UNIT FOR RESEARCH IN SCHIZOPHRENIA UPDATE

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1 UNIT FOR RESEARCH IN SCHIZOPHRENIA UPDATE INTRODUCTION The research program supported by the Alamaya Foundation focuses on a better understanding of the causes and mechanisms of schizophrenia in order to identify biological markers for early detection and intervention, and to develop preventive measures as well as new treatments. The basic concept guiding our research can be summarized as follows: the anomalies in the brain of patients involve on one hand a particular type of inhibitory neurons, called parvalbumin (PV) interneurons, and, on the other hand, nervous fibers and their protective envelope, called myelin, which connect the different parts of the central nervous system. These anomalies are responsible for various manifestations of the disease, in particular for high frequency oscillations (EEG) and cognitive disorders affecting memory, attention, concentration and action planning. It is essential to discover the origin of these anomalies. Our hypothesis proposes that a deficient control of oxidations in the human body (called "redox regulation") plays a central role in these phenomena, the deficient control itself being caused by both genetic and environmental risk factors during the development of the brain. Several genetic anomalies involved in schizophrenia lead to a redox dysregulation and to oxidative stress. Environmental risk factors such infections, inflammations, physical or psychological trauma also lead to oxidative stress. Furthermore, redox dysregulation has a tight interaction with inflammatory immune reactions and the deficient functioning of an excitatory receptor called "NMDAR". Thanks to our experimental models, we have been able to validate our hypothesis to a large extent. Figure illustrating several "hubs" on which converge genetic and environmental risk factors during the development of the brain, and which lead to deficiencies of the excitatory/inhibitory balance (E/I) in cortical microcircuits and in macrocircuits, formed by fibers connecting the various parts of the brain; these deficiencies are responsible for the impairment of cognitive, affective and social integration at the basis of the disease symptoms. 1

2 PROGRESS AND RESULTS The present evolution of clinical psychiatry in the field of schizophrenia is aimed at an early detection of individuals who are at risk of the disease since experience shows that the prognosis is all the more favorable if treatment begins early. It is therefore essential (a) to identify biological markers, which are presently lacking, in order to be able to detect vulnerable persons; (b) to develop preventive treatments devoid of serious side effects. These are two of the main objectives of the Unit for Research in Schizophrenia (URS, Centre for Psychiatric Neuroscience, Lausanne University Hospital), which strives to bridging clinical aspects with experimental research, and whose program has reached significant international visibility and acknowledgement (see p. 7, Conclusion, video of the American Psychiatric Association). The URS develops following main working lines: Data collection in patients; Investigation of the disease mechanisms in experimental models; Exploration of biological markers and new potentially preventive treatments. The translational approach of the URS has led to new discoveries in patients; among these are the following: MYELIN: Aline Monin (doctoral student, URS) has studied the role of redox regulation in oligodendrocyte cultures; these are the cells responsible for the formation of myelin, which is essential to the proper functioning of nervous fibres. She has demonstrated that a glutathione deficit causes a delay in the maturation of oligodendrocytes, and that myelin is deficient in the prefrontal cortex of young mice with a low level of glutathione. She has also shown that this delay in the formation of myelin is linked to the excess of a particular enzyme, called "FYN kinase", probably due to the oxidation of regulatory proteins. Following this observation, Margot Fournier (post-doc, URS) has shown that FYN is also increased in the cells of patients with a genetic anomaly hindering the formation of glutathione. The FYN factor could thus be developed as a potential marker for the disease, and pave the way for early intervention. These results demonstrate the impact of oxidative stress on macrocircuits (see figure Hubs in schizophrenia, p.1), namely on the integrity of nerve fibres which are essential for the coordinated action of different brain regions. They thus confirm the role of oxidative stress in the impairment of myelin, a key factor for the development of the disease, which could be considered as a potential biomarker. The results of this study have been published in Molecular Psychiatry, the scientific journal with the highest impact factor in the field of psychiatry and neuroscience (see Annex I, press release of the Lausanne University Hospital). 2

