinatal period and as adults. The effect of SAM effect on at least 5 neurodevelopment- and AD-related genes in mice was determined. SAM supplementation to pregnant mice (3 pregnant females per strain, corresponding to about 10-15 embryos) allowed the evaluation of the effect on gene methylation and expression during brain development in embryos through the collection of embryonic brain areas. 3 additional pregnant females per strain were treated in parallel, but were not sacrificed, in order that perinatally-treated pups could be analysed as adults (3 months), when amyloid plaques normally appear. Analyses were repeated on these mice versus untreated mice. Moreover, DNA methylation, gene and protein expression were evaluated in these perinatally-treated mice at the adult stage for those AD-related genes, already known to have altered DNA methylation and gene expression in these mice (PSEN1).
WP5 Genetic influences on normal and pathological brain ageing
Age-related cognitive decline and dementia is one of the major threats to the quality of life of the elderly. Definition of factors in the background of dementia in the elderly is hindered by the fact that humans differ in their cognitive abilities. It is far from clear how to distinguish non-pathological from pathological brain ageing. Age-related cognitive decline forms a continuum and people’s cognitive level in old age depends also on their intelligence and cognitive abilities. Thus, studies performing cross-sectional analysis must be interpreted with caution, either in the sense that non-pathological cognitive ageing is not invariably non-pathological, or vice versa. One problem in distinguishing normal and abnormal ageing is that AD-like changes are also seen in the elderly without cognitive decline or dementia. Thus, the most critical issue for progress in genomic studies of cognitive ageing is the identification with strict criteria and selection of appropriate phenotypes.
This WP was intended to provide whole genome sequencing of elderly individuals selected according to morphological criteria of healthy ageing in the brain and compare these with AD type brain ageing. The major advantage of our approach was that we evaluated very strictly defined cases that lacked morphological/biochemical evidence of neurodegeneration-related alterations.
To fulfil these criteria we identified brains from individuals above the age of 80-85 years without or with very mild AD-related neuropathological alterations, and brains from individuals with end-stage AD (Braak & Braak stage V-VI). Cases were selected from the Vienna Trans-Danube Ageing (VITA) study. Since the year 2000, the VITA study has followed longitudinally a community-based cohort of every inhabitant of the Vienna area on the left shore of the river Danube (districts 21 and 22) born between May 1925 and June 1926. Neuropathological evaluation included a systematic evaluation of 2x2.5 cm sized paraffin embedded tissue block sections from at least 15 anatomical regions. In sum we examined approximately 4,600 immunostained sections. The neuropathological examination demonstrated the high variability of pathological alterations in the ageing brain, which supported our concept of using very strict criteria to select cases for genome analysis.
During the evaluation of this cohort we noticed that there is a difference in the subregional tau pathology of the hippocampus in tauopathies when compared to Alzheimer’s disease. Thus, we performed an additional study in which we investigated the spatial and temporal distribution of hyperphosphorylated tau protein in a cohort of 105 patients stratified according the neuropathological diagnosis. Some diseases revealed unique combination of deposits within different cell types and neuropil in certain subregions. This may be the morphological background for clinical symptoms. In addition, some other diseases showed very similar patterns of the protein distribution. This explains similar clinical symptoms and difficult clinical diagnosis. Our data suggests that stratification of the patients with dementia, according to spatial and temporal distribution of pathologic proteins, is a key feature for correct diagnosis and possibly therapy development. We developed a mathematical model to compare tau constellations in the hippocampus. Our data suggest that some diseases are ‘closer’ to each other, whereas others are more distant.
Whole genome sequencing from neuropathologically characterised individuals without or with very mild AD-related pathological alterations (28 cases) and with end-stage AD-related pathology (12 cases) was performed using the Illumina HiSeq 2000 sequencing system platform. Bioinformatic analysis was then performed by Thrace University (Prof. Peristera Paschou, Thessaloniki, Greece). In order to enrich for functional variants, we focused on non-synonymous, splicing, stoploss and stopgain variants, which affect the transcript. We identified 41,493 functional variants before filtering for unique variants in cases and control individuals.
Our initial analysis focused on genes that are known to be involved in longevity phenotypes.
1. ApoE Haplotype and variants that have been previously implicated in longevity
The Apolipoprotein E (ApoE) haplotype has been widely studied and found to be one of the most consistent associations in human longevity studies. We were able to replicate published findings of the significant association with SNPs on TOMM40, ApoE and APOC1 genes, all found on chromosome 19.
2. Association tests of the sirtuin genes
In the next step we focused on the sirtuins. We evaluated sirtuins due to their association with longevity. Sirtuins have been implicated in influencing a wide range of cellular processes like ageing, transcription, apoptosis, inflammation and stress resistance, as well as energy efficiency and alertness during low-calorie situations. We were able to recover indicative association with markers on SIRT1, SIRT2, SIRT5 and SIRT6.
