Final Report Summary - NEURINOX (NOX enzymes as mediators of inflammation-triggered neurodegeneration: modulating NOX enzymes as novel therapies)
Executive Summary:
Neuroinflammation is a key process associated with neurodegenerative diseases (ND). The underlying hypothesis of the NEURINOX project is that NADPH oxidases (NOX) are mediators of neuroinflammation and that NOX are promising drug targets for the development of therapies against ND.
NEURINOX focuses on several diseases with strong neuroinflammatory components and other autoimmune neuropathies, including multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and mesial temporal lobe epilepsy (MTLE) and other rare autoimmune peripheral neuropathies (APN), such as recurrent Guillain-Barré Syndrome (rGBS) and chronic inflammatory demyelinating polyneuropathy (CIDP).
The project is a multi-disciplinary research programme with the following specific scientific and translational objectives:
• Identify NOX-dependent signalling pathways: to understand how, when and in which cells NOX control the neuroinflammatory component of ND, and to elucidate their role in the progression of neuroinflammation and ND.
• Provide proof of principle for NOX inhibition or activation in animal models.
• Identify redox-dependent biomarkers of disease progression and severity in human samples from patients affected by ND.
• To develop small molecules NOX inhibitors and activators as therapeutics.
NEURINOX has made significant contribution for developing in vitro and in vivo models of neuroinflammation, identifying redox-dependent pathways regulated by NOX in ND novel mouse models for studying cell specific expression of NOX2, innovative methods for specific ROS detection, and development of new specific inhibitors/activators of NOX enzymes. At the clinical level, new oxidative biomarkers were identified for ALS and MS patients.
Most relevant results were either published or are in preparation for publication in scientific journals. These results obtained during the last year of the project consolidated previous results and have focused on identification of (i) genes controlling NOX2-derived oxidative burst and (ii) testing NOX inhibitors and anti-neuroinflammatory drugs in animal models of ND and (iii) regulation of NOX expression and activity as well as downstream targets of NOX activity.
Project Context and Objectives:
Description of the Project Context and Objectives
Description of the Project Context
The family of NADPH oxidases (NOX) contains seven members NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2. They are membrane proteins comprising 6 transmembrane domains (7 for DUOX1 and 2), which use NADPH and oxygen as substrates to catalyze the formation of superoxide anion (O2•–) and hydrogen peroxide (H2O2).
Each NOX isoform is characterized by specific mechanism of activation and tissue distribution. Under physiological conditions, NOX are essential mediators of host defense and different biosynthetic processes, including thyroid hormogenesis and formation of otoconia. Mutations affecting the NOX2 enzymatic system cause a disease known as chronic granulomatous disease, which is characterized by increased susceptibility to infections by certain pathogens. Interestingly NOX2 lack of function complex is also associated with the development of several autoimmune chronic inflammatory diseases. Thus absence of NOX-derived oxidants has pathological consequences, excessive oxidant production by NOX enzymes also contribute to oxidative damage observed in cardiovascular diseases, pancreatitis and CNS pathologies (Nayernia 2014).
The most frequent neurodegenerative diseases (ND) are Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Huntington disease (HD) and multiple sclerosis (MS). Symptomatic treatments exist for PD and MS, but no disease modifying therapies are available for other ND. The symptoms of ND are largely due to the progressive impairment of neuronal function and their specificity depends on the neuronal cell types and brain regions affected. Several features are commonly shared in ND: abnormal aggregation of key proteins, spatio-temporal spreading of aggregated proteins, neuroinflammation, and oxidative modifications of macromolecules. MS differs from other ND, as it is an autoimmune disease characterized by infiltration of leukocytes in the brain and spinal cord, which destroy the myelin sheath and lead to secondary neuronal death. Neuroinflammation in other ND is mostly sustained by astrocytes and microglia. Similarly to inflammation, neuroinflammation is a primary response of the host aiming at removing harmful stimuli and initiating healing processes.
However in ND, the initiating factors are not removed and Neuroinflammation become detrimental to neuronal cells and contribute to ND progression. Identification of novel drugs controlling neuroinflammation represents a promising therapeutic approach for ND. As major source of oxidants in ND and potential regulators of neuroinflammation, NOX represent a novel and promising class of pharmacological targets for the treatment of ND. A role of excessive NOX-formed O2•–/H2O2 and secondary oxidants derived from them in CNS pathology is known since the first report that NOX2 gene knockout mice are protected from brain ischemia (Walder 1997). Since then, several NOX isoforms have been documented in the CNS and numerous studies have pointed towards of role of NOX isoforms in AD, PD, ALS and MS (reviewed in Sorce 2012; Nayernia 2014).
Increased oxidative stress resulting from increased oxidant formation is a central feature of ND. NOX-derived oxidants can directly impair neuronal function due to their toxic properties, but they are increasingly recognized as key modulators signaling pathways potentially controlling neuroinflammation. Although oxidants are key components of ND, therapeutic approaches targeting reactive oxygen species have so far been ineffective. Lack of efficacy of antioxidant strategies may be at least partly due to lack of specificity in vivo efficacy and potential concomitant attenuation of the regulatory role of oxidants. Our approach consists in targeting a primary source of O2•–/H2O2 (i.e. NOX) rather than ‘scavenging’ oxidants after they have been formed.
The NEURINOX concept
NOX present paradoxical features in ND:: NOX-derived O2•–/H2O2 lead to oxidative damage and resulting pathology, or are beneficial by regulating key physiological functions, including the resolution of inflammation in autoimmune diseases such as MS. Thus, depending on the pathology, the therapeutic approach should either enhance or mitigate NOX activity
Based on this concept, the NEURINOX consortium was formed in 2012 (http://www.NEURINOX.eu). It received funding from the European commission for 5 years and comprised researchers specialized in NOX, oxidants, inflammation and ND from the academic as well as clinical and industry settings.
The principal objectives of NEURINOX were to:
(i) Determine precise localization, levels of expression and specificity of NOX isoforms in the CNS;
(ii) Evaluate the role of NOX during ND progression in animal models of ND and patients;
(iii) Identify and characterize small molecules targeting NOX as potential therapeutics for NDs;
(iv) Develop reliable approaches to measure NOX activity in vivo; and
(v) Identify the molecular pathways controlled by NOX during neuroinflammation;
(vi) Address the correlation of NOX activity and disease progression and severity in prospective clinical trials (biomarkers).
Selected diseases with a strong neuroinflammatory contribution were selected to address the above-mentioned questions.