3 FORNIX: Alberto Corcoba (doctoral student, URS, in collaboration with Prof. R. Gruetter, CIBM- EPFL) has developed a new, non-invasive and cutting-edge imaging method (MRI-DTI) allowing the observation of the integrity of nervous fibres in the brain of glutathione deficient mice; he recorded a reduced integrity in a nervous pathway originating in the hippocampus, called the fornix, which is linked to the formation and consolidation of memory. This observation in the animal model has prompted Philipp Baumann (clinician and researcher, URS & Service of General Psychiatry) and Alessandra Griffa (collaboration with Dr P. Hagmann, Radiology, Lausanne University Hospital, and Dr J.-P. Thiran, Signal Processing Laboratory 5, EPFL) to analyze with the same method the integrity of the fornix in patients during their first psychotic episode; as a result and for the first time, they have revealed a deficiency of this integrity (decreased gfa) in young patients. As explained below, the investigations concerning a structure which is central to the functioning of memory are of particular interest. HIPPOCAMPUS: this brain structure plays an important role in the recording of memory; people in whom this structure is defective have difficulties in consolidating their memories. In the hippocampus of glutathione deficient mice, Pascal Steullet and Jan Cabungcal (senior researchers, URS) have shown that PV cells and oscillations are deficient. In patients, the volume of the hippocampus is diminished, and Philipp Baumann has demonstrated that its decrease is proportionate to the decrease of gfa in the fornix (measure of the integrity of nervous fibers). Furthermore, the volume of the hippocampus is linked to a blood marker of redox dysregulation: the higher the state of oxidation, the smaller the hippocampus. This represents a direct evidence of the impact of redox balance, measured in the periphery, on a brain structure. Finally and in parallel to these findings, recent data established by Luis Alameda (clinician and researcher, URS & Service of General Psychiatry) show that patients who were subject to traumatisms during adolescence have a smaller hippocampus. These observations have to be correlated with those made in mice by Jan Cabungcal, indicating that an additional stress in the young animal, and only in the young one, causes persistent anomalies of PV cells in the frontal cortex. This combination of results on the hippocampus complex and the fornix confirms the important role of the redox balance in the development of these structures, which are 3

4 essential for key functions such as memory and the organization of space, and explain their deficit in schizophrenia patients. OTHER ANIMAL MODELS: Jan Cabungcal has studied the markers of oxidative stress in other animal models of schizophrenia and other diseases, such as autism. In collaboration with Patricio O Donnell (University of Maryland, USA), he showed that a neonatal lesion of the ventral hippocampus, a classical model of schizophrenia in rats, also causes an oxidative stress in the prefrontal cortex. As in mice with a genetically induced low level of glutathione, the multiple morphological, physiological and behavioral effects of this intervention can be prevented by a treatment with N- acetyl-cysteine. This is all the more interesting since the intervention does not involve a manipulation of the redox system and clearly shows that diverse impacts can converge on a redox disturbance. Moreover, a model of autism displays the same oxidation characteristics as the glutathione deficient model of the URS. These results suggest that a variety of pathological impacts could lead to oxidative stress, which appears thus as a core mechanism or common final path, i.e. a site of convergence of diverse causalities (see figure Hubs in schizophrenia, p.1). This concept could lead to a new approach to the prevention of schizophrenia. Antioxidant drugs given before overt clinical manifestations and at an early stage to people at risk may be able to prevent the emergence of the disease. The results of the study have been published in Neuron, a highly and internationally renowned scientific journal (see Annexes II and III, press release of the Lausanne University Hospital, and editorial of the Schizophrenia Research Forum Website). The article rose major interest among the scientific and medical communities. In a Neuron Previews Editorial, Prof. Akira Sawa (Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, USA) and Prof. Larry J. Seidman (Department of Psychiatry, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA) wrote an article entitled Is Prophylactic Psychiatry around the Corner? Combating Adolescent Oxidative Stress for Adult Psychosis and Schizophrenia, which refers to the study mentioned above. The mention of "prophylactic psychiatry" by these distinguished scientists indicates that a major step forward has been achieved by the URS team as regards the prevention of psychosis. IMMUNE REACTIONS: several data suggest that immune reactions and inflammation can play a role in the development of schizophrenia. It is well-known that immune phenomena lead to oxidative stress. We have explored the reverse possibility that redox dysregulation could initiate immune reactions. Daniella Dwir (doctoral student, URS) has observed in the prefrontal cortex of glutathione deficient mice, thus exposed to oxidative stress a significant increase of activated immune "microglia" cells, which demonstrates the redox influence on immune reactions. She showed that a factor called RAGE plays a critical role in this interaction. Consistent with observations made in patients, these results show, in an animal model, the close interaction between redox dysregulation and inflammation, and 4