3. Association tests with SNPs that have been previously implicated in AD
We investigated the association of previously implicated Alzheimer’s disease related SNPs in our own sample of AD patients and normal brain ageing controls. Besides the APOE4 signal there was an indicative signal at SLC24A4.
Our further analysis revealed genes implicated in healthy ageing, genes involved with the nervous system like cell adhesion molecules, and genes that had functional roles in neurodegenerative diseases.
In addition to the whole genome analysis to define protective genes, we had the opportunity to study a family with neurodegenerative disease to define novel genes implicated in dementia. The family examined comprised of two affected siblings and one control aunt without symptoms. One sibling was clinically diagnosed with Alzheimer-type dementia and had a neuropathologically proven neurodegenerative disease with tauopathy, but not amyloid beta deposition. The other sibling was a clinically demented patient, who clinically resembled Alzheimer’s dementia. Neuropathology and biochemical examinations were performed. Our combined ranked method highlighted a number of interesting genes and their interactions in the background of the complex phenotype.
We examined the expression of sirtuins in healthy and diseased elderly, as well as in developing brain (hippocampus). Sirtuins were selected according to literature reports on their association with longevity and healthy ageing. Our whole genome analysis also confirmed that sirtuins do indeed play a role in healthy ageing. We microdissected and evaluated the hippocampus white matter and the entorhinal cortex using western blotting and immunohistochemistry for Sirt1, Sirt3, and Sirt5. We found that the protein level (western blot) show that with the progression of Alzheimer-related changes (Braak & Braak stages), the expression of Sirt1 and Sirt3 significantly decreases. Interestingly, the protein expression of Sirt5 showed positive correlation with the progression of Alzheimer’s disease pathology and showed an inverse correlation with the expression of Sirt1 and Sirt3. Based on these findings, we interpreted that Sirt5 could be upregulated as a compensatory response to the loss of Sirt1. Using a combination of immunohistochemical observations and immunoblotting on subcellular fractions of two selected cases (Braak & Braak stage II and stage VI) we were able to characterise the subcellular localisation of Sirt1, Sirt3, and Sirt5. With our unique approach we were able to define the precise subcellular localisation of sirtuins and their consequences on the development of brain pathology. These revelations can be used as rationale for developing therapies targeting the sirtuin system.
Furthermore, we focused on proteins related to age-related neurodegenerative diseases and we were able to perform additional experiments and studies. First, we focused on dementia with Lewy bodies (DLB) and Parkinson's disease (PD), which are frequent age-related neurodegenerative conditions characterised by the deposition of disease-associated α-synuclein. Our findings suggested a prion-like cell-to-cell spread of α-synuclein in neurons by uptake from surrounding structures. Second, we performed a study to evaluate whether epilepsy-related changes can be detected in Alzheimer’s diseased brains. In order to achieve this we evaluated the expression of Calbindin (Cb), one of the major Ca2+ binding proteins, that exhibits neuromodulatory functions such as long-term potentiation (LTP), synaptic plasticity, and memory functions. Cb is expressed in hippocampal interneurons, pyramidal cells and granule cells of the dentate gyrus (DGCs). A recent study suggested a link between Amyloid beta-induced Alzheimer's disease-related cognitive deficits and neuronal depletion of Cb. To evaluate whether this is specific for Alzheimer's disease, we performed a comparative study of Cb immunoreactivity of DGCs in cases with Alzheimer's disease-related neuropathologic change, grouped according to the stages of Braak & Braak, Creutzfeldt-Jakob-disease, FTLD (frontotemporal lobar degeneration)-tau Pick's disease type, argyrophilic grain disease, and FTLD-TDP types A and B. We concluded, that late stage Alzheimer's disease-neuropathologic change (Braak & Braak stages V and VI) associates with significantly higher ratios of Cb negative DGCs and this correlates with advanced Braak & Braak stage. This might suggest an accumulative effect of an epilepsy-like pathway on the Cb expression or the direct influence of local pathological protein deposits on the DGCs.
Finally, we analysed genome-wide sequencing data for one healthy old mouse lemur (MIM) and one animal with an Alzheimer phenotype. The initial part of this project focused on quality control and pre-processing of the raw sequence data obtained from the high throughput-sequencing instrument. We are now focusing on the comparison of the sequence with those of humans.
In summary, we achieved the goals and tasks for WP5. We were able to perform whole genome analysis using very strict neuropathology-based criteria and a longitudinal ageing cohort. We were able to select a gene product (sirtuins) that plays a role in healthy ageing, which was confirmed by literature data and our own whole genome studies. We were also able to evaluate and characterise their expression in healthy and pathological ageing in normal and Down syndrome developing brains. In addition, we were able to expand the literature data on the pathogenic role of neurodegeneration-related proteins, in particular of -synuclein and we were able to show morphological data on the pathogenesis of Alzheimer’s disease (epilepsy-like alterations). Finally, we were able to perform the first comparative genomic analysis of humans and the non-human primate model.