These included ND related to aggregated proteins, such as ALS, mesial temporal lobe epilepsy and autoimmune demyelination of the CNS (MS) and the peripheral nerves (Guillain-Barre or chronic inflammatory demyelinating polyneuropathy).
The NEURINOX consortium consisted of 13 entities, mostly composed of biomedical researchers active in the fields of ND and NOX, clinical centers and SMes involved in the development of NOX therapeutics.
Project Results:
Description of the main S&T results/foregrounds
(i) NOX2 and NOX4 are the main CNS NOX isoforms. Combination of qPCR, immunostaining and available RNAseq databases indicate that under physiological conditions most NOX isoforms are expressed only to a negligible extent in the CNS.
NOX1, NOX3, NOX5, DUOX1 and DUOX2 are undetectable or at the limit of detection in the human and mouse CNS (Jaquet V 2016) (Zhang Y, 2014) Only NOX4 is detectable at basal conditions, and RNAseq data indicate that it is expressed mostly in the brain endothelium.
In terms of expression, immunostaining and RNAseq data show that the most prevalent NOX isoform in the CNS is NOX2. NOX2 is very specific for microglia although adult neural stem cells also express it.
In ND microglia is activated in the CNS of patients and mouse models of ND and NOX2 expression is enormously enhanced in several models of ALS (Seredenina 2016) and other ND (Sorce 2014).
(ii) Proof of concept animal models using NOX knockout mice in ND models. Two models of ND known for protein aggregation and strong neuroinflammatory reaction were evaluated for NOX genetic inhibition: ALS and Creutzfeld’s Jakob disease (CJD).
We have shown that the expression of microglial NOX2 is strongly increased in both patients and animal models of ALS, specifically in affected regions of the spinal cord (Seredenina 2016). We crossed a well-characterized model of ALS (the SODG93A mice) with NOX2, NOX1 and NOX4 gene knockout mice.
However none of the genetic deletion of NOX isoforms did improve disease incubation time. In NOX2 deficient mice bred with ALS mice, neither microgliosis, nor astrogliosis nor motoneuron survival were changed (Seredenina 2016).
These results thus question initial reports (Marden 2007), showing a prolonged lifespan in ALS models related to NOX2 and NOX1 deficiency. Creutzfeld Jakob disease (CJD) is an incurable ND characterized at the neuropathological level by a massive neuronal loss conferring a spongiform aspect of the brain and a neuroinflammatory environment showing increased microglial NOX2 expression (Sorce 2014).
A NOX2-deficient CJD mouse model showed a transient improvement of motor function and decreased vacuoles formation and brain oxidant levels, but microgliosis was not affected significant survival improvement and improvement of vacuoles formation. Similarly, in another model of aggregation-induced ND, it was shown that NOX2 greatly contributes to neurotoxicity (Sonati 2013).
(iii) NOX inhibitors. Most reported NOX inhibitors are unspecific (Heumuller 2008; Hirano 2015): they act as oxidant scavengers, inhibit upstream activators of NOX (Gatto 2013) or interfere with the assays used to measure NOX activity (Dikalov 2014).
Thus, when testing potential small molecules NOX inhibitors, it is essential to use methods measuring both consumption of the substrates (NADPH, O2) and the products (O2•–,H2O2) of the reaction catalyzed by NOX (Jaquet 2009) to exclude false positives including those resulting from interference with oxidant-measuring systems (e.g. amplex red) Using this approach, we identified that a class of anti-psychotic agents have NOX inhibitory activity. Indeed a subset of N-substituted phenothiazines - but not non-substituted phenothiazines – are bone fide NOX inhibitors (Seredenina 2015).
These molecules are brain permeant and are already used as human therapeutics and can potentially be repurposed in ND. We also characterized a novel NOX2 inhibitor GSK2795039 and showed that it is competitive for the NADPH binding site of NOX2. GSK2795039 is orally available, non-toxic, CNS-permeable and inhibits NOX2 activity in vivo (Hirano 2015), Administration of thioridazine and perphenazine to SODG93A mice did not improve survival, although they showed some benefits in secondary read-outs, such brain oxidant levels, motor function for thioridazine and weigh loss for perphenazine (Seredenina 2015).
(iv) NOX activators. In the context of autoimmune diseases NOX2 activity is anti-inflammatory. This unexpected fact was originally discovered in a genetic study performed to identify polymorphic loci controlling autoimmune chronic inflammatory diseases, using models for rheumatoid arthritis and multiple sclerosis (Olofsson 2007). This led to the identification of a polymorphism in the coding sequence of Ncf1 (neutrophil cytosolic factor 1), the gene coding for p47phox, a subunit essential for the generation of O2•– by NOX2 (Olofsson 2003, Hultqvist 2011) . NOX2-derived oxidants generated by macrophages and other antigen presenting cells regulate activation of autoreactive T cells (Gelderman 2007)
Since then, a protective role of NOX2-derived oxidants has been reproduced in several different models of autoimmune disorders, including models for MS and Guillain Barré (Becanovic 2006), (Hultqvist 2004). The NOX2-dependent anti-inflammatory effect is mediated by several different pathways: (i) downregulation of autoreactive T cells during antigen presentation (Gelderman 2007); (ii) autocrine downregulation of inflammatory macrophages (Holmdahl, 2016), (Khmaladze 2014) (iii) downregulation of STAT1 mediated activation of the interferon pathway (Kelkka 2014), (Madhzal 2014); and (iv) promotion of a protective effect by neutrophil extracellular traps (NETS) formed by neutrophils (Schauer, 2014)
Further studies aimed to identify both the chemical nature of the oxidants involved and the respective roles played by these potentially protective pathways in MS and peripheral neuropathies are needed. Indeed, it is still unclear at present if excess oxidants in the CNS are counteracting the protective effect of the peripheral inflammatory attack, thereby promoting neurodegeneration (Schuh C 2014). Thus although the NOX inhibitory approach appears valid to decrease neuroinflammation, NOX activation may have therapeutic benefit for autoimmune-mediated neurodegeneration, including MS and other peripheral demyelinating neuropathies.
The NEURINOX partner, Redoxis AB has developed and characterised novel molecules able to enhance NOX2 activity with the objective to treat CNS autoimmune diseases (Hultqvist 2015) (Holmdahl 2004), (Wallner 2012). Identified NOX agonists have anti-inflammatory properties and are able to decrease the pro-inflammatory role of TNF-α in the low nanomolar range and are currently being optimised for regulatory safety studies and selection of candidate drug for clinical evaluation in ND.