5 demonstrate that they potentiate each other in a damaging positive feedback (see figure Hubs in schizophrenia, p.1). METABOLOMICS: Margot Fournier has studied the effect of oxidative stress on the various metabolites in the fibroblasts of patients. She observed that the reaction to stress of different metabolic pathways is affected. As expected, the redox system is affected but also, and these aspects are new, certain lipids, the arginine/nitric oxide system as well as the extracellular matrix (protective envelope of nerve cells). These results have been published in the journal Schizophrenia Bulletin. The finding concerning the extracellular matrix is of particular interest since a specific form of this matrix, called "perineuronal net", essential for the synaptic plasticity, is affected in the brain of patients and in the animal model, as demonstrated by Jan Cabungcal and Pascal Steullet (publication in the journal Proceedings of the National Academy of Sciences USA in May 2013). The multiple interactions described above suggest the existence of a scheme with several entries, involving retroactive and combined effects of NMDAR hypofunction, inflammation and redox dysregulation. It is probably by focusing on these three elements that preventive measures should be envisioned (see figure Hubs in schizophrenia, p.1). A review article summarizing these results and their conceptual consequences has been prepared by Pascal Steullet and published in Schizophrenia Research. MAJOR INTERNATIONAL COLLABORATIONS Scientists and researchers worldwide are interested in collaborating with Prof. Kim Do Cuénod and her research group. Present collaborations include among others: Harvard Medical School, Boston, USA, Department of Molecular & Cellular Biology, Prof. Takao Hensch Harvard Medical School, Boston, USA, Department of Psychiatry, Prof. Larry J. Seidman University of Maryland School of Medicine, Baltimore, USA, Prof. Patricio O Donnell Mental Health Centre Sct. Hans, Capital Region of Denmark, Institute of Biological Psychiatry, Prof. Thomas Werge PUBLICATIONS It has to be noted that Molecular Psychiatry and Neuron are two of the most influential and selective scientific journals worldwide in the field of biological psychiatry and neuroscience. Biological Psychiatry and Proceedings of the National Academy of Sciences USA also have a very high impact factor. The article published in Biological Psychiatry in 2013 has raised major interest among the international scientific community as well as among the national and international media (see Annexes IV, V VI). 5

6 ORIGINAL ARTICLES Kulak A, Steullet P, Cabungcal JH, Werge T, Ingason A, Cuenod M, Do KQ. Redox Dysregulation in the Pathophysiology of Schizophrenia and Bipolar Disorder: Insights from Animal Models. Antioxidants & Redox Signaling, 18 (12), (2013) Cabungcal JH, Steullet P, Kraftsik R, Cuenod M, Do KQ. Early-Life Insults Impair Parvalbumin Interneurons via Oxidative Stress: Reversal by N-Acetylcysteine. Biological Psychiatry, 73 (6), (2013) Cabungcal JH, Steullet P, Morishita H, Kraftsik R, Cuenod M, Hensch TK, Do KQ. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proceedings of the National Academy of Sciences USA, 110 (22), (2013) Baumann PS, Crespi S, Marion-Veyron R, Solida A, Thonney J, Favrod J, Bonsack C, Do KQ, Conus P. Treatment and Early Intervention in Psychosis Program (TIPP-Lausanne): implementation of an early intervention programme for psychosis in Switzerland. Early Interv Psychiatry, 7 (3), (2013) Fournier M, Ferrari C, Baumann PS, Polari A, Monin A, Bellier-Teichmann T, Pappan K, Wulff J, Cuenod M, Conus P, Do KQ. Impaired metabolic reactivity to oxidative stress in early psychosis patients. Schizophrenia Bulletin, 40 (5), (2014) Monin A, Baumann PS, Griffa A, Xin L, Mekle R, Fournier M, Butticaz C, Klaey M, Cabungcal JH, Steullet P, Ferrari C, Cuenod M, Gruetter R, Thiran JP, Hagmann P, Conus P, Do KQ. Glutathione deficit impairs myelin maturation: relevance for white matter integrity in schizophrenia patients. Molecular Psychiatry, doi: /mp [Epub ahead of print] (2014) Cabungcal JH*, Counotte DS*, Lewis E*, Tejeda HA, Piantadosi P, Pollock C, Calhoon GG, Sullivan E, Presgraves E, Kil JJ, Hong LE, Cuenod M, Do KQ*, O Donnell P*. Juvenile antioxidant treatment prevents adult deficits in a developmental model of schizophrenia. Neuron, 83 (5), (2014) * Equal contribution Duarte JM, Do K., Gruetter R. Longitudinal neurochemical modifications in the aging mouse brain measured in vivo by (1)H magnetic resonance spectroscopy. Neurobiology of Aging, 35 (7), (2014) O'Donnell P, Do KQ, Arango C. Oxidative/Nitrosative stress in psychiatric disorders: are we there yet? Schizophrenia Bulletin, 40 (5), (2014) Steullet P, Cabungcal JH, Cuenod M, Do KQ. Fast oscillatory activity in the anterior cingular cortex: dopaminergic modulation and effect of perineuronal net loss. Front Cell Neurosci, doi: /fncel ecollection (2014) REVIEW ARTICLES Kim Q. Do. Gènes, environnement et neurodéveloppement: le cas de la schizophrénie. Revue Médicale Suisse, 9: (2013) Steullet P, Cabungcal JH, Monin A, Dwir D, O Donnell P, Cuenod., Do KQ. Redox dysregulation, neuroinflammation, and NMDA receptor hypofunction: a central hub in schizophrenia pathophysiology? Schizophrenia Research, doi: /j.schres [Epub ahead of print] (2014) 6

