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Memory loss in Alzheimer disease: underlying mechanisms and therapeutic targets

Final Report Summary - MEMOSAD (Memory loss in Alzheimer disease: underlying mechanisms and therapeutic targets)

Dementia is a brain disease characterised by memory loss, personality change and impaired intellectual functions. Alzheimer disease (AD) is the most common type of dementia. Dementias constitute a major public health problem. The risk of developing the disease increases with age and life expectancy and we anticipate a rise in number of cases. This will have impact on the European healthcare system and the societal and economic burdens. AD cannot be prevented or cured. The only available treatments temporarily alleviate symptoms. Development of effective treatments has been limited by a lack of knowledge. The disease is characterised by extracellular plaques composed mostly of Abeta peptide and intracellular neurofibrillary tangles composed of the abnormally phosphorylated microtubule-binding protein tau. Toxic pathways induced by Abeta and tau are unknown. The nature of the toxic Abeta and tau species remains elusive. Disease-modifying therapies are only possible when a detailed understanding of the molecular basis of the disease process is available. The consortium was created with the aim of defining the molecular mechanisms of Abeta- and tau-induced synaptotoxicity and of developing disease-modifying therapeutics for the prevention of memory loss in AD. MEMOSAD brought together 10 expert groups from six European countries that are recognised leaders in the field of memory and AD research. The project was structured in four scientific work packages (WPs).

Project context and objectives

Dementia is a brain disease characterised by memory loss, personality change and impaired intellectual functions which are caused by diseases or trauma to the brain. AD is the most common type of dementia accounting for 50 - 70 % of all cases. Dementias constitute a major public health problem. In 2008 there were about 7.2 million people with dementia in EU-27, with a cost of EUR 160 billion, of which 56 % was informal care. Because the risk of developing the disease increases with age, we anticipate a rise in the number of cases. This will have huge impact in the European health care system and the societal and economic burden. One characteristic symptom in AD is memory loss. As the disease progresses, the patient becomes increasingly dependent. Currently, AD cannot be prevented or cured. Available treatments only temporarily alleviate symptoms. Development of effective treatments is limited by a lack of knowledge. AD is characterised by two extracellular plaques composed mostly of Abeta peptide and intracellular neurofibrillary tangles (NFT) composed of abnormally phosphorylated microtubule-binding protein tau. A large body of evidence linked Abeta accumulation to neurotoxicity, supporting a causative role of Abeta accumulation in AD and providing the rationale for a number of AD therapies that target Abeta. There is no genetic evidence linking tau and AD.

Mutations in the tau gene cause a different form of dementia known as fronto-temporal dementia with Parkinson linked to chromosome 17, characterised by the presence of NFT in the absence of amyloid plaques. Tau accumulation is sufficient to trigger neurodegeneration. Pathological tau accumulation occurs in AD and FTDP-17 and a number of neurodegenerative diseases known as tauopathies. A mechanistic link between Abeta and tau pathologies was identified. A successful approach for prevention and treatment of AD required an understanding of the relationship between Abeta and tau. Prophylactic therapy should consider both pathologies. Soluble Abeta oligomers seemed responsible for most of Abeta-associated toxicity. The nature of oligomers has been described as neurotoxic. The underlying mechanism of Abeta toxicity was unknown. The exact sequence of Abeta-induced events, their causal relationship and their relevance to AD pathology and the role of tau as a possible mediator was largely unknown. Little was known on toxic mechanisms induced by abnormal tau. Tau is a neuronal, microtubule-bound protein that under pathologic conditions becomes hyperphosphorylated, dissociates from microtubules, gets misrouted into the somatodendritic compartment and self-aggregates to form oligomers which progress to the tangles. The high capacity of tau as a phosphate acceptor could upset signal transduction pathways based on phosphorylation. In other amyloidogenic proteins, the process of aggregation involves intermediates or by-products, which may be more toxic than polymeric end products. The toxicity of tau might be due to a direct toxic effect of tau oligomers or to altered microtubule-dependent processes, which may lead to starvation of synapses, oxidative stress and energy crisis. The stereotypic progression of tau pathology in AD could be caused by direct transfer of toxic tau species to neighbouring cells in a prionoid fashion. Oligomers and higher aggregates might be treatable by brain-penetrant aggregation inhibitors. Experimental evidence suggests that abnormal Abeta accumulation triggers tau pathology and tau has been proposed as an essential mediator of Abeta-induced neurotoxicity. The steps connecting Abeta to tau remained undefined.

Objectives

Memosad aimed at defining molecular mechanisms of Abeta and tau and at developing disease-modifying therapeutics for the prevention of memory loss in AD. The objectives were:

- to define the toxic Abeta and tau species responsible for memory loss in AD;
- to elucidate the mechanisms of Abeta-mediated and tau-mediated toxicity at the basis of memory loss;
- to define the mechanistic link between Abeta and tau that brings about memory loss and to translate the biological findings into therapeutic strategies.

The work started in January 2008 and ran for 42 months.

The project was structured in four WPs:

WP1: Effects of Abeta oligomers on memory loss and synaptic function
WP2: Effect of wild-type tau and tau mutants on memory loss and synaptic function
WP3: Combined effects of Abeta and tau on memory loss and synaptic function
WP4: Translation of fundamental findings in therapeutic strategies.

Project results

WP1: Effects of Abeta oligomers on memory loss and function

The aim of WP1 was to define the toxic Abeta species and the mechanism of toxicity.

1.1 Biochemical and biophysical characterisation of Abeta preparations

Using synthetic Abeta as a source for oligomers beneficiary 2 (B2) generated in vitro mature Abeta fibrils and showed that incubation of the fibrils with natural lipids was sufficient to destabilise and solubilise the fibrils in a way that oligomers were generated. Amyloid fibrils usually considered as stable and inert can be destabilised and easily reverted to toxic oligomers by lipids present in the brain. The group showed that different ratios of Abeta40/Abeta42 can affect the kinetics of amyloid fibril formation. There is a sigmoid curve with a lag phase corresponding to nucleus formation followed by a slope for fibril growth. Pathologic Abeta40/Abeta42 ratio of 7:3 has a short lag phase and slower extension rate compared to physiologic 9:1 ratio and delayed fibril formation. The pathological 7:3 ratio preparations produced unstructured networks of aggregated peptide compared to fibrils obtained with Abeta preparations at 9:1 ratio. B5 demonstrated that Abeta in the medium from APP-expressing cultured cells is monomer, dimer, trimer and tetramer. They found that SDS-stable Abeta dimers and trimers cannot be detected with an Asp1-directed antibody. It is likely that the free N-terminus is either absent or masked by an N-terminal extension. B5 has made progress in the characterisation of Abeta species isolated from human brain. The presence of Abeta monomer in TBS and TBST extracts of temporal cortex seems to be specific for AD.