(v) Measurement of NOX activity in vivo. Measuring NOX activity in tissues is a challenging task. Hydroethidine or dihydroethidium (Kalyanaraman 2010) is the ‘gold standard’ for O2•– determination in biological systems (Dikalov 2007). 2-hydroxyethidium is a specific oxidative product of O2•–. 2-hydroxyethidium can be measured by a combination of chromatographic separation of ethidium and 2-hydroxyethidine coupled with fluorescent or mass spectrometry. Using the LC-MS/MS approach, we showed that following hydroethidine into the spinal cord revealed that thioridazine decreased O2•– in the spinal cord of SOD1G93A mice in vivo (Seredenina 2016)
(vi) NOX and oxidative biomarkers in NDs. A large part of NEURINOX was dedicated to ND patients with the objective to evaluate NOX activity and oxidized biomarkersduring ND progression and severity. In a prospective clinical study, NOX2 activity from peripheral neutrophils and monocytes was directly measured in fresh whole blood of a cohort of 83 ALS patients, and age- and gender-matched healthy controls. Upon addition of a specific activator, no difference was observed between patients and healthy controls, however, inside the ALS group, low NOX2 activity in leukocytes was significantly associated with a longer survival (Marrali, 2014).
This represents an important finding in the field of ALS because this may be a prognosis biomarker of the severity of ALS.
More importantly, such a measure could be implemented as surrogate biomarker to address the benefit of a drug in clinical studies. A similar approach was used with patients affected by chronic inflammatory demyelinating polyneuropathy (CIDP) a neurological disorder characterized by damaged myelin sheath of the peripheral nerves. Intravenous immunoglobulin (IVIg) therapy is used as a first-line therapy and usually provides substantial benefit to patients. A prospective clinical study enrolled 30 CIDP patients treated with IVIg and 30 control subjects for whom NOX2 activity was measured in neutrophils and monocytes from freshly collected blood. At diagnosis NOX2 activity was significantly increased in CIDP patients compared to controls. However, following IVIg therapy, NOX2 activity was even more increased compared to basal levels (Marrali, 2016).The exact cause of this observation is unclear, but the results are consistent with therapeutic improvement in autoimmune demyelination being associated with enhanced NOX2. Because of the simplicity and robustness of this assay, it should be included systematically in clinical settings for ND.
This would potentially provide key information on inclusion criteria and the response to a drug. Another approach was used to determine levels of F2-isoprostanes by LC-MS/MS in cerebrospinal fluid and plasma of patients with progressive MS. Compared with controls, plasma concentrations of F2-isoprostanes and prostaglandin F2 (PGF2) were decreased with increasing disability score (Lam 2016) This was in contrast to the situation in cerebrospinal fluid, where the concentrations of PGF2, but not F2-isoprostanes, were significantly higher in patients with progressive disease than controls. Cerebrospinal fluid PGF2 was reduced with natalizumab and methylprednisolone treatment, suggesting that PGF2 levels in the CSF represents reliable surrogate biomarkers for evaluation of the efficacy of a drug. These results suggest that MS progression is associated with low rather than high systemic oxidative activity, and that this may play a role in immune dysregulation with central nervous system inflammation accompanied by increased local cyclooxygenase-dependent lipid oxidation.
Altogether the results obtained by the NEURINOX consortium led to numerous findings related to ND, oxidative stress in mouse models of ND and patients affected by ND. These findings are documented in 78 papers and 2 patents. More specifically, NEURINOX clarified NOX localization in the CNS, identified novel small molecule NOX inhibitors/activators and state-of-the-art methods to measure O2•– in vivo, showed a strong association between NOX2 and disease progression in ND, and indicated that NOX2 expression and activity parallels microgliosis and neuroinflammation in ND. However, inhibition of NOX provided only limited beneficial effects in ND. NOX2 up-regulation is certainly a common feature of ND and a sign of a neuroinflammatory response, but NOX2 inhibition is not a disease-modifying treatment. NOX2 upregulation, increased oxidant generation, microgliosis and neuroinflammation are all associated factors of ND, and rather represent a consequence of the neurodegenerative process. One of the key findings of our studies is the correlation between NOX2 activity and ALS progression, which makes NOX2 a promising biomarker for future evaluation of therapies for ND.
To advance the development of efficient drugs for ND-targeting redox systems it will be essential to use and propagate reliable molecular probes to identify:
(i) The pattern of expression (RNAseq, antibodies);
(ii) Inhibit specific oxidant-generating systems (small molecules, CRISPR-CAS);
(iii) Localize and quantify specific reactive oxygen species in vivo to understand how and which therapeutics should be used and,
(iv) Identify the impact of oxidative modifications of target proteins on cell functioning by redox proteomics. Understanding the kinetics of oxidant formation and metabolism, the relative role of various oxidant-generating systems and their inter-dependence in the fine redox regulation will pave the way for long awaited therapeutics targeting oxidative stress in CNS disorders.
Potential Impact:
Potential impact
Conclusions to date regarding the role of NOX in ND and impact on future research
The results obtained by the NEURINOX consortium clarified NOX localization in the CNS, identified novel small molecule NOX inhibitors/activators and state-of-the-art methods to measure O2•– in vivo, showed a strong association between NOX2 and disease progression in ND, and indicated that NOX2 expression and activity parallels microgliosis and neuroinflammation in ND. Importantly, however, inhibition of NOX provided at best only limited beneficial effects in ND. NOX2 up-regulation is indeed a common feature of ND and a sign of a neuroinflammatory response, but NOX2 inhibition is not a disease-modifying treatment. One of the key findings of our studies shows that a correlation between NOX2 activity and disease progression or response to treatments in patients is measurable in the blood, which makes NOX2 a promising biomarker for future evaluation of therapies for ND.
Description of the expected final results, potential impact and use
The NEURINOX consortium is the first concerted effort to gain a comprehensive view of the implication of ROS-generating NOX enzymes in neuroinflammation. NEURINOX contributes to better understand brain dysfunction and more particularly the link between neuroinflammation NOX enzymes and aims at identifying new therapeutic targets for neurodegeneration. A successful demonstration of the benefits of NOX modulating drugs in ALS, MS and CIPD animal models, and in ALS pre-clinical trials can validate novel high potential therapeutic targets for ALS and also other neurodegenerative diseases. Final expected results are the following:
• A better understanding of common mechanisms of brain diseases, and in particular oxidative stress-mediated neurodegeneration and the link between the different NOX isoforms and neuroinflammation and how their activities control the neuroinflammatory process in neurodegenerative disease.