WP6 Animal models for ageing
Adequate animal models are crucial to study human brain ageing. Most of the previously used models have been genetically modified rodents and have proved highly important for modelling specific aspects of neuropathological disorders including AD. Unfortunately, results obtained in these models cannot necessarily be extrapolated to humans. Thus, the use of non-human primates has been recommended for the testing of drugs before developing a therapeutic approach in humans. An interesting alternative non-human primate model for human ageing and ageing related neuropathologies is one of the 18 species of mouse lemurs, the gray mouse lemur, Microcebus murinus (MIM). It is small, nocturnal, non-human primate, genetically much closer to humans than rodents. Its lifespan is no more than 8 years under natural conditions, but up to 15 years in captivity. It has been maintained and bred successfully in captivity since the 1960s. Comparable to humans during ageing, some, but not all captive mouse lemurs naturally display some of the neuropathological signs of AD with the presence of amyloid beta plaques and/or neurofibrillary tangles, the development of cerebral atrophy and sensory, motoric and cognitive deficits.
DEVELAGE has unique access to two of the world’s largest colonies of the MIM model with a total of more than 350 individuals, representing all different age cohorts as well as matrilines with AD-like brain disorders. This allowed us to use the colonies in France and Germany to pursue the establishment of a standardised and integrative emotional, hormonal, cognitive, neuro-imaging and genomic approach: to disentangle for the first time healthy ageing from AD-like phenotypes at an early stage and to compare molecular pathways linked to the development of AD in the human and non-human primate brain.
This WP was designed to promote interdisciplinary collaboration to increase our understanding of the causes of AD and translate this knowledge into innovative approaches for preventive therapeutics of neurodegenerative disease. This was achieved through the development of genetic and genomic aspects, brain imaging and a behavioural and cognitive phenotyping platform. The integrative approach applied to this new animal model has led to novel possibilities for modelling human ageing and exploring new avenues for therapeutic intervention of related ageing disorders such as Alzheimer's disease.
We performed a genomic analysis to establish groups for phenotyping and to identify candidate genes for AD. We sequenced the whole genome of one AD-like carrier (showing numerous amyloid beta deposits in the brain) and one wild-type MIM matched for age. The phenotypic selection of animals by in vivo diagnosis of cerebral atrophies (using MRI) and by ex vivo diagnosis of amyloid deposits (by immunohistochemistry) was performed to increase the number of AD-like animals. We established in vivo Magnetic Resonance Imaging (MRI) protocols for MIM to discriminate between healthy ageing and AD.
We aimed to establish and validate behavioural and hormonal tools to phenotype subjects in both colonies, to apply them to discriminate in the long run between wild-type phenotypes and AD-like carriers and thereby to validate the mouse lemur as a primate brain ageing model.
Previous research on AD in humans and animal AD models had discussed if the prevalence of AD is linked to an emotional phenotype reflected in the personality traits of anxiousness and novelty-seeking and to chronic stress hormones. To measure phenotypic markers of emotionality in mouse lemurs and to validate them for repeated usage in the same individuals, we established a behavioural test battery, commonly used in biomedical research, the ‘sleeping box emergence’ test, the ‘open-field’ test and the ‘novel object’ test. With the behavioural traits measured, it was then possible to characterise the emotional phenotype of an animal by the two personality traits ‘anxiety’ and ‘novelty-seeking’. We performed cognitive phenotyping of animals in order to explore how this linked to potential predictors of diseases. Furthermore, we collected stool samples from MIM, fed with a standardised diet, and phenotyped emotionally and/or cognitively. Additionally, a MIM validated ELISA test, in collaboration with the German Primate Center in Göttingen, to determine the glucocorticoid metabolite levels in the stool samples, as an indicator for chronic stress. For cognitive evaluation we aimed to translate cognitive tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) to the model MIM. The CANTAB battery, developed 25 years ago by the Cambridge University, is a well renowned set of tasks, which allows the assessment of the cognitive abilities of humans. By testing subjects with these rapid, computerised and standardised tasks, disorders in cognition may be detected at an early stage. Performance failure in a specific test may help to characterise which brain region is affected and may help in diagnosing age-related disorders like AD.
We programmed, explored and optimised training and testing protocols of three cognitive tasks of the CANTAB battery for the model MIM.