7 INVITED CONFERENCES & SYMPOSIA PROF. KIM DO CUÉNOD Prof. Kim Do Cuénod is regularly invited to share the results and perspectives of her research program at major international events, research centres and universities. Plenary Speaker, Neuroscience th Annual Meeting of the Society for Neuroscience: "Genes and Environment Interaction during Development: Redox Imbalance in Schizophrenia", 17 November 2014, Washington DC, USA Keynote speaker, Central Institute of Mental Health, Department of Psychiatry: "Oxidative stress, NMDA hypofunction and neuroinflammation in schizophrenia: a translational approach", 7 October 2014, Mannheim, Germany Keynote speaker, Pfizer Inc., Neuroscience Research Unit, Psychiatry and Behavioral Disorders: "Oxidative stress in psychosis: a translational approach towards new targets for stage specific treatment", 13 May 2014, Cambridge, USA Keynote speaker, Harvard Medical School, Department of Psychiatry: "Redox dysregulation in schizophrenia: functional anomalies prevented by N-acetyl-cysteine in translational models", 12 May 2014, Boston, USA Symposium (co-chair, speaker), Society of Biological Psychiatry, 69 th Annual Scientific Meeting: "Redox Dysregulation Affects Myelination And Parvalbumine Interneurons In Schizophrenia Models", 10 May 2014, New York, USA Symposium (speaker), Society of Biological Psychiatry, 69 th Annual Scientific Meeting: "Redox Dysregulation Models of Schizophrenia: Functional Anomalies Prevented By N-acetylcysteine", 8 May 2014, New York, USA Satellite Symposium (organizer, speaker) "Schizophrenia: could it be prevented?", Society of Biological Psychiatry, 69th Annual Scientific Meeting: "Oxidative stress in psychosis: a translational approach", 7 May 2014, New York, USA Keynote speaker, Kings College London, Institute of Psychiatry, Department of Psychosis Studies: "Search for biomarkers and redox dysregulation in early psychosis: a translational approach", 28 April 2014, London, UK Inaugural Lesson, Lausanne University Hospital (CHUV): "Neurosciences et psychiatrie: le développement du cerveau, toutes les chances et tous les risques", 8 April 2014, Lausanne, Switzerland Symposium (speaker), Swiss Society for Neuroscience, Annual Meeting: "Oxidative stress in psychosis: a translational approach", 25 January 2014, Bern, Switzerland Lecture, PhD course on "Advanced biomedical imaging methods and instrumentation", Center for Biomedical Imaging (CIBM), Swiss Federal Institute of Technology (EPFL): "Multimodal imaging in humans and animal models: towards early detection and novel drug targets in psychosis", 13 December 2013, Lausanne, Switzerland Workshop, Brocher Foundation, Neuroscience, Ethics and Law: New Challenges for Human Identity, Freedom and Responsibility: "Neuroscience of the self in health and disease", 6 November 2013, Hermance, Switzerland Symposium (speaker), 2nd SFCNS Congress, Swiss Federation of Clinical Neuro-Societies: "Oxidative stress in psychosis: a translational approach", 6 June 2013, Montreux, Switzerland 7

8 Symposium (speaker), Society of Biological Psychiatry, 68th Annual Scientific Meeting: "The Perineuronal Net Protects Fast-Spiking Parvalbumine Interneurons Against Oxidative Stress", 18 May 2013, San Francisco, USA Conference, Le Relais Association genevoise de soutien aux proches de personnes souffrant de troubles psychiques: "Schizophrénie: la recherche progresse", 13 May 2013, Geneva, Switzerland Symposium (speaker), CINP Thematic Meeting, Pharmacogenomics and Personalised Medicine in Psychiatry: "Redox imbalance in schizophrenia and its improvement by N-acetylcysteine", 21 April 2013, Jerusalem, Israel Conference, Institute of Anatomy and Cell Biology, Università Cattolica del S. Cuore: "Redox dysregulation in schizophrenia: a translational approach", 5 April 2013, Rome, Italy Conference, l'îlot Association vaudoise de proches de personnes souffrant de schizophrénie: "Schizophrénie: la recherche progresse", 6 March 2013, Lausanne, Switzerland OBJECTIVES On the clinical side, research has been conducted in a cohort of first episode psychosis (FEP) patients in order to confirm the presence of neurobiological markers observed in chronic patients. In the coming years, the aim is: 1) to consolidate results in a larger cohort of FEP patients, and 2) to extend the study to an At-Risk Mental State (ARMS/prodrome) cohort of subjects, in order to explore whether previously identified multilevel markers in FEP are also present in UHR (Ultra High Risk) patients and whether they represent stable markers or markers of progression during the prodromal phase; 3) to identify new and more efficient molecules for the development of innovative treatments and preventive measures. The main research questions are the following: a) Are phenomena that are well documented at the chronic stage also present at the beginning of illness? b) Are there stage specific phenomena that could be used as bio-markers and treatment targets? c) What are the underlying mechanisms driving the modifications of brain structure? We thus aim to provide valid neurobiological markers for: 1) the early identification of patients; 2) the definition of the various stages of the disease; 3) the assessment of illness progression; 4) the development of new therapeutic tools. 8