Abeta monomer in formic acid extracts was not specific for AD. A similar pattern was seen in frontal cortex. SDS-stable dimers were frequently present in brain extracts with 9/14 AD cases exhibiting appreciable dimer in the TBS and TBST fractions of temporal cortex with virtually no dimer in corresponding extracts of non-demented (ND) or non-AD dementia (DNAD) brains. The amount of dimer in the formic acid extract appeared higher in AD compared with DNAD and ND, but was not as consistent in the frontal cortex. Two differences were observed between 7PA2 and brain oligomers. The extreme N-terminus of Abeta in brain dimers is intact and accessible under native conditions. C-terminal specific antibodies cannot IP brain oligomers, whereas they can IP brain monomer. All antibodies tested can detect brain dimers by Western blot indicating that dimers are composed of full-length Abeta, but that under native conditions their C-terminus is inaccessible. Only one oligomer-specific antibody was capable of recognising Abeta in the water-soluble phase. Abeta monomers and dimers in AD human brain could be phosphorylated at one or two sites. B5 characterised Abeta in extracts of 100 brain samples. This revealed an interesting correlation between TBS-soluble Abeta monomer and SDS-stable Abeta dimer and Braak staging, suggesting that both SDS-stable dimer and water-soluble Abeta monomer are linked to aberrant tau metabolism. B5 and B8 analysed Abeta species present in the brain of APP J9 mice, which overexpressed human APP harbouring at different ages. Species of Abeta oligomers ∼ 30 and ∼ 50 kDa were detected in the TBS fraction in the hippocampus of APP mice by 6 months of age. These species increased at 12 months and decreased at 18 months. Insoluble Abeta species were detected in APP mice at 12-18 months of age. To correlate changes in memory and the presence of specific Abeta species in vivo, the behaviour of APP J9 mice was analysed.

1.2 The toxic effects of Abeta oligomers

The effect of Abeta species on memory is investigated using injection of natural or synthetic Abeta oligomers directly into the brain of naïve animals and analysis of APP-expressing transgenic mice that constitutively produce their own Abeta. B2 and B9 studied the acute biological effects caused by injected synthetic backward Abeta oligomers in exploratory and memory tests. Injection of backward oligomers prevented successful memory formation. One week after oligomer injection animals have recovered and could no longer be distinguished in open-field activity from control or untreated animals. Forward and backward oligomers are indistinguishable biochemically and cause similar pathophysiological defects. The injection of pathological Abeta preparations before the shock event prevented animals from successful memory formation. B5 and B9 analysed the effects of intracerebroventricular (icv) injection of natural Abeta oligomers on memory consolidation in rats using an avoidance learning paradigm. Rats were trained in an avoidance test and at 0, 3, 6, 9 or 12 hours post-training were icv injected with 5 μl of Abeta-containing 7PA2 CM or 7PA2 CM immunodepleted of Abeta (ID-7PA2). When tested for recall at 24 h post-training there was no significant difference between groups receiving 7PA2 CM or the ID-7PA2 control. Rats receiving injections of Abeta oligomers at 6 or 9 hours post-training showed a significant impairment in memory consolidation at the 48 h recall point. Animals injected at 9 h had significantly fewer synapses in the hippocampal dentate gyrus than rats that showed no memory impairment. B5 showed that injection of soluble AD brain-derived Abeta in naïve rats 1 h post-training caused an impairment of memory consolidation when animals were tested for recall at 24 h and 48 h. B9 tested exploratory behaviour in mice that received a bilateral icv injection with Abeta-containing- or Abeta-immunodepleted extract 1 h-1.5 h before the experiment. Mice that received an injection with AD brain-derived Abeta covered a significantly longer distance.

This increase in activity was reflected in a higher frequency of centre and corner visits in these mice. B5 analysed the effect of icv injection of the Abeta-containing AD brain extracts on long-term potentiation (LTP). Injection of AD brain-Abeta 15 min before high frequency stimulation (HFS) strongly inhibited LTP. The same brain extracts immunodepleted of Abeta failed to inhibit LTP, whereas a mock-immunodepleted extracts strongly inhibited LTP. It was recently demonstrated that the prion protein (PrPC) is required for plasticity impairment mediated by synthetic Abeta assemblies. The group tested the effect of D13 on LTP. Pre-injection of D13 abrogated the inhibition of LTP, supporting the role of PrPC in mediating the Abeta-induced LTP impairment. B8 and B9 analysed the hippocampal-dependent memory deficits of APP J9 mice at 2, 6 and 12 months of age in the Morris water maze (MWM) spatial memory test. Control mice at all ages and APP J9 mice at 2 months improved the latencies and learned to locate the platform during the probe trial. APP J9 mice at 6 months displayed early deficits in long-term spatial reference memory. At 12 and 18 months of age APP J9 mice were not able to learn the spatial task and showed significant impairments on memory retention. These deficits were independent of amyloid plaque deposition and were associated with accumulation of intraneuronal Abeta in the mouse brains.

1.3 Toxic effects of Abeta oligomers

It has been hypothesised that Abeta oligomers induce reversible changes in synaptic morphology and composition. B2 investigated the effect of lipid-destabilised synthetic Abeta fibrils on cultured mouse neurons. The Abeta / lipid mixture containing backward oligomers resulted highly toxic to neurons inducing cell death. Preparations of Abeta at pathological ratios cause neuronal cell death and increased staining of apoptotic markers. No effect on synapses was observed with physiological Abeta40/42 preparations and with pure Abeta40. Treatment with sub-lethal concentration of pathological Abeta40/42 ratio and pure Abeta42 preparations suppressed spontaneous neuronal activity. The data indicate that pathological but not physiological Abeta40/42 ratios generate cytotoxic species that inhibit synaptic activity without affecting cell viability. Double immunostaining with the anti-synaptophysin antibody and the A11 antibody showed co-localisation when the Abeta40/42 ratio was 7:3 or 0:10. No such co-localisation was observed at 9:1 and 10:0 ratios. Extensive washing of neurons did not modify the A11 staining and did not restore synaptic activity for several hours. Partial synaptic recovery was observed 18-24 h after Abeta washing. It is likely that the partial neuronal activity restoration is due to the generation of novel synaptic contacts rather than recovery of existing synapses, suggesting that pathological situations can be triggered by Abeta species that are quantitatively similar but qualitatively different. B5 investigated the effect of natural Abeta oligomers on synapses in vivo. This revealed an approximately 40 % lower synaptic density in animals exhibiting an oligomer-mediated behavioural deficit. Data suggest that Abeta oligomers target specific temporal facets of consolidation-associated synaptic remodelling whereby loss of functional synapses results in impaired consolidation.