• Novel oxidation biomarkers, molecular pathways, genes and SNPs correlating with NOX activity and ND for neurodegenerative and autoimmune-mediated neuroinflammation.
• Small molecules (NOX inhibitors and NOX activators), validated by NEURINOX partners in animal models for efficacy and mechanism of action (MOA).
• Validation of NOX as viable targets for the development of therapeutics for selected neurodegenerative diseases.
With these results, the NEURINOX research have the following impacts:
• Impact on better understanding of brain function and redox regulation. The NEURINOX results allow a better understanding of the role of NOX in ND, including ALS, MS and MTLE-HS. It also helps identifying more general redox mechanisms involved in brain function and ND
• Impact on better management of neuroinflammatory and subsequent neurodegenerative diseases. Costs of ND to society are huge and a breakthrough in the treatment of ND will allow great economic gains. By exploring a new therapeutic approach in ALS and NOX activity as new biomarkers of ND progression, NEURINOX contributes to the improvement in clinical management of ND and hence to a reduction of health care costs.
• Impact on public health. NOX-mediated therapeutics may be used for slowing progression of neurodegenerative diseases, which are so far untreatable
• Impact on ND research and for structuring European research efforts. NEURINOX brings together international experts in neuroinflammation and related areas and also seeks collaboration with other European initiatives in the area of ND research, thus contributing to structuring European research efforts..
• Impact on competitiveness of European industry. NEURINOX aims at creating new knowledge and translating it into novel therapeutic targets through the involvement of a number of SMEs who are well positioned to derive new therapeutic products from the project results. Through industrial collaboration, the proposed work is increasing the competitiveness and is boosting the innovative capacity of European health-related enterprises, which is a global priority of the FP7 HEALTH programme.
Dissemination and Exploitation Activities
To deal with the questions related to dissemination of results, exploitation and IPR a specific work package (WP9) was setup. The aim of the dissemination actions was to reach the identified target audience and to transfer correct and incisive information about the project activities and achievements. It was also the objective of the consortium to communicate around the collaborative actions made feasible thanks to the support of the European Commission.
The consortium has therefore designed a plan for disseminating knowledge with the objectives:
• To raise public participation and awareness of the progress made within NEURINOX
• To enhance exchanges with scientific world
• To prepare exploitation of results, create market opportunities
• To spread the knowledge gained beyond the consortium
The dissemination activities have been organised in four large parts by target group: Dissemination towards:
• the public
• the scientific world
• the LifeSciHealth Programme
• the industry
For each of the target groups the aim, content, specific target, main message, detailed activities and timing of activities are described in the report attached.
Exploitation of NEURINOX results
The WP9 leader, in collaboration with the Executive Board, has supported partners in their respective IP procedures, making sure that the access of NEURINOX results etc. are dealt with on a fair and viable basis for all. This task has included patent searches, filing of patent (or other IPR) applications, etc.
The research activities undertaken under the umbrella of the project have generated results with commercial potentials. This has raised the issues of intellectual property rights (IPRs), of protection of the property rights (confidential IP as well as patenting).
The NEURINOX consortium has been composed of research groups and clinical institutions, Universities and SME with extensive experience in NADPH oxidases (NOX) research and neurodegenerative diseases. They have exploited the results of the project in various ways.
The Research Centres and Universities have been mostly benefited from the advance in knowledge which have strengthen their position as leading research institutions in Europe and brought new opportunities for future partnerships. Concrete plans for exploitation covered mainly publications in peer-reviewed international journals and filing of patent applications. The general principles for IPR ownership and IPR protection have been established in the Consortium Agreement.
The NEURINOX results are used in three main ways by individual partners:
• Continuous research
• Dissemination by public presentations and publication
• Commercial development by confidential IPR and exploitation of patents
Shown in 2 tables detailed in the attached report
List of Websites:
NEURINOX partners and contact
NEURINOX Coordinator
1) Dr Vincent Jaquet
Department of Pathology and Immunology
Faculty of Medicine
University of Geneva
1, rue Michel-Servet
1211 Geneva 4, Switzerland
Phone: +41 22 379 4136
E-mail: Vincent.Jaquet@unige.ch
NEURINOX public website: http://www.NEURINOX.eu
Partners
2) University of Zürich (UZH)
Neuropathology Institute
Schmelzbergstrasse 12
8091 Zurich, Switzerland
http://www.en.neuropathologie.usz.ch/
3) GenKyoTex SA (GKT)
16, Chemin des Aulx
CH-1228 Plan - Les-Ouates – Geneva, Switzerland
http://www.genkyotex.com
4) Redoxis AB (RDX)
Medicon Village
Scheelevägen 2
223 81 Lund, Sweden
http://www.redoxis.com/
5) ARTTIC (ART)
Dominique Wasquel / Sara Skogsäter
58A rue du Dessous des Berges
75013 Paris, France
http://www.arttic.eu
6) University of Torino (UT)
Department of Neuroscience
University of Torino
Via Cherasco 15
10126 Torino, Italy
http://www.unito.it/
7) Karolinska Institute (KI), Sweden.
a) Section for Medical Inflammation Research (MIR)
Dept of Medical Biochemistry and Biophysics
b) Neuroimmunology Unit (NU)
Department of Clinical Neurosciences
Center for Molecular Medicine
http://www.ki.se/
8) Université Grenoble Alpes (UGA) previously called Joseph Fourier University (UJF)
621 avenue Centrale
38400 Saint-Martin-d'Hères
http://www.univ-grenoble-alpes.fr/en/
9) SynapCell SAS (SYN)
Bâtiment Biopolis
5 avenue du Grand Sablon
38700 La Tronche, France
http://www.synapcell.fr/
10) Biomedical Research Foundation Academy of Athens (BRFAA)
4 Soranou Ephessiou Street,
Athens 115 27, Greece
http://www.bioacademy.gr/
11) University of Athens (UOA)
National and Kapodistrian University of Athens,
Clinical Genomics and Pharmacogenomics Unit,
4th Department of Internal Medicine,
Attikon Hospital, Medical School,
75 Mikras Asias
115-27 Athens, Greece
http://en.uoa.gr/about-us.html
12) University of Sydney (USYD) terminated in October 2012
Parramatta Road, 92-94
Camperdown NSW 2006, Australia
www.usyd.edu.au
13) NEURIX (NEURIX)
14 Chemin des Aulx
Plan les Ouates 1228
Switzerland
http://www.neurix.ch/
14) Victor Chang Cardiac Research Institute Limited (VCCRI)
School of Medical Sciences,
University of New South Wales, Australia.