9 CONCLUSION The work carried out with a long-term vision by the team of the Unit for Research in Schizophrenia (URS) is highly fruitful. The translational research program that has been developed step by step over the last 15 years is presently recognized as a scientific approach unique in its kind. The interactions between clinicians and researchers, as well as the confrontation of observations made in patients and in experimental models, lead to essential developments in the exploration and knowledge of schizophrenia, and more generally of psychosis. Such an approach represents a pioneer achievement in the field of psychiatry. The impact and visibility of the studies conducted by the research group directed by Prof. Kim Do Cuénod was confirmed by the following events in 2014: Given her exceptional scientific accomplishments, Prof. Kim Do Cuénod was elected Individual Member of the Swiss Academy of Medical Sciences (SAMS) at the session held by the SAMS Senate on 20 May This election represents a very high distinction and emphasizes the excellence of Kim Do Cuénod's career in the field of biological psychiatry. A video clip on the translational research program directed by Prof. Kim Do Cuénod and her clinician colleague Prof. Philippe Conus was produced at the request of the American Psychiatric Association (APA), and broadcasted during its Annual Meeting, which took place at the beginning of May 2014, in New York. APA had chosen 5 research centres outside the United States for such a presentation: 1 in Japan, 1 in Shanghai and 3 in Europe, among which the Unit for Research in Schizophrenia (URS) in Lausanne. APA's choice represents a great honor and a first-rate acknowledgment of the work carried out at the URS. The clip can be accessed on the Website of the Alamaya Foundation, (page Videos). Prof. Kim Do Cuénod was interviewed on the French Channel of the Swiss National Radio (Radio Suisse Romande la 1ère) in the context of a scientific program called CQFD. The edition of September 4 th, 2014, focused on "News about schizophrenia"; the results of two studies conducted by the URS were presented and commented by Kim Do Cuénod. The interview can be accessed on the Website of the Alamaya Foundation (page Media). Prof. Michel Cuénod, Chair of the Alamaya Foundation, invited professor at the University of Lausanne and scientific adviser of the URS, was interviewed on the French Channel of the Swiss National Radio (Radio Suisse Romande la 1ère) in the context of the CQFD program broadcasted on March 17 th, The program entitled "People with schizophrenia have a life" was aired on the occasion of the 11 th edition of the "Journées de la Schizophrénie" (Days of Schizophrenia), a yearly event to raise awareness on schizophrenia among the general public, which, in 2014, took place from 17 to 23 March. The interview can be accessed on the Website of the Alamaya Foundation (page Media). 9

10 ANNEX I CHUV Centre Hospitalier Universitaire Vaudois COMMUNIQUÉ DE PRESSE GLUTATHIONE DEFICIT AFFECTS WHITE MATTER INTEGRITY IN PREFRONTAL CORTEX AND IMPAIRS BRAIN CONNECTIVITY IN SCHIZOPHRENIA Aline Monin, Philipp S. Baumann, Alessandra Griffa, Lijing Xin, Ralf Mekle, Margot Fournier, Christophe Butticaz, Magali Klaey, Jan-Harry Cabungcal, Pascal Steullet, Carina Ferrari, Michel Cuenod, Rolf Gruetter, Jean-Philippe Thiran, Patric Hagmann, Philippe Conus, Kim Q. Do. Molecular Psychiatry, 2014 Chez les patients souffrant de schizophrénie on observe, entre autres, des anomalies des fibres qui assurent les connexions entre différentes parties du cerveau. Le groupe de recherche de la Prof. Kim Do Cuénod (Centre de neurosciences psychiatriques, Département de psychiatrie, CHUV-UNIL), en collaboration avec le Prof. Philippe Conus (Service de psychiatrie général, Département de psychiatrie CHUV-UNIL), le Prof. Patric Hagmann (Département de radiologie CHUV) et le Prof. Rolf Gruetter (Centre d imagerie biomédicale EPFL), vient de publier un article dans le prestigieux journal Molecular Psychiatry dans lequel les mécanismes responsables de ces défauts de connectivité ont été étudiés et leur cause en partie élucidée. Ces résultats démontrent qu une mauvaise régulation de l équilibre entre oxydations et réductions pendant le développement du cerveau pourrait jouer un rôle important dans le développement de la schizophrénie. Les fibres nerveuses sont entourées d une gaine isolante (la myéline) formée par des cellules spéciales (les oligodendrocytes). Ces cellules sont particulièrement sensibles à un excès d oxydation. Une doctorante travaillant sous la supervision de la Prof. Kim Do Cuénod, Mme Aline Monin, a montré que le déficit d'une molécule normalement abondante et protectrice contre les oxydations (le glutathion) entraîne une insuffisance des cellules formatrices de myéline et de la myélinisation du cortex. Elle a également montré que l'anomalie dans le développement de ces cellules formatrices de myéline est liée à une dérégulation de l activité d un enzyme appelée "Fyn kinase". L intérêt majeur de cette recherche est le fait que les résultats obtenus dans des modèles expérimentaux ont également été identifiés chez les patients: la Fyn kinase est également dérégulée dans les cellules de peau provenant de patients porteurs d une anomalie génétique entravant la formation du glutathion et exposées à un stress oxydatif. D autre part, plus le taux de glutathion (mesuré par résonance magnétique spectroscopique) dans le cortex préfrontal médian est élevé, meilleure est l intégrité des fibres nerveuses d un faisceau appelé "cingulum" (mesurée par Diffusion Spectrum Imaging), comme l ont montré les Drs Philipp Baumann, Alessandra Griffa et Lijing Xin. Ces résultats confirment le rôle déterminant de l équilibre entre oxydations et réductions lors du développement des fibres nerveuses, et son implication probable dans l origine des défauts de connectivité chez les patients souffrant de schizophrénie. Ils ouvrent la voie à l établissement de biomarqueurs et de nouveaux mécanismes d intervention précoce. 10