1.4 Signalling pathways

The toxic effects of Abeta oligomers are probably mediated by changes in molecules and signalling cascades essential for synaptic plasticity and memory. B5 analysed changes induced by icv injected Abeta on the expression of specific neuroplastic proteins. The levels of these proteins were analysed in extracts of dentate gyri from rats injected with human AD brain extract or with an Abeta-immunodepleted (ID) extract. The levels of all of proteins were similar in animals treated with AD or ID brain extracts. Phosphorylated tau was reliably detected in both groups, but did not differ between AD and ID-treated groups. pCREB was not detected. To ascertain Abeta treatment changed the processing of APP, APLP1 and APLP2 B5 compared the ratio of C-terminal fragments (CTFs) to Full-length proteins. No difference was observed. Results demonstrate that proteins believed to be centrally involved in AD are not altered at early time intervals following administration of an Abeta-containing extract that is known to impair memory consolidation. Administration of AD brain-derived Abeta did not alter markers of synaptic integrity nor the plasticity-associated transcriptional factor CREB at early stage. B8 investigated the cAMP-response element binding protein (CREB) pathway. They showed that the age-dependent spatial memory deficits observed in the MWM test coincided with reduced expression of specific CREB-dependent target genes in a time and region-specific manner. MWM training induced an increase of CREB-target gene expression in control mice that was reduced in APP J9 mice. These deficits were associated with accumulation of intraneuronal/extracellular Abeta oligomeric species but not amyloid plaques in the hippocampus and cortex. Treatment of cortical or hippocampal neurons with forskolin (FSK) or with a depolarising agent (KCl) resulted in increased neuronal activity of a CRE reporter gene.

In combination, they induced a synergistic effect. Activation of CRE-dependent transcription induced by KCl or KCl/FSK was reduced in cortical neurons from APP J9 mice. CREB-dependent transcriptional activity induced by depolarisation was reduced in wt cortical neurons treated with Abeta oligomers. CRE-luciferase transcription was blocked by the calcium-activated Ser/Thr phosphatase calcineurin/PP2B inhibitors cyclosporine (CsA) or FK-506, indicating that this phosphatase is required for activity-induced CRE-dependent transcription. Similar results were obtained for endogenous CREB-dependent genes. Ca2+ and cAMP signals increased the mRNA levels of BDNF, c-fos and Nr4A2 in control neurons. The induction of these genes was reduced in APP J9 neurons. Treatment of cultures with an anti-Abeta specific antibody or a gamma-secretase inhibitor increased CRE-transcriptional activation to that of control neurons. This confirms that extracellular / intracellular Abeta causes down-regulation of CREB-transcriptional activity in APP J9 neurons. Binding of CREB to its co-activators may be involved in the observed transcriptional deficits. Overexpression and activation of CREB transcriptional co-activators reversed the transcriptional deficits in APP J9 neurons. The level and phosphorylation status of TORC1 was investigated in total and nuclear extracts of WT and APP J9 neurons upon synaptic stimulation with KCl and forskolin. Dephosphorylated TORC1 was induced by neuronal activity in WT control neurons. The level of dephosphorylated TORC1 was reduced in APP J9 neurons indicating lower levels of active TORC1 as a result of Abeta accumulation. The levels of dephosphorylated TORC1 induced by KCL and forskolin was increased in nuclear extracts from control neurons, whereas the level of dephosphorylated TORC1 was reduced compared to control neurons in nuclear extracts from APP J9 neurons. Phosphorylation of CREB induced by KCl/FSK was enhanced in both control and APP neurons. These results indicate that deficient dephosphorylation of the CREB transcriptional co-activator TORC1 as a result of Abeta accumulation results in CREB-dependent transcriptional deficits in primary neurons from APP J9 mice. The group performed Western blot analysis of hippocampal lysates of control and APP J9 mice at 6 months and demonstrated a ∼2-fold increase in phosphorylated TORC1 in APP J9 brain lysates. This suggests that deficient dephosphorylation of TORC1 that is active TORC1, is responsible in part for hippocampal-dependent memory deficits in APP J9 mice.

WP2 Effect of wild-type tau and tau mutants

WP2 aims to identify toxic tau species and their effects on neuronal signalling cascades, synaptic and brain physiology, memory and behaviour.

2.1 Cellular models

To investigate the effect of tau mutants and tau kinases in cellular and animal models, B3 and B4 generated a number of viral vectors encoding such constructs. B3 used these constructs to express tau and tau kinases in retinal ganglion cells. Expression of tau led to inhibition of axonal traffic and decay of synapses. This could be prevented by MARK2 expression. SADK and p70S6K had a similar effect, suggesting that a number of kinases from the CaMK group can regulate tau. There seems to be interplay between MARK2 and GSK-3 kinases. GSK-3 can phosphorylate MARK2 at position 212 resulting in MARK2 inhibition. Expression of MARK2 in CHO cells led to disruption of microtubules, whereas co-expression with GSK-3 kinase rescued microtubules. Pharmacological inhibition of GSK-3 kinase might result in MARK2 activation as an unwanted side effect. B3 demonstrated that N2a neuronal cell is indeed a unique model to study tau aggregation and the generation of tau toxicity. Robust tau aggregation is obtained when the expression of the tau construct TauRD K280 is induced in N2a cells, but not with anti-aggregation mutants. The model showed several unexpected features: toxicity is linked to tau's competence to aggregate, since non-aggregatable forms of Tau do not cause toxicity; toxicity is correlated with phosphorylation in the repeat domain; the pathway of aggregation involves several sequential steps of proteolytic cleavage which generate fragments of high amyloidogenic potential. The cleavage involves cytosolic proteases and lysosomal proteases. At least two distinct mechanisms of autophagy are involved: Chaperone-mediated autophagy, responsible for the delivery of tau to the lysosomal receptor, but ingestion of tau and elimination is incomplete so that fragments of tau remain in the cytosol which then aggregate; and Macroautophagy, involved in taking up tau aggregates and delivering them to the lysosome via autophagosomes. This process can be overloaded, leading to the accumulation of tau aggregates in the cytosol. It illustrates the synergy and at the same time antagonism of macroautophagy and chaperone-mediated autophagy in this cell model. B3 studied the effect of tau mutants expressed in hippocampal neurons.

Expression of mutant tau was induced by the withdrawal of doxycycline. As tau increased, there was a parallel increase in toxicity and a decrease in cell density and dendritic spines density. Tau missorting of transgenic and endogenous mouse tau set in around DIV 16-19, concomitant with pathological phosphorylation at several sites, especially at the KXGS motifs in the repeat domain of tau. The phosphorylation of the KXGS motifs caused tau detachment from microtubules. Insoluble neurofibrillary tangles could not be detected, probably due to insufficient tau expression during the life time of the cells. B3 has set up a method to monitor the expression of tau(RD) by bioluminescence in living mice, making use of the co-expressed luciferase gene from a bicistronic plasmid. With this approach it was possible to image hippocampal and cortical cell cultures with high expression of tau. These cells (21 DIV) revealed some aggregated tau by the Sarkosyl-extraction assay, spine loss and toxicity. B4 investigated the retrograde axonal transport of the septohippocampal pathway in vivo in their tau transgenic mice that display tau pathology in the absence of motor dysfunction. In order to evaluate whether tau pathology could affect not only cholinergic neurons but also their projection to hippocampus, they evaluated AT8 immunoreactivity on sagittal sections. Septohippocampal fibres were immunoreactive, further indicating that tau pathology affects basal forebrain cholinergic neurons (BFCNs) and supporting a role of transgenic tau in impairing axonal transport within cholinergic neurons.