Two learning and memory tasks, named the visual pairwise discrimination task (PD) and its reverse (PDR), explored appetitive, visual associate learning as well as cognitive flexibility. They are claimed to be sensitive to alterations of the medial temporal lobe (visual discrimination learning) and the prefrontal cortex and interconnected subcortical areas (cognitive flexibility). Tasks were performed in an experimental chamber, specifically adapted to MIM and based on an operant conditioning paradigm with positive reinforcement. We trained 48 mouse lemurs in the PD pretask training. 34 subjects entered the actual PD task with 32 of them reaching the desired 80% criterion in both PD acquisition and the reversal phase. Among the 14 animals, which failed to be trained in the pretask training, most of the animals (N=9) could not reach the criterion in the ‘Must Touch’ training phase. This was probably due to a lack of motivation to touch the screen.
The performance in the PD acquisition and PDR task of one group of 20 young adults (10 males, 10 females; mean age = 2.6 years; range 1.1 to 4.1) and one group of 10 aged adults (4 males, 6 females; mean age = 7.9 years; range 6.9 to 9.5 years) was compared. In the PD acquisition we found a significant difference in the number of sessions and trials required to reach the criterion between young and aged adults. Young mouse lemurs needed fewer trials than aged ones to reach the criterion. Great inter-individual variability in the number of trials to reach the criterion was found: the fastest animals (2 young: 1.1 and 2.4 years old) reach the criterion within only 120 trials (4 sessions), the slowest (the oldest: 9.5 years old) needed 780 trials (26 sessions). Lastly, we checked whether there was a difference in the attention to the task and in the motivation between the age groups. We found that young and aged mouse lemurs did not significantly differ in their latency to respond after the stimuli were displayed, or in their latency to collect the reward. Within each age group, we did not find any significant differences in these variables between the sexes.
In the PDR task, we found a statistical trend (p < 0.1) towards a difference in the number of sessions and a significant difference in the number of trials required to reach the 80% learning criterion between young and aged adults. Young mouse lemurs needed fewer trials than aged ones to reach the criterion. Again, great inter-individual variability in the number of trials to reach the 80% criterion was found: the fastest animal (1 young: 3.1 years old) reached the criterion within only 208 trials (7 sessions), the slowest (the oldest: 9.5 years old) needed 1800 trials (60 sessions) which was a far outlier (young range: 208-960; aged range: 300-1020). Among young adults, we found that females tended to require fewer trials to reach the criterion than males. We did not find this difference among aged adults, but the sample size was smaller though. Young and aged mouse lemurs did not significantly differ in their latency to respond after the stimuli display or in their latency to collect the reward. Within each age group, we did not find any differences in these variables between sexes.
We explored in a subgroup of animals, for which data on the hormonal, emotional and cognitive level were available by linear mixed models, whether different factors such as emotional phenotype and hormones predict cognitive performance, so that AD-like cognitive decline may be identified at an early stage. Findings showed that faecal glucocorticoids were more strongly related to cognitive performance than to personality traits.
All in all, our findings provide the first evidence in MIM of age-related effects on learning and memory, comparable to humans. Our integrative approach shows that MIM mimics the complex phenomena of human brain ageing and AD-like dysfunctions much closer than other known primate models. The DEVELAGE approach thus presents promising avenues to identify cognitive decline at an early stage in MIM, and to relate them to potential AD-predictors such as dysfunctions in brain biochemical signalling pathways, candidate genes, brain atrophies, chronic stress hormones or personality traits. DEVELAGE has made important steps towards further characterisation of this novel animal model for ageing and AD-research on the cognitive level.
Potential Impact:
Potential impact (including socio-economic impact and wider societal implications)
The increasing number of elderly people, resulting from an ever increasing life-expectancy, will have a major impact on the prevalence of age-related diseases in the near future leading to enormous costs and challenges for health systems in Europe. These diseases have dramatic repercussions on our society and health systems resulting from the involved nature of patient care and the fact that there is currently no treatment to halt their progress. DEVELAGE has contributed to all of the expected impacts as listed in the work programme and the technical annex. DEVELAGE has used a multidisciplinary basic biomedical approach to perform world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s disease (AD). DEVELAGE has addressed issues starting from understanding brain development and the molecular pathways underlying age-related neurodegenerative disorders, through the use of methylation sensitive genes to understand the influence of gender on Alzheimer’s disease and characterising the mouse lemur as a new experimental model of ageing. DEVELAGE ultimately aimed to produce basic research with the potential to inform new treatments and prevention schemes by uncovering drug targets or regulatory mechanisms to interfere with. In this sense, DEVELAGE has contributed a wealth of new findings to address the urgent need for novel strategies to reduce the burden of age-related common degenerative disorders on society.