11 ANNEX II CHUV Centre Hospitalier Universitaire Vaudois COMMUNIQUÉ DE PRESSE JUVENILE ANTIOXIDANT TREATMENT PREVENTS ADULT DEFICITS IN A DEVELOPMENTAL MODEL OF SCHIZOPHRENIA Jan-Harry Cabungcal, Danielle S. Counotte, Eastman M. Lewis, Hugo A. Tejeda, Patrick Piantadosi, Cameron Pollock, Gwendolyn G. Calhoon, Elyse M. Sullivan, Echo Presgraves, Jonathan Kil, L. Elliot Hong, Michel Cuenod, Kim Q. Do, Patricio O Donnell Neuron, 2014 Le laboratoire de la Prof. Kim Do Cuénod (Centre de neurosciences psychiatriques, Département de psychiatrie, CHUV-UNIL) avait montré dans un modèle animal de la schizophrénie qu une insuffisance de la lutte contre les oxydations entraîne des anomalies de certains neurones essentiels pour les activités cognitives (appelés "parvalbumine interneurones" ou PVI). Ces anomalies ont également été observées dans le cerveau des patients. Cela suggérait que ce contrôle déficient pourrait être une des causes de la schizophrénie. Comme l intervention portait au niveau génétique sur le système de contrôle des oxydations lui-même, il était intéressant d observer qu un stress oxydatif affectait sélectivement ces neurones PVI. Une nouvelle étape dans la recherche a maintenant été franchie, grâce à une collaboration avec l équipe du Prof. Patricio O Donnell de l Université du Maryland à Baltimore (USA). L article publié dans la revue Neuron le 3 septembre 2014 porte sur un autre modèle bien établi de la schizophrénie qui consiste à faire une petite lésion d une structure du cerveau, l hippocampe ventral, chez le rat nouveau-né. Ces animaux présentent eux aussi, à l âge adulte, des manifestations pathologiques typiques de la maladie. L équipe de Lausanne a observé que ces animaux présentent également un stress oxydatif et des anomalies des neurones PVI, bien que leur système de régulation des oxydations n ait pas été impliqué, prouvant ainsi que le stress oxydatif peut avoir des origines variées. Le plus fascinant, c est qu un traitement avec un antioxydant, la N-acétyle-cystéine, permet non seulement de protéger l animal contre l oxydation des PVI, mais corrige aussi les autres anomalies physiologiques et comportementales connues de ce modèle. Il semble donc que, pendant le développement du cerveau, des causes multiples sont susceptibles d induire un stress oxydatif neuronal sélectif conduisant au développement de la maladie. Cette découverte ouvre la voie à de nouveaux traitements. 11