FluoroGold was injected within the hippocampus of Thy-tau22 mice and WT littermate controls. There was a 50 % decrease in FluoroGold-positive neurons in medial septum in Thy-tau22 compared to controls. Probably as a consequence of loss of retrograde axonal transport, NGF tended to increase in hippocampus of Thy-tau22 mice, suggesting it may not be fully available for cholinergic neurons. There was 33 % decrease in the number of ChAT-immunoreactive neurons in medial septum, suggesting that both loss of cholinergic neurons and retrograde axonal transport are involved in cholinergic dysfunction following Tau pathology in Thy-tau22 mice. Work of B4 suggested a possible role of tau in protecting neurons from DNA damage under stress conditions. Tau is lightly detected in the nucleus of neurons and in vitro studies have shown that it is able to bind to DNA. Tau could protect DNA from damage induced by hydroxyl free radicals. B4 investigated the possible role of tau in protecting neurons from DNA damage under stress conditions. The group showed that heat stress induces a reversible accumulation of dephosphorylated tau in nuclei of neurons in both primary cultures and brain slices of mouse cerebral cortex. Using chromatin immunoprecipitation assays, they could show that endogenous tau interacted with neuronal DNA in situ both under control and under heat shock conditions, but binding of nuclear tau to cellular DNA increased after a heat stress. A comparative genotoxicity test showed that tau fully protected neuronal genomic DNA against damage induced by heat stress. Heat stress-induced DNA damage observed in tau-deficiency cells could be rescued by adenoviral vectors encoding htau bearing a nuclear localisation signal. Oxidative stress increased nuclear localisation of tau. The data highlight a protective role of nuclear tau on neuronal DNA integrity under heat stress condition. This study highlights a function for nuclear tau as a nuclear key player in the neuronal early stress response.

2.2 Organotypic brain slice models

B3 investigated effects of tau constructs in the semi-native environment of organotypic brain slices. The group tested different transfection protocols of viral vectors and different promoters to achieve neuron-specific protein expression. Methods were optimised to evaluate slice viability, cell morphology and neurotoxicity. There was high variability in the levels of tau protein expression which hampered interpretation of data. The group established slice cultures from inducible transgenic mice expressing pro- or anti-aggregation variants of the tau repeat domain. Brain slices were prepared from regulatable tau mice expressing a pro-aggregation variant of the human tau repeat domain. At eight days, tau expression and phosphorylation at the KXGS motifs and tau mossorting became detectable. Tangle formation visualised by thioflavin S (ThS) staining was seen in two-week cultured slices and was associated with toxicity. Sarcosyl-extractions indicated co-aggregation of endogenous mouse tau and the human transgenic tau. There was extensive spine loss. Analysis of neurons by NeuN staining revealed a reduced number of granule and pyramidal neurons in cultures expressing pro-aggregation tau, most prominent within the CA3 region. Tau pathology could be prevented by switching off tau(RD)K280 expression or by tau aggregation inhibitors.

2.3 Mouse models of tau toxicity

Thy-tau22 mice generated by B4 express human mutant tau under a Thy1.2-promoter and display tau pathology in the absence of motor dysfunction. Using brain slices, B4 and B9 showed that LTP and long-term depression (LTD) are both affected. In-depth behavioural characterisation of Thy-tau22 mice showed clear learning and memory impairments. Apart from memory deficits, B9 addressed the possibility that non-cognitive behaviours are affected in these mice. AD is characterised by neuropsychiatric symptoms that are critical in primary care giving of AD patients and that have been relatively ignored in murine models. Six-month-old Thy-tau22 mice had higher levels of obsessive-compulsive behaviours than non-transgenic controls as measured in the marble burying assay. Tau transgenic mice showed a higher duration of immobility in forced swimming and tail suspension tests than controls. Male tau transgenic mice displayed enhanced aggression relative to WT controls, as assessed by the social dominance tube test.

These results indicate increased obsessive-compulsive behaviour, aggression and depression-like behaviour in AD and AD mouse models. B3 generated and characterised tau transgenic mouse lines with inducible up- and down-regulation of expression of different forms of tau: htau40 K280; htau40 K280/2P; K18 K280 and K18 K280/2P. The Pro-RD mice showed the most pronounced tau pathology from 3 months onwards, whereas Pro-FL tau showed only a pathological conformation of tau in a pre-tangle state in the first year. There was co-aggregation of endogenous mouse tau and exogenous human tau. Anti-aggregation mice of both variants showed no pathology up to two years. In collaboration with B9, LTP and behaviour of Pro-RD mice were evaluated before and after switching off the tau gene. Pro-RD mice display impairments in hippocampus-dependent learning and memory tests as well as hippocampal LTP and these deficits are reversed by switching off the expression of the human tau transgene. Histopathologically, during the tau expression phase the hyperphosphorylation and aggregation of tau is accompanied by a loss of neurons and synapses. Almost no pathology is observed in anti-aggregation mice. When the expression of pro-aggregant tau is discontinued, there is no visible recovery of tau aggregation and neuronal loss. There is partial recovery of synapses, which may explain the recovery of learning and memory (MWM) as well as LTP. The remaining tau aggregates in switched-off mice consist only of endogenous mouse tau whereas the aggregated human tau is no longer visible. This suggests that the tau aggregates are in a dynamic equilibrium with their subunits and that normal tau can form aggregates if poisoned, except that the mouse tau aggregates and oligomers are less toxic to synapses. More recent analysis of mice expressing full-length versions of mutant tau gave qualitatively similar results.

B4 and B8 investigated CREB and BDNF signalling in Thy-tau22 mice. BDNF is important for neuronal survival, synaptic plasticity, learning and memory. CREB signalling is involved in neuronal survival and memory consolidation and modulates the expression of BDNF and both pathways have been implicated in AD. The groups could show that there is no major loss of BDNF/TrkB expression in Thy-tau22 mice with the development of the hippocampal tau pathology and that there is no evidence of changes in expression of proteins involved in the classical BDNF/TrkB pathway. There is a lack of responsiveness to BDNF-mediated synaptic facilitation in Thy-tau22 mice that is observed as early as four months, while TrkB receptor remains activatable and forskolin-induced facilitation is not affected. BDNF-mediated synaptic facilitation in the electrophysiology experiments is dependent on NMDA receptors. Further data indicate that NMDA subunits are trapped intro sarkosyl-insoluble fractions, suggesting that tau aggregates may sequester these receptors. Depression of the field excitatory post-synaptic potentials fEPSP slope induced by direct NMDA application on WT slices is significantly reduced in Thy-tau22 mice. The data suggest that tau pathology induces alteration of NMDA-dependent synaptic response to BDNF, possibly contributing to memory alterations.