405 Liverpool Street
Darlinghurst, 2010, Australia
www.victorchang.com.au
Neuroinflammation is a key process associated with neurodegenerative diseases (ND). The underlying hypothesis of the NEURINOX project is that NADPH oxidases (NOX) are mediators of neuroinflammation and that NOX are promising drug targets for the development of therapies against ND.
NEURINOX focuses on several diseases with strong neuroinflammatory components and other autoimmune neuropathies, including multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and mesial temporal lobe epilepsy (MTLE) and other rare autoimmune peripheral neuropathies (APN), such as recurrent Guillain-Barré Syndrome (rGBS) and chronic inflammatory demyelinating polyneuropathy (CIDP).
The project is a multi-disciplinary research programme with the following specific scientific and translational objectives:
• Identify NOX-dependent signalling pathways: to understand how, when and in which cells NOX control the neuroinflammatory component of ND, and to elucidate their role in the progression of neuroinflammation and ND.
• Provide proof of principle for NOX inhibition or activation in animal models.
• Identify redox-dependent biomarkers of disease progression and severity in human samples from patients affected by ND.
• To develop small molecules NOX inhibitors and activators as therapeutics.
NEURINOX has made significant contribution for developing in vitro and in vivo models of neuroinflammation, identifying redox-dependent pathways regulated by NOX in ND novel mouse models for studying cell specific expression of NOX2, innovative methods for specific ROS detection, and development of new specific inhibitors/activators of NOX enzymes. At the clinical level, new oxidative biomarkers were identified for ALS and MS patients.
Most relevant results were either published or are in preparation for publication in scientific journals. These results obtained during the last year of the project consolidated previous results and have focused on identification of (i) genes controlling NOX2-derived oxidative burst and (ii) testing NOX inhibitors and anti-neuroinflammatory drugs in animal models of ND and (iii) regulation of NOX expression and activity as well as downstream targets of NOX activity.
Project Context and Objectives:
Description of the Project Context and Objectives
Description of the Project Context
The family of NADPH oxidases (NOX) contains seven members NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2. They are membrane proteins comprising 6 transmembrane domains (7 for DUOX1 and 2), which use NADPH and oxygen as substrates to catalyze the formation of superoxide anion (O2•–) and hydrogen peroxide (H2O2).
Each NOX isoform is characterized by specific mechanism of activation and tissue distribution. Under physiological conditions, NOX are essential mediators of host defense and different biosynthetic processes, including thyroid hormogenesis and formation of otoconia. Mutations affecting the NOX2 enzymatic system cause a disease known as chronic granulomatous disease, which is characterized by increased susceptibility to infections by certain pathogens. Interestingly NOX2 lack of function complex is also associated with the development of several autoimmune chronic inflammatory diseases. Thus absence of NOX-derived oxidants has pathological consequences, excessive oxidant production by NOX enzymes also contribute to oxidative damage observed in cardiovascular diseases, pancreatitis and CNS pathologies (Nayernia 2014).
The most frequent neurodegenerative diseases (ND) are Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Huntington disease (HD) and multiple sclerosis (MS). Symptomatic treatments exist for PD and MS, but no disease modifying therapies are available for other ND. The symptoms of ND are largely due to the progressive impairment of neuronal function and their specificity depends on the neuronal cell types and brain regions affected. Several features are commonly shared in ND: abnormal aggregation of key proteins, spatio-temporal spreading of aggregated proteins, neuroinflammation, and oxidative modifications of macromolecules. MS differs from other ND, as it is an autoimmune disease characterized by infiltration of leukocytes in the brain and spinal cord, which destroy the myelin sheath and lead to secondary neuronal death. Neuroinflammation in other ND is mostly sustained by astrocytes and microglia. Similarly to inflammation, neuroinflammation is a primary response of the host aiming at removing harmful stimuli and initiating healing processes.
However in ND, the initiating factors are not removed and Neuroinflammation become detrimental to neuronal cells and contribute to ND progression. Identification of novel drugs controlling neuroinflammation represents a promising therapeutic approach for ND. As major source of oxidants in ND and potential regulators of neuroinflammation, NOX represent a novel and promising class of pharmacological targets for the treatment of ND. A role of excessive NOX-formed O2•–/H2O2 and secondary oxidants derived from them in CNS pathology is known since the first report that NOX2 gene knockout mice are protected from brain ischemia (Walder 1997). Since then, several NOX isoforms have been documented in the CNS and numerous studies have pointed towards of role of NOX isoforms in AD, PD, ALS and MS (reviewed in Sorce 2012; Nayernia 2014).
Increased oxidative stress resulting from increased oxidant formation is a central feature of ND. NOX-derived oxidants can directly impair neuronal function due to their toxic properties, but they are increasingly recognized as key modulators signaling pathways potentially controlling neuroinflammation. Although oxidants are key components of ND, therapeutic approaches targeting reactive oxygen species have so far been ineffective. Lack of efficacy of antioxidant strategies may be at least partly due to lack of specificity in vivo efficacy and potential concomitant attenuation of the regulatory role of oxidants. Our approach consists in targeting a primary source of O2•–/H2O2 (i.e. NOX) rather than ‘scavenging’ oxidants after they have been formed.
The NEURINOX concept
NOX present paradoxical features in ND:: NOX-derived O2•–/H2O2 lead to oxidative damage and resulting pathology, or are beneficial by regulating key physiological functions, including the resolution of inflammation in autoimmune diseases such as MS. Thus, depending on the pathology, the therapeutic approach should either enhance or mitigate NOX activity
Based on this concept, the NEURINOX consortium was formed in 2012 (http://www.NEURINOX.eu). It received funding from the European commission for 5 years and comprised researchers specialized in NOX, oxidants, inflammation and ND from the academic as well as clinical and industry settings.
The principal objectives of NEURINOX were to:
(i) Determine precise localization, levels of expression and specificity of NOX isoforms in the CNS;
(ii) Evaluate the role of NOX during ND progression in animal models of ND and patients;
(iii) Identify and characterize small molecules targeting NOX as potential therapeutics for NDs;
(iv) Develop reliable approaches to measure NOX activity in vivo; and
(v) Identify the molecular pathways controlled by NOX during neuroinflammation;
(vi) Address the correlation of NOX activity and disease progression and severity in prospective clinical trials (biomarkers).
Selected diseases with a strong neuroinflammatory contribution were selected to address the above-mentioned questions.
These included ND related to aggregated proteins, such as ALS, mesial temporal lobe epilepsy and autoimmune demyelination of the CNS (MS) and the peripheral nerves (Guillain-Barre or chronic inflammatory demyelinating polyneuropathy).