Specific impact of DEVELAGE actions and results
In the DEVELAGE project we evaluated proteins characterising age-related neurodegenerative disorders and their modifications. We confirmed that brain development and ageing are linked, and provided evidence that pathways, which are important for the developing brain, are altered in epilepsy or Down syndrome (DS) and could also contribute to the pathogenesis of Alzheimer’s disease. In particular we provide the first description of the expression pattern and cellular distribution of components of mTORC1 in human hippocampus during the pre- and early postnatal development in DS patients. These findings suggest a critical role for mTOR signalling pathway in the DS hippocampus. They support the idea that a dysregulated mTOR pathway may contribute to the early disease pathogenesis and that hippocampal functional impairment, involving this pathway, may develop before neurodegeneration. Future investigation targeting mTOR signalling in DS experimental models might be worthwhile to further develop our current understanding of the role of this pathway, as a link between neurodevelopmental disorders and neurodegeneration.
We investigated the role of miR-146a, (which is upregulated in Alzheimer’s disease) in the modulation of astrocyte-mediated inflammation. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. With this result we defined an important role for miR-146a in human neurological disorders associated with chronic inflammation, which may represent a novel target for therapeutic strategies. Implementation of anti-inflammatory miRNAs at brain levels by synthetic mimics may represent a strategy for preventing the pathologic consequences of neuroinflammation associated with neurodegeneration, which is worth pursuing in experimental models.
We characterised key mechanisms involved in neurogenesis including the role of neurotrophic factors. We have made significant steps towards an improved understanding of the development and ageing of the hippocampus and the entorhinal cortex, which are crucial structures for human memory, where Alzheimer’s disease originates. The mechanisms of hippocampal neurogenesis are mainly investigated in adult animal models, providing preclinical models for therapeutic strategies. However, it is well known that differences between species exist. Our studies provide a gold standard for the knowledge of various progenitor subtypes and the mechanisms involved in neurogenesis at various stages of life, in both healthy people and during neurodegenerative diseases, and will help in improving the design of potential therapeutic strategies when translated from experimental studies.
DEVELAGE mapped the transcriptome and metabolome of the entorhinal cortex and hippocampus in the very same individuals affected by first stages of AD-related pathology. We established what happens in the brains of aged individuals before clinical symptoms related to Alzheimer’s disease arise. We identified a major shift in brain energy metabolism and increased oxidative stress damage. In addition an altered composition of lipids and an altered neuroinflammatory response precede the outbreak of symptoms. The study provided a wealth of data, enabling further study, and providing novel targets for therapeutic strategies. Olfactory and taste receptors have been implicated for the first time as new chemoreceptors in the brain, which are deregulated in neurodegenerative diseases. We recognised early neuroinflammatory responses in the aged brain, which are linked to the presence of soluble oligomers and precede the appearance of tau deposits and amyloid plaques. Together these serve as a rationale for developing treatments geared to the prevention of age-related molecular changes that fuel neurodegenerative diseases in old age.
We characterised methylation sensitive genes in normal ageing and the early phases of AD-like neurodegeneration We explored the pattern of DNA methylation in many neurological disorders, such as Alzheimer’s disease. Based on our DNA methylation maps of mouse and human brain we identified new target genes in Alzheimer’s disease. Our study of the methylation and expression patterns of our selected candidate genes (BDNF, Reelin and PSEN1) showed that Reelin and BDNF mRNA and protein levels are independent on DNA methylation of their gene promoters. The higher PSEN1 expression in females seems to be dependent on DNA hypomethylation at specific CpG sites
The finding that Reelin, BDNF and PSEN1 expression, in addition to age and genotype, are also influenced by gender represents a major breakthrough. Our findings highlight gender as a novel and important variable to be considered for further studies aimed at characterising molecular mechanisms underlying AD pathogenesis. In fact, it offers a novel framework for a better understanding of the higher AD incidence observed in the females of our species.
We used precise morphological definitions and whole genome sequencing to evaluate genetic constellations of healthy state brain in oldest-old individuals. In the DEVELAGE project we have compared a group of individuals who demonstrated normal brain ageing to individuals with Alzheimer’s disease in search of a protective factor. This work also has the potential to serve as basis for the development of personalised therapy. Indeed, we were able to define a target molecule for the family of sirtuins that we evaluated in brain tissue to assess their role in the progression of disease. We observed complex patterns of sirtuin expression in correlation with tissue damage, which has the potential to inform the development of therapies interacting with sirtuins. In addition, we demonstrated that human brain cells internalise disease-associated α-synuclein reminiscent of that of prion protein seen in prion diseases. This observation in humans supported the notion of prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures based on experimental studies. Demonstration of this mechanism in the human brain is crucial for the development of therapies against Parkinson’s disease or dementia with Lewy bodies since it suggests that therapies, which block the uptake of pathological α-synuclein, may prevent the damage of neurons affected in these disorders. In this study we used a novel antibody that selectively binds to the pathological but not to the physiological normal form of α-synuclein. We used a complex approach based on electron microscopic examination, which enabled us to visualise how the pathological molecules are transported in the cells.