2.4 Zebrafish as a model for Alzheimer tauopathy

B6 has developed a novel transgenesis system that allows the expression of genes of interest in zebrafish at high levels and that greatly facilitates identification of the transgenic fish by the simultaneous expression of a fluorescent reporter (DsRed). Tau constructs cloned in this system were provided by B3: htau40, htau40 K280 (Pro-FL), htau40 K280/2P (Anti-FL), K18 K280 (Pro-RD), K18 K280/2P (Anti-RD), F3 K280, F3 K280/2P and htau40P301L (pro-aggregation). Transgenic fish were generated for each of these constructs. In collaboration with B3, the group performed an in-depth characterisation of stable transgenic fish carrying the pro-aggregation FTDP-linked mutation P301L (htau40P301L). The fluorescently labelled tau transgenic zebrafish rapidly recapitulate key pathological features of tauopathies including phosphorylation and conformational changes of human tau protein, tangle formation and cell death as well as altered motor neuron morphology and behavioural disturbances. To analyse neurodegeneration in detail and to demonstrate the suitability of zebrafish larvae to study cellular processes in a whole living animal in vivo, B6 invested in the development of in vivo imaging techniques applied to zebrafish. In a first set of experiments the group monitored the neurodegeneration in tau transgenic fish by confocal time-lapse imaging. Neurons in the spinal cord which express DsRed were recorded over a period of 12 hours and dying cells were detected by monitoring Acridine Orange uptake. It was possible to visualise an intact neuron that first altered its shape and started to round up. The cell fragmented and took up Acridine Orange, indicating a breakdown of the cellular membranes and began to disappear. This is the first demonstration of in vivo cell death imaging in the field of neurodegeneration. B6 improved the quality and resolution of their in vivo imaging system by generating a transgenic zebrafish line that in addition to tau also expresses simultaneously a membrane-bound YFP fluorochrome (mYFP) and a mitochondrially targeted CFP fluorochrome (mitoCFP).

The system offers the opportunity to measure axonal transport of labelled mitochondria in living zebrafish. Using this system and in collaboration with B3, the group first showed that neuronal morphology is not altered in the stable tau transgenic fish. However, axonal transport was strongly reduced in these animals. Preliminary results indicated that the density of both static and moving mitochondria in peripheral axons is highly reduced, while the speed of the moving mitochondria is not dramatically altered. The difference in axonal transport rates is caused by tau, since this effect is not observed in fish expressing only DsRed (without tau). Areas outside of the repeat domain are required for tau effect, since the expression of repeat-domain-only tau (htau40 K280) does not seem to affect axonal transport. To further validate that the transport inhibition is mediated by the microtubule binding of tau, B6 co-expressed MARK kinase, which phosphorylates tau at the microtubule-binding domain and subsequently detaches tau from the microtubules. Upon MARK expression axonal transport was restored in tau transgenic zebrafish. Microtubule destabilisation through nocodazole treatment of WT larvae could fully phenocopy the transport inhibition phenotype of the tau transgenic larvae. The tau-fish model is suitable for screening compound libraries and was used to test compounds targeting the tau-kinase GSK3. High throughput screening for inhibitors of the tau-kinase GSK3 resulted in the identification of approximately 2000 compounds. Several compounds from the pyrazine chemical series were co-crystallised with GSK3 and subsequently optimised for potency and selectivity. In tissue culture cells two novel compounds exhibited highly specific and selective inhibitory profiles for GSK3. For in vivo validation the group used the htau40P301L transgenic fish. While two of the GSK3 inhibitors were highly active in cultured cells, only one of them displayed in vivo activity. The in vivo active GSK3 inhibitor lowered abnormal tau phosphorylation by as much as 70 %. The compound was not capable of reducing tau-induced toxicity in the transgenic zebrafish htau40P301L model.

2.5 C. elegans as a model for Alzheimer tauopathy

C. elegans is an organism for the investigation of human diseases. It has a simple and stereotyped nervous system, containing 302 neurons with a nearly complete map of all axons and synapses. To investigate the pathological mechanisms of tau protein in vivo B7 got a number of tau mutants cloned in C. elegans vectors from B3 and used these constructs to generate various worms' transgenic lines. Lines expressing the pro-aggregation variants show slower growth compared to the anti-aggregation lines. The pro-aggregation F3 K280 line showed progressive uncoordinated movement after reaching adulthood. Data integrated strains were obtained for F3 K280 (pro) and F3 K280/2P (anti) mutants. Both transgenic lines show retarded development, reduced body length and impairment of movement. These defects are more pronounced for the pro-aggregation mutant. The impairment of movement is progressive. The group invested in the generation of new strains exhibiting stronger phenotype. A strain has been generated that displays stronger defects than the anti-aggregation variant. The strain shows progressive neuronal loss, loss of synaptic terminals and organelle and vesicle accumulation. Tau aggregates detected in neurons are most likely the cause of the strong paralysis phenotype in these worms. In the transgenic line, there was a reduced number of synaptic vesicles and more diffuse staining of synapses suggesting defects in neuronal cargos. GABAergic and cholinergic neurons display morphological defects. The expression of pathogenic tau results in displaced neuronal projection and gaps between neurons.

Using synaptobrevin-GFP as marker, they could show that the expression of the pro-aggregation F3 K280 variant results in reduced number of synapses along the axons. The expression of F3 K280 resulted in accumulation of mitochondria in the proximal part of the axons. The transport dynamics of mitochondria was tested and was significantly reduced in the strain expressing F3 K280. This further substantiates the aforementioned model that the tau-induced pathogenesis is partially caused by axonal transport defects. B7 analysed if the tau phenotype in transgenic worms could be modified by affecting the expression of relevant proteins. The phenotype was improved upon down-regulation of known suppressors. The group crossed the transgenic htau mutant with loss-of-function strains of LRK-1, the C. elegans homologue of LRRK2. A reduction of the phenotype in the lrk-1 mutant background was observed. The group crossed the transgenic line with mutants of genes that were identified in the suppression screen. One of the most promising candidates, CYLD-1, encodes a de-ubiquitinase in C. elegans and has a clear orthologue in the human genome. Cyld-1 mutant was combined with the tau transgenes. The mutant was able to suppress both the movement phenotype and neuronal morphological defects. De-ubiquitinases represent attractive therapeutic targets and are in clinical trials to treat some types of cancer. B7 used the Split-Ubiquitin system to screen a human foetal brain library to identify interaction partners of the pro- and anti-aggregation mutants of htau40 and the F3 fragment. The screen was performed twice. Potential interaction candidates identified are functionally related to cytoskeleton regulation, transcriptional regulation and stress response.

WP3 Combined effects of Abeta and tau on memory loss and synaptic function

The aim is to understand the synergy between amyloid and tau pathologies and its consequences on neurodegeneration, memory loss and cognitive dysfunction.

3.1 Toxic Abeta oligomers on tau metabolism

Toxic Abeta oligomers identified in WP1 are tested for their effect on tau metabolism. B3 investigated early effects of different Abeta preparations on endogenous tau in primary hippocampal neurons. The Abeta preparations were ADDL preparations; cross-linked Abeta dimers obtained from B5 or synthetic Abeta at different Abeta40/42 ratios. When exposing cultured hippocampal neurons to ADDL preparations there was missorting of endogenous tau into the soma and dendrites. Redistribution of endogenous tau is one of the earliest signs of neuronal degeneration in AD. In the missorted dendritic regions there was a depletion of spines and spine-related proteins. Tau in these regions shows elevated phosphorylation at sites that are diagnostic of AD-tau and local elevation of the MARK kinase activity. These effects occur without major changes in tau, tubulin, kinase levels or activities. Missorting affects not only tau, but also other axonal markers. Abeta oligomers evoke responses that disrupt the axonal sorting machinery; they allow endogenous tau to enter dendrites and to destroy spines and microtubules locally.