The NEURINOX consortium consisted of 13 entities, mostly composed of biomedical researchers active in the fields of ND and NOX, clinical centers and SMes involved in the development of NOX therapeutics.
Project Results:
Description of the main S&T results/foregrounds
(i) NOX2 and NOX4 are the main CNS NOX isoforms. Combination of qPCR, immunostaining and available RNAseq databases indicate that under physiological conditions most NOX isoforms are expressed only to a negligible extent in the CNS.
NOX1, NOX3, NOX5, DUOX1 and DUOX2 are undetectable or at the limit of detection in the human and mouse CNS (Jaquet V 2016) (Zhang Y, 2014) Only NOX4 is detectable at basal conditions, and RNAseq data indicate that it is expressed mostly in the brain endothelium.
In terms of expression, immunostaining and RNAseq data show that the most prevalent NOX isoform in the CNS is NOX2. NOX2 is very specific for microglia although adult neural stem cells also express it.
In ND microglia is activated in the CNS of patients and mouse models of ND and NOX2 expression is enormously enhanced in several models of ALS (Seredenina 2016) and other ND (Sorce 2014).
(ii) Proof of concept animal models using NOX knockout mice in ND models. Two models of ND known for protein aggregation and strong neuroinflammatory reaction were evaluated for NOX genetic inhibition: ALS and Creutzfeld’s Jakob disease (CJD).
We have shown that the expression of microglial NOX2 is strongly increased in both patients and animal models of ALS, specifically in affected regions of the spinal cord (Seredenina 2016). We crossed a well-characterized model of ALS (the SODG93A mice) with NOX2, NOX1 and NOX4 gene knockout mice.
However none of the genetic deletion of NOX isoforms did improve disease incubation time. In NOX2 deficient mice bred with ALS mice, neither microgliosis, nor astrogliosis nor motoneuron survival were changed (Seredenina 2016).
These results thus question initial reports (Marden 2007), showing a prolonged lifespan in ALS models related to NOX2 and NOX1 deficiency. Creutzfeld Jakob disease (CJD) is an incurable ND characterized at the neuropathological level by a massive neuronal loss conferring a spongiform aspect of the brain and a neuroinflammatory environment showing increased microglial NOX2 expression (Sorce 2014).
A NOX2-deficient CJD mouse model showed a transient improvement of motor function and decreased vacuoles formation and brain oxidant levels, but microgliosis was not affected significant survival improvement and improvement of vacuoles formation. Similarly, in another model of aggregation-induced ND, it was shown that NOX2 greatly contributes to neurotoxicity (Sonati 2013).
(iii) NOX inhibitors. Most reported NOX inhibitors are unspecific (Heumuller 2008; Hirano 2015): they act as oxidant scavengers, inhibit upstream activators of NOX (Gatto 2013) or interfere with the assays used to measure NOX activity (Dikalov 2014).
Thus, when testing potential small molecules NOX inhibitors, it is essential to use methods measuring both consumption of the substrates (NADPH, O2) and the products (O2•–,H2O2) of the reaction catalyzed by NOX (Jaquet 2009) to exclude false positives including those resulting from interference with oxidant-measuring systems (e.g. amplex red) Using this approach, we identified that a class of anti-psychotic agents have NOX inhibitory activity. Indeed a subset of N-substituted phenothiazines - but not non-substituted phenothiazines – are bone fide NOX inhibitors (Seredenina 2015).
These molecules are brain permeant and are already used as human therapeutics and can potentially be repurposed in ND. We also characterized a novel NOX2 inhibitor GSK2795039 and showed that it is competitive for the NADPH binding site of NOX2. GSK2795039 is orally available, non-toxic, CNS-permeable and inhibits NOX2 activity in vivo (Hirano 2015), Administration of thioridazine and perphenazine to SODG93A mice did not improve survival, although they showed some benefits in secondary read-outs, such brain oxidant levels, motor function for thioridazine and weigh loss for perphenazine (Seredenina 2015).
(iv) NOX activators. In the context of autoimmune diseases NOX2 activity is anti-inflammatory. This unexpected fact was originally discovered in a genetic study performed to identify polymorphic loci controlling autoimmune chronic inflammatory diseases, using models for rheumatoid arthritis and multiple sclerosis (Olofsson 2007). This led to the identification of a polymorphism in the coding sequence of Ncf1 (neutrophil cytosolic factor 1), the gene coding for p47phox, a subunit essential for the generation of O2•– by NOX2 (Olofsson 2003, Hultqvist 2011) . NOX2-derived oxidants generated by macrophages and other antigen presenting cells regulate activation of autoreactive T cells (Gelderman 2007)
Since then, a protective role of NOX2-derived oxidants has been reproduced in several different models of autoimmune disorders, including models for MS and Guillain Barré (Becanovic 2006), (Hultqvist 2004). The NOX2-dependent anti-inflammatory effect is mediated by several different pathways: (i) downregulation of autoreactive T cells during antigen presentation (Gelderman 2007); (ii) autocrine downregulation of inflammatory macrophages (Holmdahl, 2016), (Khmaladze 2014) (iii) downregulation of STAT1 mediated activation of the interferon pathway (Kelkka 2014), (Madhzal 2014); and (iv) promotion of a protective effect by neutrophil extracellular traps (NETS) formed by neutrophils (Schauer, 2014)
Further studies aimed to identify both the chemical nature of the oxidants involved and the respective roles played by these potentially protective pathways in MS and peripheral neuropathies are needed. Indeed, it is still unclear at present if excess oxidants in the CNS are counteracting the protective effect of the peripheral inflammatory attack, thereby promoting neurodegeneration (Schuh C 2014). Thus although the NOX inhibitory approach appears valid to decrease neuroinflammation, NOX activation may have therapeutic benefit for autoimmune-mediated neurodegeneration, including MS and other peripheral demyelinating neuropathies.
The NEURINOX partner, Redoxis AB has developed and characterised novel molecules able to enhance NOX2 activity with the objective to treat CNS autoimmune diseases (Hultqvist 2015) (Holmdahl 2004), (Wallner 2012). Identified NOX agonists have anti-inflammatory properties and are able to decrease the pro-inflammatory role of TNF-α in the low nanomolar range and are currently being optimised for regulatory safety studies and selection of candidate drug for clinical evaluation in ND.