We characterised and evaluated the gray mouse lemur, a non-human primate model of ageing, using an integrative approach. Firstly, we sequenced the whole genome of one AD-like carrier of numerous amyloid beta deposits. Our analysis of the genetic variations of the APP gene, which is involved in Alzheimer’s disease, seems to show that this gene is also associated with the AD-like trait in the mouse lemur. This result supports the case for this species being a very good primate model in which to study Alzheimer’s disease.
We have succeeded in establishing for the first time a validated behavioural test battery for the model mouse lemur with which boldness, neophobia and general activity can be measured reliably. These are phenotypic markers of emotionality implicated as indicative of the presence of AD. We successfully implemented a CANTAB comparable cognitive test battery and translated three tasks with which we could compare cognitive performance of cohorts of young and aged mouse lemurs.
All in all, the establishment of these methods is the first step towards the further validation of brain imaging, behavioural and cognitive phenotyping tools, in this new primate brain ageing model across laboratories, an important issue for the usage of this primate model for therapeutic interventions.
There are numerous direct beneficiaries from the knowledge generated by this research project. These include the investigators working on the project and their staff and students, who have benefited from training and experience and are likely to continue investigating age-related common degenerative disorders under additional peer-reviewed external funding. Additional beneficiaries are the DEVELAGE partners, whose awareness of the role of specific issues involved in age-related common degenerative disorders has been substantially enhanced by regular discussion in DEVELAGE project meetings, and the global scientific community, who will benefit from access to data when these are published in peer-reviewed journals.
Impact on health care issues
Population ageing is a highly generalised process. It is most advanced in the most highly developed countries. With the ageing of population, diseases such as AD type dementia will affect more and more people. AD affects 3 to 5% of the population aged between 65 and 74, with this figure increasing to around 50% in those over 85 years old. Society problems linked to dementia are numerous and affect not only patients, but also those in their surroundings, as most patients require long-term care. Dementia is a progressive brain dysfunction, leading to a restriction of daily activities. People with dementia suffer mainly from impaired memory and orientation, limitations of concentration, planning and judgement, personality changes and later also perceptual, speech and walking disorders. During the course of dementia, patients often lose their independence in managing everyday life. There are effects on perception and social relationships; people become more and more dependent on care. The average annual financial cost of dementia in the UK has recently been estimated by the Alzheimer’s Society (UK) to be £32,250 (approximately €44,400) per person (including costs of healthcare and social care at home or hosted in long stay establishments). If no progress is achieved in the understanding of how preventive therapy could be initiated, the consequences could be dramatic; even the collapse of European and world-wide health-systems.
DEVELAGE results could lead to improvements in the health of European citizens by having addressed a major health problem with a particularly effective approach. The results of the DEVELAGE project will enable researchers to define the first alterations in the human brain preceding the development clinical symptoms. These observations could be translated either into biomarkers that can predict cognitive decline or help to define targets for neuroprotection. This is crucial, since currently the effectiveness of the therapies used is reduced due to their relatively late application, i.e. when the symptoms are already present. This concept is also supported by genomic studies defining protective and risk constellations. In summary, the definition of ‘protective’ and ‘risk’ genomic and transcriptomic constellations can be used for predicting when preventive therapy should be initiated and the characterisation of proteins that may be used as biomarkers with predictive and prognostic value for age-related disorders. This could lead to a reduction in the costs of nursing of patients and reduce the time lost from active work, since therapy can be initiated at an early phase of disease. Elongation of a patient’s active life period by even 3-5 years can dramatically decrease the workload and emotional distress of family members and society. DEVELAGE has presented further findings, which represent the first important steps towards developing effective therapies. The demonstration of the internalisation process of α-synuclein in neurons provides support for the development of vaccination strategies to block the uptake of pathological α -synuclein. The description of the role of sirtuins could help to develop better targeted therapies acting on sirtuins, increasing the likelihood of therapeutical trial success by providing the exact knowledge of what happens in the human brain during the progression of Alzheimer’s disease. The discovery of microRNA disturbances opens up an innovative novel approach to modify harmful aspects of brain disease. In addition, the studies on the developing brain clarify many open questions as to how some brain structures are formed. This is important for individuals with mental retardation and in particular for individuals with Down syndrome. Several observations in the DEVELAGE project defined differences which are already present during the brain development of normal and Down syndrome individuals. These aspects can be used to define strategies leading to the improved mental capability of individuals with Down syndrome and could also help to retard the development of Alzheimer’s disease in these individuals. Our findings on the methylation of genes in Alzheimer’s disease supports the notion that therapies or even food supplements (like SAM) could be effective in hindering harmful pathways in Alzheimer’s disease. Finally, for the development of any therapies a reliable animal model is needed. DEVELAGE has been able to make the first steps towards the validation of a highly promising non human primate animal model of brain ageing.