This is in analogy to the loss of spines and microtubules observed in AD. Missorted tau had increased phosphorylation at pS202/pT205 in the repeat domain of tau which causes the detachment of tau from microtubules. The reaction with antibody PHF1 was not enhanced. Abeta oligomers seem to induce tau kinases specific for the KXGS motifs in tau repeats, the consequent tau phosphorylation and dissociation from microtubules. Apart from cdk5, most proline-directed kinases tested showed little change upon Abeta treatment. Changes included an increase in cytosolic Ca++ which could be responsible for microtubule disassembly. This rise in Ca++ could contribute to the inhibition of mitochondria transport. Consistent with this hypothesis, treatment of hippocampal neurons with Abeta oligomers and Taxol rescued the microtubules, the missorting of tau and prevented the loss of spines and mitochondria. Other cell stressors caused a similar effect, namely tau missorting, local disappearance of spines and increased tau phosphorylation. Missorting of tau occurs as a response to diverse types of stress and exposure to Abeta appears to be equivalent to stressing the cells. Similar experiments with Abeta dimer preparations produced in general less pronounced dendritic targeting and less deposition. Missorting of endogenous tau into the somatodendritic compartment was observed to a lesser extent. There were no changes in global tau levels and only minor changes in phosphorylation. The higher oligomers found in ADDL preparations seem more toxic than Abeta dimers. The groups analysed Abeta-induced changes using neurons derived from tau knock-out mice.

There was a notable difference in response of neurons to Abeta and other cell stressors. Tau KO neurons were less inhibited by Abeta with regard to spontaneous activity, they were less affected in terms of loss of dendritic microtubules or missorting of neurofilaments, even though Abeta oligomers were similarly directed to bind to synapses. A major difference was that the loss of spines was less pronounced, arguing that tau plays a role in the Abeta-induced synaptic loss. B2 and B4 investigated the effect of Abeta on tau metabolism in vivo, upon injection of toxic synthetic Abeta oligomers into the brain of Thy-tau22 mice. Abeta oligomers injections in free-moving Thy-tau22 animals caused a consistent increase in tau phosphorylation at different epitopes including AD2, AT8, AT180 and AT270 in all injected animals.

3.2 Combined effects of Abeta and tau pathology

Animal models are used to study the combined effect of Abeta and tau pathologies. B6 generated transgenic fish expressing AD-related proteins (APPswe, tau P301L and presenilin 1 L166P) with the aim of crossing them to obtain double and triple transgenics. APPswe fish do not produce any detectable Abeta. As an alternative to transgenic fish, B6 developed a new method to inject directly toxic Abeta oligomers into the fish ventricles. No increase in neuronal cell death was observed in tauP301L transgenic zebrafish injected with Abeta. This might be due to the instability of the Abeta peptide in zebrafish. The group crossed the APPswe and the tau transgenic fish to evaluate if the APPswe transgene increases the number of dead cells in the spinal cord of the tau transgenic fish. There was no increase in cell death in APPswe transgenic fish crossed to tau transgenic fish. A combined effect of Abeta and tau could not be demonstrated. It cannot be excluded that the lack of effect is due to the low levels of Abeta. B8 and B9 used the triple transgenic AD model that expresses APPSw, PS1 and tau to investigate the combined role of Abeta and tau on memory loss and CREB-mediated transcription. Analysis of learning curves indicated significant learning deficits in 3xTg-AD at six months but not at two months of age. In the probe trial, control mice displayed significantly higher occupancy of the target quadrant relative to other quadrants, whereas 3xTg-AD mice failed to show such a preference. The number of target platform crossings by 3xTg-AD was significantly lower than those of non-transgenic mice. No significant differences in spatial memory were detected in mice at two months of age. Immunohistochemical staining in brain coronal sections were performed using the anti-Abeta antibody 6E10 and the anti-phospho-tau antibodies CP13 and AT180.

Results indicate that hippocampal-dependent spatial memory deficits are associated with abnormal accumulation of Abeta and phosphorylated tau in the hippocampus of 3xTg-AD mice in an age-dependent manner. B8 purified total RNA from hippocampus of non-transgenic control and 3xTg-AD mice at six months. Quantitative mRNA analyses indicated significant reduction of c-fos and Arc, two CREB-target genes that are dependent on the co-activator CRTC1 in the hippocampus of trained 3xTg-AD mice. Transcript analysis of the neurotrophin BDNF showed an increase of BDNF mRNA levels in the hippocampus of WT mice after MWM training. BDNF mRNA levels were not significantly enhanced in the hippocampus of 3xTg-AD mice after MWM training. These results indicate a deregulation of CREB-dependent genes related to synaptic plasticity and memory associated with early spatial memory deficits in 3xTg-AD mice. The combined effect of Abeta and tau was investigated upon injection of toxic Abeta species into the brain of tau-transgenic mice. The toxic Abeta oligomers generated by B2 were tested on the Thy-tau22 mice by B4 and B9. Repeated hippocampal injections of small soluble Abeta1-42 oligomers in freely moving mice were able to induce marked neuronal loss in the vicinity of oligomers deposition. No significant difference was observed between all mice groups collaterally injected with control vehicle or Abeta1-42 oligomers during the training phase in the passive avoidance test. Decreased memory performance was observed in the Abeta1-42 oligomers-injected mice group.

3.3 Tau on the Abeta-induced synaptotoxic effects

B2 and B4 evaluated the effect of toxic Abeta oligomers on hippocampal neurons derived from tau knock-out mice. Abeta preparations with different Abeta42/40 ratios were evaluated in the MEA chips assay. Tau WT and KO cultures at 9 DIV and at 19 DIV demonstrated significant differences in spontaneous activity rate during development in vitro, but in both, spontaneous firing was sensitive to NMDAR and AMPAR inhibitors. After two weeks in vitro, both cultures on the chip were acutely challenged with toxic Abeta oligomers pre-aggregated for 2 h, and the rate of neural firing was compared before and after the treatment. Tau KO networks demonstrated approximately 50 % less susceptibility to Abeta42/40 10:0 and 3:7 inhibitory effects than the WT cultures and were not sensitive to Abeta42/40 0:10 and 1:9. The group analysed the dose-dependent cytotoxic effect of toxic and non-toxic ratios of Abeta42/40 on both tau WT and KO cultures. Tau KO neurons were less sensitive to toxic Abeta preparations than WT cultures, thus corroborating the MEA data. These data are in line with the results of B3, showing that tau KO hippocampal neurons are less sensitive to Abeta-induced loss of dendritic spines, loss of dendritic microtubules and missorting of neurofilaments. The data highlight a role of tau in the Abeta-induced synaptotoxic effects.