(v) Measurement of NOX activity in vivo. Measuring NOX activity in tissues is a challenging task. Hydroethidine or dihydroethidium (Kalyanaraman 2010) is the ‘gold standard’ for O2•– determination in biological systems (Dikalov 2007). 2-hydroxyethidium is a specific oxidative product of O2•–. 2-hydroxyethidium can be measured by a combination of chromatographic separation of ethidium and 2-hydroxyethidine coupled with fluorescent or mass spectrometry. Using the LC-MS/MS approach, we showed that following hydroethidine into the spinal cord revealed that thioridazine decreased O2•– in the spinal cord of SOD1G93A mice in vivo (Seredenina 2016)
(vi) NOX and oxidative biomarkers in NDs. A large part of NEURINOX was dedicated to ND patients with the objective to evaluate NOX activity and oxidized biomarkersduring ND progression and severity. In a prospective clinical study, NOX2 activity from peripheral neutrophils and monocytes was directly measured in fresh whole blood of a cohort of 83 ALS patients, and age- and gender-matched healthy controls. Upon addition of a specific activator, no difference was observed between patients and healthy controls, however, inside the ALS group, low NOX2 activity in leukocytes was significantly associated with a longer survival (Marrali, 2014).
This represents an important finding in the field of ALS because this may be a prognosis biomarker of the severity of ALS.
More importantly, such a measure could be implemented as surrogate biomarker to address the benefit of a drug in clinical studies. A similar approach was used with patients affected by chronic inflammatory demyelinating polyneuropathy (CIDP) a neurological disorder characterized by damaged myelin sheath of the peripheral nerves. Intravenous immunoglobulin (IVIg) therapy is used as a first-line therapy and usually provides substantial benefit to patients. A prospective clinical study enrolled 30 CIDP patients treated with IVIg and 30 control subjects for whom NOX2 activity was measured in neutrophils and monocytes from freshly collected blood. At diagnosis NOX2 activity was significantly increased in CIDP patients compared to controls. However, following IVIg therapy, NOX2 activity was even more increased compared to basal levels (Marrali, 2016).The exact cause of this observation is unclear, but the results are consistent with therapeutic improvement in autoimmune demyelination being associated with enhanced NOX2. Because of the simplicity and robustness of this assay, it should be included systematically in clinical settings for ND.
This would potentially provide key information on inclusion criteria and the response to a drug. Another approach was used to determine levels of F2-isoprostanes by LC-MS/MS in cerebrospinal fluid and plasma of patients with progressive MS. Compared with controls, plasma concentrations of F2-isoprostanes and prostaglandin F2 (PGF2) were decreased with increasing disability score (Lam 2016) This was in contrast to the situation in cerebrospinal fluid, where the concentrations of PGF2, but not F2-isoprostanes, were significantly higher in patients with progressive disease than controls. Cerebrospinal fluid PGF2 was reduced with natalizumab and methylprednisolone treatment, suggesting that PGF2 levels in the CSF represents reliable surrogate biomarkers for evaluation of the efficacy of a drug. These results suggest that MS progression is associated with low rather than high systemic oxidative activity, and that this may play a role in immune dysregulation with central nervous system inflammation accompanied by increased local cyclooxygenase-dependent lipid oxidation.
Altogether the results obtained by the NEURINOX consortium led to numerous findings related to ND, oxidative stress in mouse models of ND and patients affected by ND. These findings are documented in 78 papers and 2 patents. More specifically, NEURINOX clarified NOX localization in the CNS, identified novel small molecule NOX inhibitors/activators and state-of-the-art methods to measure O2•– in vivo, showed a strong association between NOX2 and disease progression in ND, and indicated that NOX2 expression and activity parallels microgliosis and neuroinflammation in ND. However, inhibition of NOX provided only limited beneficial effects in ND. NOX2 up-regulation is certainly a common feature of ND and a sign of a neuroinflammatory response, but NOX2 inhibition is not a disease-modifying treatment. NOX2 upregulation, increased oxidant generation, microgliosis and neuroinflammation are all associated factors of ND, and rather represent a consequence of the neurodegenerative process. One of the key findings of our studies is the correlation between NOX2 activity and ALS progression, which makes NOX2 a promising biomarker for future evaluation of therapies for ND.
To advance the development of efficient drugs for ND-targeting redox systems it will be essential to use and propagate reliable molecular probes to identify:
(i) The pattern of expression (RNAseq, antibodies);
(ii) Inhibit specific oxidant-generating systems (small molecules, CRISPR-CAS);
(iii) Localize and quantify specific reactive oxygen species in vivo to understand how and which therapeutics should be used and,
(iv) Identify the impact of oxidative modifications of target proteins on cell functioning by redox proteomics. Understanding the kinetics of oxidant formation and metabolism, the relative role of various oxidant-generating systems and their inter-dependence in the fine redox regulation will pave the way for long awaited therapeutics targeting oxidative stress in CNS disorders.
Potential Impact:
Potential impact
Conclusions to date regarding the role of NOX in ND and impact on future research
The results obtained by the NEURINOX consortium clarified NOX localization in the CNS, identified novel small molecule NOX inhibitors/activators and state-of-the-art methods to measure O2•– in vivo, showed a strong association between NOX2 and disease progression in ND, and indicated that NOX2 expression and activity parallels microgliosis and neuroinflammation in ND. Importantly, however, inhibition of NOX provided at best only limited beneficial effects in ND. NOX2 up-regulation is indeed a common feature of ND and a sign of a neuroinflammatory response, but NOX2 inhibition is not a disease-modifying treatment. One of the key findings of our studies shows that a correlation between NOX2 activity and disease progression or response to treatments in patients is measurable in the blood, which makes NOX2 a promising biomarker for future evaluation of therapies for ND.
Description of the expected final results, potential impact and use
The NEURINOX consortium is the first concerted effort to gain a comprehensive view of the implication of ROS-generating NOX enzymes in neuroinflammation. NEURINOX contributes to better understand brain dysfunction and more particularly the link between neuroinflammation NOX enzymes and aims at identifying new therapeutic targets for neurodegeneration. A successful demonstration of the benefits of NOX modulating drugs in ALS, MS and CIPD animal models, and in ALS pre-clinical trials can validate novel high potential therapeutic targets for ALS and also other neurodegenerative diseases. Final expected results are the following:
• A better understanding of common mechanisms of brain diseases, and in particular oxidative stress-mediated neurodegeneration and the link between the different NOX isoforms and neuroinflammation and how their activities control the neuroinflammatory process in neurodegenerative disease.
• Novel oxidation biomarkers, molecular pathways, genes and SNPs correlating with NOX activity and ND for neurodegenerative and autoimmune-mediated neuroinflammation.