12 ANNEX III Model of Schizophrenia Cortical Deficits Reversed With Antioxidants August 6, In a new study published online August 6 in Neuron, an international collaboration offers up evidence linking the reported alterations in cortical interneurons in schizophrenia to oxidative stress. Kim Do and colleagues at Lausanne University Hospital in Switzerland teamed up with Patricio O'Donnell's group at the University of Maryland in Baltimore to test the Lausanne group's longstanding hypothesis of oxidative stress in schizophrenia by using the neonatal ventral hippocampal lesion (NVHL) model that O'Donnell has employed to probe subtle miswiring of the prefrontal cortex. The authors report evidence that NVHL produces oxidative stress in rat cortical interneurons, particularly those expressing parvalbumin, and that giving the animals an antioxidant throughout most of postnatal development, or even just in the last stages before adulthood, can reverse this finding, along with various deficits in neuronal chemistry and electrophysiology and brain function. Linking up hypotheses For many years, the two research groups have been working on separate hypotheses: O'Donnell's lab has investigated the delayed effects of neonatal hippocampal lesions in rats on the development of wiring in the prefrontal cortex. The researchers have proposed that the abnormalities that arise only in rat "adolescence" can model some aspects, particularly in the dopaminergic system, of schizophrenia pathophysiology (see SRF related news report and SRF related conference report). For their part, Do and Michel Cuenod have pursued evidence that the nervous system of people with schizophrenia is awash in reactive oxygen species that can harm neurons in various ways. In particular, they have focused on the possibility of a shortage of glutathione, which scavenges these oxidative molecules (reviewed in SRF related conference report). In their joint study, co-first authors Jan-Harry Cabungcal, Danielle Counotte, Eastman Lewis, and their colleagues lesioned the ventral hippocampus in rat pups (postnatal days 7-9) and exposed them to the antioxidant N-acetyl cysteine (NAC) from P5 until P60, first through their mothers and later directly via drinking water. Consistent with previous reports, the lesions resulted in changes to interneurons in prefrontal cortex (PFC), specifically to the expression of parvalbumin (PV) in NVHL rats versus sham-operated animals. NAC treatment reversed these changes, and in support of the idea that oxidative stress was a factor in the reduction in PV, the authors found significantly higher levels of the DNA oxidation marker 8- oxo-dg in cortical pyramidal cells and interneurons of the NVHL rats at P21. The immunohistochemistry data were supported by electrophysiology in cortical slice and in-vivo preparations. Well-known abnormalities in PFC pyramidal cell synaptic function of NVHL mice were also corrected by the NAC treatment. Finally, Cabungcal and colleagues zoomed out to look at information processing and sensorimotor gating, using mismatch negativity and prepulse inhibition (PPI) tests to probe for deficits that have 12

13 been found in people with schizophrenia. In both cases, NAC treatment normalized the alterations stemming from the neonatal lesions. The authors also addressed one possible critique that the beneficial effects were due to effects on glutamate neurotransmission via the cysteine-glutamate transporter. However, both ebselen and apocynin antioxidants that do not affect glutamate levels rescued the PPI deficits in NVHL animals. Questions for the future "Our data suggest that oxidative stress in PFC is a core feature mediating alterations induced by the NVHL, and antioxidant treatment prevents these alterations," conclude the researchers. How would the NVHL have produced oxidative stress? The authors speculate that the reduction in glutamatergic input during development places the PV-expressing interneurons in PFC under oxidative stress, citing evidence that NMDA receptor blockade can have this effect (see SRF related news report). However, they also note that this model of prefrontal dysfunction does not have to accurately reflect processes of schizophrenia in order to reproduce the pathophysiology of the disease. The results are certainly intriguing, given that they present a possible therapeutic option that could be applied early in the disease, or even in the prodrome Cabungcal and colleagues were able to significantly improve prepulse inhibition by giving NAP beginning in rat adolescence (P35). The compound appears to have few major side effects, and it is currently used as a non-regulated supplement, though it would be hard to predict its effects on the developing nervous system. Hakon Heimer. Reference: Cabungcal JH, Counotte DS, Lewis EM, Tejeda HA, Piantadosi P, Pollock C, Calhoon GG, Sullivan EM, Presgraves E, Kil J, Hong LE, Cuenod M, Do KQ, O'Donnell P. Juvenile Antioxidant Treatment Prevents Adult Deficits in a Developmental Model of Schizophrenia. Neuron Aug 12. Abstract 13

14 ANNEX IV Science News... from universities, journals, and other research organizations Neuron Loss in Schizophrenia and Depression Could Be Prevented, Study Suggests Mar. 13, 2013 Gamma-aminobutyric acid (GABA) deficits have been implicated in schizophrenia and depression. In schizophrenia, deficits have been particularly well-described for a subtype of GABA neuron, the parvalbumin fast-spiking interneurons. The activity of these neurons is critical for proper cognitive and emotional functioning. It now appears that parvalbumin neurons are particularly vulnerable to oxidative stress, a factor that may emerge commonly in development, particularly in the context of psychiatric disorders like schizophrenia or bipolar disorder, where compromised mitochondrial function plays a role. Parvalbumin neurons may be protected from this effect by N-acetylcysteine, also known as Mucomyst, a medication commonly prescribed to protect the liver against the toxic effects of acetaminophen (Tylenol) overdose, reports a new study in the current issue of Biological Psychiatry. Dr. Kim Do and collaborators, from the Center for Psychiatric Neurosciences of Lausanne University in Switzerland, have worked many years on the hypothesis that one of the causes of schizophrenia is related to vulnerability genes/factors leading to oxidative stress. These oxidative stresses can be due to infections, inflammations, traumas or psychosocial stress occurring during typical brain development, meaning that at-risk subjects are particularly exposed during childhood and adolescence, but not once they reach adulthood. Their study was performed with mice deficient in glutathione, a molecule essential for cellular protection against oxidations, leaving their neurons more exposed to the deleterious effects of oxidative stress. Under those conditions, they found that the parvalbumin neurons were impaired in the brains of mice that were stressed when they were young. These impairments persisted through their life. Interestingly, the same stresses applied to adults had no effect on their parvalbumin neurons. Most strikingly, mice treated with the antioxidant N-acetylcysteine, from before birth and onwards, were fully protected against these negative consequences on parvalbumin neurons. "These data highlight the need to develop novel therapeutic approaches based on antioxidant compounds such as N-acetylcysteine, which could be used preventively in young at-risk subjects," said Do. "To give an antioxidant from childhood on to carriers of a genetic vulnerability for schizophrenia could reduce the risk of emergence of the disease." "This study raises the possibility that GABA neuronal deficits in psychiatric disorder may be preventable using a drug, N-acetylcysteine, which is quite safe to administer to humans," added Dr. John Krystal, Editor of Biological Psychiatry. 14