WP4 Therapeutic strategies

WP4 focused on the validation of the targets identified in WP1, WP2 and WP3, preclinical evaluation of novel therapeutic strategies targeting Abeta-aggregates and preclinical evaluation of novel therapeutic strategies targeting tau-aggregates.

4.1 Therapeutic targets

B2 identified the Aph1B subunit of gamma-secretase as a candidate target to treat AD. Gamma-Secretase is responsible for the final cleavage of APP causing release of the Abeta peptide. The same activity cleaves Notch, which complicates the development of useful inhibitors of gamma-secretase. Gamma-Secretase activity is mediated by a multiprotein complex consisting of Presenilin (PS), Aph1, Pen2 and Nicastrin (NCT). B2 generated knock-out mice deficient in Aph1 genes alone or in combination and made the observation that inactivation of the Aph1B gamma-secretase in a mouse AD model led to improvements of AD-relevant phenotypic features without Notch-related side effects. The Aph1B complex contributes to total gamma-secretase activity in the human brain. B9 tested TUDCA compound as a possible modulator of gamma-secretase activity in vivo. TUDCA is a bile acid found in liver, with proposed neuroprotective properties. TUDCA seems to decrease CTGF expression in hepatocytes. B9 tested the TUDCA compound in the APP/PS1 model for possible effect on mice behaviour. Two-month old APP/PS1 mice were treated with TUDCA-supplemented food pellets. Memory rescue was observed. No difference between treated transgenic and control WT mice could be detected. There was significant decrease in Abeta plaques in hippocampus and cortex in treated mice. Gamma-secretase modulation by TUDCA compound might be beneficial for AD treatment. B3 demonstrated that aggregated tau is cleared mainly by the process of macroautophagy. The group analysed several modulators of autophagy in the N2a cell model. Of interest was the investigation of trehalose. The group showed that trehalose treatment induces activation of autophagy as seen by increase of autophagosomes and autophagy markers. There was a concomitant reduction in levels of soluble tau, tau aggregates and toxicity.

These results suggest that trehalose has the potential to be used as a treatment of tauopathies, including AD. B4 showed that voluntary exercise in the Thy-tau22 mice improves their cognitive performance. The loss of cholinergic neurons was prevented by voluntary exercise NPC1 and NPC2 genes were identified as upregulated in the exercised animals. Since NPC enhances cholesterol efflux from neuronal cells, the group tested the role of an enzyme involved in cholesterol brain elimination. The major exportable form of brain cholesterol is generated by an enzyme encoded by the CYP46A1 gene. Increasing brain CYP46A1 gene expression through AAV-mediated gene transfer reduced the level of hyperphosphotylated tau and improved spatial memory defects. The data suggest that playing on cholesterol metabolism may be of interest in AD treatment. CYLD-1 encodes a de-ubiquitinase in C. elegans and has a clear orthologue in the human genome. To validate de-ubiquitinases in general and CYLD-1 gene in particular as candidate therapeutic targets the group downregulated the CYLD-1 gene in tau-transgenic worms by either RNAi treatment or by crossing this strain with the cyld-1 mutant. These data validate the de-ubiquitinases as attractive therapeutic targets. De-ubiquitinases are in clinical trials to treat types of cancer. The CREB-pathway plays an important role in Abeta- and tau-induced toxic effects. The group performed gene therapy assays in vivo using adeno-associated vectors expressing GFP or mouse TORC1 (CRTC1). The performance of all groups improved significantly during spatial training, although the latencies of APP mice injected with the AVV-GFP were higher on days 2, 3 and 4 than those injected with AVV-TORC1 or non-transgenic control groups.

In the probe trial, control GFP- and TORC1-injected mice displayed significantly higher occupancy of the target quadrant relative to other quadrants, whereas GFP-injected APP mice failed to show such a preference. TORC1-injected APP J9 mice showed a preference for the target platform location similar to the control groups. The number of target platform crossings by TORC1-injected APP mice was different to the rest of platform locations and to target platform crossings of GFP-injected APP mice. This result indicates that gene delivery of TORC1 directly into the hippocampus efficiently reverses spatial and learning memory impairments in APP J9 mice. The data validate the activation of CREB-pathway as a possible therapy for AD treatment. We validated the modulation of tau phosphorylation as possible target for AD therapy. Tau kinases and phosphatases are known to regulate the phosphorylation, conformation and aggregation of tau. Counter players of the kinases are PP-1 and 2A, 2B, modulated by accessory proteins. The aim is to demonstrate that modulation of tau phosphorylation brings a therapeutic benefit. B9 evaluated the modulation of tau phosphorylation in brain slices of Thy-tau 22 mice. Acute application of CDCK5- and GSK3-antagonists restored a late-phase LTD (L-LTD) in the tau slices. Acute application of selenium (Se2+) restored the L-LTD in the transgenic slices without affecting the WT L-LTD. Studies by B4 identified a down-regulation of total protein phosphatase activity in Thy-Tau22 mice. These data indicate that loss of total phosphatase activity may be contributing to tauopathies and validate the modulation of tau phosphorylation as candidate target in AD treatment. B9 tested Se2+ in vivo in AD mouse models.

In behavioural assays, the Se2+-treated AD mice exhibited neuro-cognition at similar levels as control mice. Tau mice with no Se2+-supplement maintained deficits in neuro-cognition. Analysis of hippocampal slices showed comparable synaptic plasticity in treated transgenic mice. The group tested brain slices from APP/PS1 mice. Chronic Se2+ application rescued the L-LTD and had a marked effect on synaptic transmission. The beneficial effects of modulators of tau phosphorylation are apparent in the tau-transgenic mouse model and in another AD model that does not overexpress tau. B2 and B4 investigated the role of microRNA-regulated pathways on tau phosphorylation and neurodegeneration. They used a conditional knockout approach to remove Dicer from pyramidal neurons in the adult mouse cortex and hippocampus. Dicer is a type III RNase enzyme responsible for the processing of microRNA precursors into mature microRNAs. Dicer deletion caused neurodegeneration in hippocampus and cortex that coincided with tau hyperphosphorylation. Systematic analysis of enzymes involved in tau phosphorylation identified ERK1 as one of the candidate kinases responsible for this event in vivo. The groups found that miRNAs belonging to the miR-15 family are potent regulators of ERK1 expression in mouse neuronal cells and are co-expressed with ERK1/2 in vivo. B6 contributed to the validation of tau phosphorylation as possible AD therapy by using the zebrafish tau-model. The group overexpressed in tau-zebrafish MARK kinase or an inactive mutant (dnMARK) provided by B3. MARK overexpression could rescue the transport deficit in tau-transgenic fish to normal levels. The number of motile mitochondria was higher than in WT fish. These data demonstrate that the phenotype induced by tau can be modulated by modulating tau phosphorylation.