Cross-thematic impact
Currently, researchers either work in the field of developmental neuroscience, the field of age-related neurodegeneration or in behavioural neuroscience, and the cross-talk between these disciplines is rare. Our project is well positioned to have an impact on all of these fields and opens the pathways for collaborations. DEVELAGE provided several results on the neurogenesis in brain structures important for cognition and memory. These are important for researchers in the field of Alzheimer’s disease, since they allow us to better understand which cell populations might be disease-sensitive and need support during age related cognitive decline. The issue of neurogenesis in the ageing brain is still an open question; accordingly our observations contribute to this field by showing how the cell layers develop in the hippocampus and related structures, and which cell populations may have the capacity to provide new cells, even in the ageing brain. Conversely, our strategy to evaluate neurodegeneration-related protein in the developing brain also revealed important results, in particular the demonstration of different patterns of tau phosphorylation in the developing brain as compared to the developing Down syndrome brain. It is important that we evaluated factors important for the brain development in the ageing brain, which was an important link between the two disciplines. Findings of ageing-protective genetic constellations also provide information for researchers in the field of development; in fact we found several genes which are important in brain development and show up as protective in brain ageing. These concepts were also implemented in a non-human primate model where we provided the first rationale to evaluate the animal model from developmental aspects and not only from the ageing aspect. These results render the DEVELAGE project as a successful model to bring researchers working on seemingly different fields together in highly collaborative research.
Competitiveness and European added value
DEVELAGE has improved the competitiveness of European Universities and research-based SMEs by collaboration on methodological and thematic aspects that will bring forth added value to all participants. DEVELAGE has hence increased the innovative capacity of European health-related industry and business by facilitating tight interactions with world-class European researchers that will probably last beyond the end of the project. DEVELAGE collaborations have thereby strengthened the international competitiveness of European academies and SMEs that will generate added European value for SMEs and academic research.
By bringing together internationally renowned experts in closely interacting work packages, DEVELAGE has achieved world-class collaborative research on age-related common degenerative disorders, including Alzheimer’s disease. The complementary expertise of partners in project relevant fields of basic and clinical research has been successfully organised. DEVELAGE facilitated sharing of methodologies between a critical mass of partners thereby potentiating their scientific impact. Moreover, DEVELAGE has made technically sophisticated methodologies available to its partners, such as: the exchange of protocols for brain imaging; behavioural phenotyping and cognitive phenotyping of the primate brain aging model mouse lemur (UMP2/TIHO, TIHO/UMP2) as a prerequisite to standardise methods; the exchange of protocols for the characterisation of dysfunctions of brain molecular pathways in mouse lemur and human brains (MUV/UMP2); and the characterisation of mouse lemur brains phenotyped by UMP2/TIHO with comparable immunhistochemical methods as used in humans (MUW/TIHO/UMP2). Overall, DEVELAGE has brought about breakthroughs in research on age-related common degenerative disorders. DEVELAGE has produced knowledge that will significantly contribute to the development of pharmacologic strategies to treat age-related common degenerative disorders, including Alzheimer’s disease. The results of DEVELAGE could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
The strategic importance of this research project lies in the fact that there is an important potential demand and that the European Union must be at the edge of technology in the field. Indeed all European countries are concerned by age-related neurodegenerative diseases and therefore the efforts have to be considered at the European level. Indeed, the goals of the Joint Programming in Neurodegenerative Disease project (JPND) were defined as to find cures for neurodegenerative diseases and to enable early diagnosis for early targeted treatments (http://www.neurodegenerationresearch.eu/). DEVELAGE has impact on these goals, since our aims comprise also the characterisation of the earliest stages of AD. DEVELAGE also fits well with the Innovation Union strategy, to enhance European competitiveness while tackling societal challenges (http://ec.europa.eu/research/innovation-union/index_en.cfm?section=activehealthy-ageing). With the implementation of this project the European investment into basic research of high potential for the health industry is strengthened at a time when the US is cutting investments into research. This investment will therefore create career opportunities for young researchers in Europe and strengthen an important area of research in Europe.
Main dissemination activities and exploitation of results
Professional project management and dissemination of results has also significantly contributed to the successful collaboration of the DEVELAGE beneficiaries. The Coordinator has encouraged all participants to actively disseminate their project aims and results to colleagues, stakeholders and the wider public
At the beginning of the project, a logo and an overall project design was created (see Figure 4) in order to identify the project. It is used together with the Grant Agreement number HEALTH-F2-2011-278486 and the FP7 logo on any printed and electronic issues for the public and for any other official contact.
The DEVELAGE project was very successful in attracting the attention of two major media outlets, including being featured by the EuroNews award winning science documentary Futuris in their video ‘Deep inside the brain’, which was estimated to have been viewed by approximately 1 million viewers and coincided with the European Month of the Brain initiative in May 2013. Additionally, the DEVELAGE project was selected as one of the most interesting research projects currently being funded by the EC on cognitive ageing for a documentary filming by ASAPS www.asaps-sharingage.eu (see Figure 5).