• Small molecules (NOX inhibitors and NOX activators), validated by NEURINOX partners in animal models for efficacy and mechanism of action (MOA).
• Validation of NOX as viable targets for the development of therapeutics for selected neurodegenerative diseases.
With these results, the NEURINOX research have the following impacts:
• Impact on better understanding of brain function and redox regulation. The NEURINOX results allow a better understanding of the role of NOX in ND, including ALS, MS and MTLE-HS. It also helps identifying more general redox mechanisms involved in brain function and ND
• Impact on better management of neuroinflammatory and subsequent neurodegenerative diseases. Costs of ND to society are huge and a breakthrough in the treatment of ND will allow great economic gains. By exploring a new therapeutic approach in ALS and NOX activity as new biomarkers of ND progression, NEURINOX contributes to the improvement in clinical management of ND and hence to a reduction of health care costs.
• Impact on public health. NOX-mediated therapeutics may be used for slowing progression of neurodegenerative diseases, which are so far untreatable
• Impact on ND research and for structuring European research efforts. NEURINOX brings together international experts in neuroinflammation and related areas and also seeks collaboration with other European initiatives in the area of ND research, thus contributing to structuring European research efforts..
• Impact on competitiveness of European industry. NEURINOX aims at creating new knowledge and translating it into novel therapeutic targets through the involvement of a number of SMEs who are well positioned to derive new therapeutic products from the project results. Through industrial collaboration, the proposed work is increasing the competitiveness and is boosting the innovative capacity of European health-related enterprises, which is a global priority of the FP7 HEALTH programme.
Dissemination and Exploitation Activities
To deal with the questions related to dissemination of results, exploitation and IPR a specific work package (WP9) was setup. The aim of the dissemination actions was to reach the identified target audience and to transfer correct and incisive information about the project activities and achievements. It was also the objective of the consortium to communicate around the collaborative actions made feasible thanks to the support of the European Commission.
The consortium has therefore designed a plan for disseminating knowledge with the objectives:
• To raise public participation and awareness of the progress made within NEURINOX
• To enhance exchanges with scientific world
• To prepare exploitation of results, create market opportunities
• To spread the knowledge gained beyond the consortium
The dissemination activities have been organised in four large parts by target group: Dissemination towards:
• the public
• the scientific world
• the LifeSciHealth Programme
• the industry
For each of the target groups the aim, content, specific target, main message, detailed activities and timing of activities are described in the report attached.
Exploitation of NEURINOX results
The WP9 leader, in collaboration with the Executive Board, has supported partners in their respective IP procedures, making sure that the access of NEURINOX results etc. are dealt with on a fair and viable basis for all. This task has included patent searches, filing of patent (or other IPR) applications, etc.
The research activities undertaken under the umbrella of the project have generated results with commercial potentials. This has raised the issues of intellectual property rights (IPRs), of protection of the property rights (confidential IP as well as patenting).
The NEURINOX consortium has been composed of research groups and clinical institutions, Universities and SME with extensive experience in NADPH oxidases (NOX) research and neurodegenerative diseases. They have exploited the results of the project in various ways.
The Research Centres and Universities have been mostly benefited from the advance in knowledge which have strengthen their position as leading research institutions in Europe and brought new opportunities for future partnerships. Concrete plans for exploitation covered mainly publications in peer-reviewed international journals and filing of patent applications. The general principles for IPR ownership and IPR protection have been established in the Consortium Agreement.
The NEURINOX results are used in three main ways by individual partners:
• Continuous research
• Dissemination by public presentations and publication
• Commercial development by confidential IPR and exploitation of patents
Shown in 2 tables detailed in the attached report
List of Websites:
NEURINOX partners and contact
NEURINOX Coordinator
1) Dr Vincent Jaquet
Department of Pathology and Immunology
Faculty of Medicine
University of Geneva
1, rue Michel-Servet
1211 Geneva 4, Switzerland
Phone: +41 22 379 4136
E-mail: Vincent.Jaquet@unige.ch
NEURINOX public website: http://www.NEURINOX.eu
Partners
2) University of Zürich (UZH)
Neuropathology Institute
Schmelzbergstrasse 12
8091 Zurich, Switzerland
http://www.en.neuropathologie.usz.ch/
3) GenKyoTex SA (GKT)
16, Chemin des Aulx
CH-1228 Plan - Les-Ouates – Geneva, Switzerland
http://www.genkyotex.com
4) Redoxis AB (RDX)
Medicon Village
Scheelevägen 2
223 81 Lund, Sweden
http://www.redoxis.com/
5) ARTTIC (ART)
Dominique Wasquel / Sara Skogsäter
58A rue du Dessous des Berges
75013 Paris, France
http://www.arttic.eu
6) University of Torino (UT)
Department of Neuroscience
University of Torino
Via Cherasco 15
10126 Torino, Italy
http://www.unito.it/
7) Karolinska Institute (KI), Sweden.
a) Section for Medical Inflammation Research (MIR)
Dept of Medical Biochemistry and Biophysics
b) Neuroimmunology Unit (NU)
Department of Clinical Neurosciences
Center for Molecular Medicine
http://www.ki.se/
8) Université Grenoble Alpes (UGA) previously called Joseph Fourier University (UJF)
621 avenue Centrale
38400 Saint-Martin-d'Hères
http://www.univ-grenoble-alpes.fr/en/
9) SynapCell SAS (SYN)
Bâtiment Biopolis
5 avenue du Grand Sablon
38700 La Tronche, France
http://www.synapcell.fr/
10) Biomedical Research Foundation Academy of Athens (BRFAA)
4 Soranou Ephessiou Street,
Athens 115 27, Greece
http://www.bioacademy.gr/
11) University of Athens (UOA)
National and Kapodistrian University of Athens,
Clinical Genomics and Pharmacogenomics Unit,
4th Department of Internal Medicine,
Attikon Hospital, Medical School,
75 Mikras Asias
115-27 Athens, Greece
http://en.uoa.gr/about-us.html
12) University of Sydney (USYD) terminated in October 2012
Parramatta Road, 92-94
Camperdown NSW 2006, Australia
www.usyd.edu.au
13) NEURIX (NEURIX)
14 Chemin des Aulx
Plan les Ouates 1228
Switzerland
http://www.neurix.ch/
14) Victor Chang Cardiac Research Institute Limited (VCCRI)
School of Medical Sciences,
University of New South Wales, Australia.
405 Liverpool Street
Darlinghurst, 2010, Australia
www.victorchang.com.au