4.2 Anti-Abeta aggregation compounds with therapeutic potential

B10 has molecule inhibitors of Abeta aggregation that are effective in reducing Abeta toxicity in cell culture paradigms, LTP in hippocampal slices and in an acute model of cognitive impairment. RS-0406 blocks natural oligomer formation in CHO cells stably expressing human APP751 with the V717F FAD mutation and prevents the Abeta-mediated deficit of LTP by inhibiting oligomer formation. It is effective in protecting against the deficit in cognitive behaviour induced by naturally derived oligomers of Abeta. B10 researched the potential of RS-0406 as a basis for therapeutically effective inhibitors of toxic assemblies of Abeta. This led to SEN1269, which shows a significant improvement in potency in both Abeta fibrillogenesis and cell viability assays compared to RS-0406. SEN1269 shows greater cell penetration than RS-0406 and an improved in vitro ADME profile. In vivo, SEN1269 is effective in rescuing the deficit in LTP caused by naturally secreted oligomers of Abeta. SEN1269 is effective in a dose-related manner in protecting against the deficit in cognitive behaviour induced by exogenously applied (icv) naturally secreted oligomers of Abeta. The compound lacks oral bioavailability and CNS penetration. Several compounds have been identified which have promising in vitro ADME profiles. SEN1428, SEN1449, SEN1461, SEN1500 and SEN1576 show appreciable oral bioavailability and brain penetration. B5 and B10 tested the effects of the compounds on memory in vivo. SEN1500 provided a dose-related protective effect to the lever switching errors and incorrect lever perseverations induced by icv injection of Abeta. B3 tested the Abeta anti-aggregation SEN compounds on tau-aggregation obtained from B10. The compounds were not effective in preventing tau aggregation in vitro and in the N2a cell model of tauopathy.

4.3 Anti-tau aggregation compounds with therapeutic potential

B3 explored the potential of rhodanines and PTH compounds. Both compounds crossed the blood-brain-barrier (BBB). B3 evaluated Methylene Blue (MB) in the N2a cell model of tau aggregation. MB is a potent inhibitor of Abeta and tau aggregation in vitro. Two compounds of the rhodanine family and the PTH family exhibited pronounced protection against tau-induced toxicity and resulted in a complete rescue of cell viability. B3 developed an organotypic slice model derived from regulatable pro-aggregant TauRD mice and demonstrated that compounds of the rhodanine class inhibited tau aggregation, spine loss, synapse loss, neuronal loss and toxicity in hippocampal slices. This suggests that toxicity is linked to the aggregation of tau and can be rescued by aggregation inhibitors. B6 evaluated two tau anti-aggregation compounds of in zebrafish. Both failed to reduce tau-induced neuronal death. B7 tested in the C. elegans model of tauopathy tau anti-aggregation compounds. A tau anti-aggregation compound was able to reduce the tau-mediated toxicity in C. elegans. Movement defects and the abnormal changes in neuronal morphology were improved.

Analysis of MB-treated mice showed an increase of tau-aggregation and decreased levels of autophagy-related proteins. The anti-aggregation approach appears as a promising strategy for tau-induced pathology. B7 performed a genome-wide RNAi screen to identify suppressors of tau toxicity in C. elegans. 20 candidates were identified with 10 potential therapeutic targets. One is the protease calpain, which was shown to be involved in tau pathogenesis through processing tau or through activation of tau kinase CDK5. The strongest suppressor identified is the homologue of tubulin chaperone TBCE or TBCEL that is involved in the regulation of the turnover of tubulin. The de-ubiquitinase CYLD-1 was able to suppress the tau-mediated phenotype upon RNAi treatment. B4 started active tau immunisation targeting a particular tau epitope in the Thy-tau22 mouse model. An immune response was obtained against the targeted epitope. A trend to decreasing AT100- and AP422-immunoreactivities was observed in brain sections of vaccinated animals compared to unvaccinated control Thy-tau22 mice. These data were supported by biochemical analyses showing a decrease in aggregated tau in vaccinated animals. Active immunotherapy delayed cognitive deficits in the Thy-tau22 model. Tau concentration increased in sera of vaccinated mice, suggesting that circulating antibodies sequester tau and favour tau efflux from the brain. These data suggest that tau immunotherapy may be a useful therapeutic strategy for AD and other tauopathies.

Potential impact

Our project unified leading groups of neuroscientists from Europe experienced in Abeta and tau research. We proposed that Memosad would deliver three or four validated therapeutic targets and at least two compounds with demonstrated therapeutic efficacy in mouse models. We generated non-peptidic Abeta aggregation inhibitors and demonstrated their beneficial effects on memory tasks in two murine models. We validated tau immunotherapy as a useful therapeutic strategy for AD and other tauopathies. The targets identified may have additional value as biomarkers with a potential use as diagnostic tools. The data obtained is also of relevance for other neurodegenerative disorders. The project is expected to have an impact on various societal levels: on the health of European citizens by contributing to an early diagnosis of AD and development and validation of new therapies for treatment and prevention of an incurable brain disease; on Europe's economy, if medical treatment can delay symptoms by a few years and substantially decrease the economic burden of AD measured as productivity loss by affected individuals and caregivers as well as by the burden on Europe's health care systems; on Europe's competitiveness and the innovative capacity of its health-related industries by contributing to gain a role as global centre for biomedical research.

Research into AD and related dementia is a subject of high priority within the scientific world. Despite the enormous increase of our basic knowledge resulting from the allocation of all resources, a successful therapeutic approach is missing and no breakthrough seems to be in sight. This awareness confronts our societies with the question whether or not the present approach consisting of combined fundamental and clinical research will lead to the prevention and treatment of these diseases. The response science has to provide is there is no other way than to further promote our knowledge on the pathogeneses of AD. Funding was stopped at a time when the close cooperation between participants was achieved and when the common research work started to bear abundant fruit. Progress in the complicated area of research into AD and related dementias is in the long run a matter of organisation through the national and international funding institutions. The consortium organised two symposiums, published 53 and did 54 presentations and 30 posters. More papers have been announced.

Project website: http://www.verum-foundation.de/memosad

VERUM - Foundation for Behaviour and Environment, Theresienstrasse 6-8, 80333 München, Germany
Phone: +49-892-8890235;
Fax: +49-892-8890512;
verum@verum-foundation.de

Beneficiaries

1. Franz Adlkofer, VERUM Foundation
2. Bart De Strooper, Department of Molecular and Developmental Genetics, VIB
3. Eva-Maria Mandelkow, Max Planck Unit for Structural Molecular Biology
4. Luc Buee, Centre de Recherche Jean-Pierre Aubert, Universite Lille 2
5. Dominic Walsh, Conway Institute, University College Dublin
6. Christian Haass, Adolf-Butenandt-Institut, Ludwigs-Maximilians-Universitat München
7. Ralf Baumeister, Biologie III, Albert-Ludwigs-Universitat Freiburg
8. Carlos Saura, Neurosciences Institute, Universitat Autonoma de Barcelona
9. Rudi D'Hooge, Laboratory of Biological Psychology, Katholieke Universiteit Leuven
10. David Scopes, Senexis Ltd, Cambridge.

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