On the web, public awareness of DEVELAGE has been addressed by the project website www.develage.eu. The website content was regularly updated and includes key background information about the project and its beneficiaries. Additionally, fact sheets are available in 5 languages for external stakeholder to be able to obtain information at a glance. The news and media section provides visitors with the latest publications and dissemination activities of DEVELAGE, as well as a series of DEVELAGE video clips, which were released over a period of months during the 2nd year of the project, in order to generate significantly greater exposure for the project and increased impact.
A major step for the dissemination of the project, both in this reporting period and as a lasting tribute to the achievements and impact created by the DEVELAGE project, was achieved through the creation and launch of the DEVELAGE ‘gateway’ website. The concept for the new ‘gateway’ website was to provide a project overview with a focus on: partners, results, collaboration and impact. This would require neither updates nor maintenance in the future and would represent a digital final results summary, thus providing an effective substitute to the planned print version, but with the considerable advantage of increased visibility and the possibility to update if required. The focus of the ‘gateway’ website is on large images and graphic elements with the text kept deliberately short to provide a quick summary overview to the whole project. The website was designed in a modular fashion to allow easy implementation of possible future updates, such as announcements of symposia, workshops and other public events. The new ‘gateway’ website also provided the opportunity to accommodate changing technology trends by providing full compatibility for viewing on mobile devices, including mobile phones and tablets.
The original project website was updated throughout the reporting period and is now archived but still accessible by means of a prominent link available on the new ‘gateway’ website, with a prominent reciprocal link also featured on every page of the original website redirecting back to the summary page. In this way interested parties still have access to the information available on the original DEVELAGE website.
The Consortium also took measures to provide DEVELAGE exposure to the public through conventional PR media work and new media, which complemented the public access website, particularly for those that do not have internet access.
The scientific community has a vested interest in keeping up with DEVELAGE progress and results to build on the body of knowledge that currently exists. This will be achieved through conventional scientific dissemination means, i.e. peer reviewed publications and presentations at international conferences. 53 peer-reviewed publications were published during the project funding period as part of the project and partners have presented DEVELAGE work world-wide 73 times at international scientific conferences. These publications also included a significant number with contributions from multiple partners, emphasising the impressive level of cross-consortium cooperation, which the DEVELAGE project has demonstrated. A number of additional publications are at the preparation or review stage and are expected to be published in the near future.
Major dissemination highlights during the last year of the project included the DEVELAGE satellite symposium being accepted by, and organised as part of, the XVIIIth International Congress of Neuropathy, which was held in Rio de Janeiro, Brazil in September 2014. This symposium was attended by 50 congress participants and received extremely positive feedback with the conclusion of the attendees being that the concept is very novel and highly interesting. Additionally, a further DEVELAGE symposium under the title ‘Linking Pathways in the Developing and Ageing Brain with Neurodegeneration’ was organised by the project management team, and held at the Medical University of Vienna, Austria on 27th November 2014, in month 35 of the project. The DEVELAGE Vienna symposium was held in cooperation with the Clinical Neurosciences (CLINS) Doctoral Programme, and consisted of talks presented by DEVELAGE partners and also young researchers from DEVELAGE and CLINS. In total the symposium was attended by 62 people. Three training events in Barcelona, Montpellier and Vienna were organised during the project, to provide an opportunity for young researchers from across Europe to benefit from the expertise and insights of the DEVELAGE project, these also included a ‘hands-on’ brain training course, restricted to 18 people.
List of Websites:
Website:
http://www.develage.eu/
Contact details:
Project Coordinator
Medizinische Universität Wien
Prof. Gabor Kovacs
Clin. Inst. Neurology
AKH Wien 4J
Währinger Gürtel 18-20, 1090 Vienna, Austria
E-mail: gabor.kovacs@meduniwien.ac.at
Phone: +43-1-40 400 55 07
Fax:
biolution - DEVELAGE office
Helmut-Qualtinger-Gasse 2/2
1030 Vienna, Austria
E-mail: office@develage.eu
Phone: +43-1-786 95 95
Fax: +43-1-786 95 95 20
List of DEVELAGE partners
Beneficiaries
Medical University of Vienna (Coordinator) – Gabor Kovacs - Austria
The Bellvitge Institute of Biomedical Research - Isidro Ferrer - Spain
Amsterdam Medical Center – Eleonora Aronica - Netherlands
Sapienza University of Rome – Sigfrido Scarpa - Italy
Tierärztliche Hochschule Hannover – Elke Zimmermann - Germany
University of Montpellier 2 - Jean-Michel Verdier - France
Universite Paris Diderot – Homa Adle-Biasette - France
biolution GmbH (SME) - Iris Grünert - Austria