15 ANNEX V ACTUALITES NCCR-SYNAPSY Pre venir la schizophreńie chez les enfants En intervenant tôt sur le de veloppement du cerveau, avec un traitement approprie, il serait possible de diminuer l apparition de troubles schizophre niques, relève une étude du groupe de recherche du Prof. Kim Do, publié aujourd'hui dans la version imprimée de "Biological Psychiatry". L'Unité de recherche sur la schizophrénie du CHUV, dirigée par le Prof. Kim Do, membre du NCCR Synapsy, travaille depuis plusieurs années sur l hypothèse qu une des causes de la schizophrénie est liée à des gènes de risque associés à des stress oxydatifs causés entre autres par des infections, inflammations, traumatismes et stress psychique, durant le développement du cerveau. L'étude d'un modèle animal (souris), dont la synthèse du glutathion est diminuée entrai nant ainsi un risque élevé de stress oxydatifs, permet d explorer les origines et l'évolution de certaines anomalies observées dans le cerveau des patients. Une destruction irréversible L'article publié en ligne en novembre 2012 et aujourd'hui imprimé dans la revue Biological Psychiatry porte sur une étude impliquant le modèle animal pauvre en glutathion. Cette substance produite par l'organisme est un important régulateur des oxydations et un agent protecteur essentiel pour les cellules nerveuses. Les chercheurs ont étudié une classe particulière de neurones inhibiteurs - dits "à parvalbumine - qui jouent un rôle capital dans toutes les activités du cortex, qu il s agisse de mémoire, d attention ou d émotion. Ces neurones sont moins nombreux dans le cortex frontal des patients souffrant de schizophrénie et du modèle animal. L'étude démontre que ces derniers sont particulièrement sensibles aux excès d oxydants (stress oxydatif) pendant l enfance et la jeunesse et que cette vulnérabilité disparai t à l âge adulte. De plus, la destruction de ces neurones est durable et irréversible, d ou l importance de l intervention précoce. Un antioxydant pour traitement Fait remarquable, en administrant à l'animal un antioxydant appelé N-acétyl-cystéine dès avant sa naissance, on le protège totalement contre les déficits des neurones à parvalbumine. Il apparai t donc qu un traitement préventif approprié est susceptible d éviter les conséquences défavorables qui résultent de la combinaison d une anomalie génétique (déficit en glutathion ou autres gènes de risque) avec un stress oxydatif pendant le développement du cerveau. Ces résultats revêtent une importance particulière du fait que de nombreux événements adverses au cours de l enfance et de la jeunesse, couplés à la vulnérabilité génétique peuvent provoquer un stress oxydatif. Sans l apport d un traitement préventif approprié, ces traumatismes pourraient avoir un effet délétère à long terme, s ils surviennent pendant l enfance et la période péripubertale. 15

16 ANNEX VI LAUSANNE 15 mars :34; Act: :59 Des antioxydants pour prévenir la schizophrénie D'après une étude menée par des chercheurs lausannois sur des souris, des antioxydants pourraient être administrés à titre préventif aux jeunes touchés par un risque de schizophrénie. L'équipe de Kim Do, responsable de l'unité de recherche sur la schizophrénie du CHUV et professeure associée à l'université de Lausanne, a administré à des souriceaux un médicament antioxydant bien connu, la N-acétylcystéine, utilisée notamment contre la bronchite et bien supportée par l'être humain. Les dommages causés à certains neurones par un stress oxydatif pourraient en effet être en cause dans la genèse de la schizophrénie. C'est en particulier le cas des interneurones exprimant la parvalbumine (PV), une protéine impliquée dans la régulation cellulaire et la production de substances de signalisation. On parle de stress oxydatif lorsque la cellule est agressée par un excès de certaines molécules d'oxygène et que son système de détoxification est dépassé. Cela peut être le cas lors d'infections et inflammations mais aussi lors d'évènements négatifs vécus durant l'enfance et la jeunesse. Les chercheurs lausannois ont constaté que les interneurones PV sont particulièrement sensibles au stress oxydatif, comme ils l'écrivent dans la revue «Biological Psychiatry». Effet protecteur Pour leur étude, ils ont utilisé des souriceaux privés d'un gène déterminant pour une molécule protégeant de l'oxydation et les ont soumis à des situations effrayantes. Résultat: une perturbation accrue des interneurones PV. Chez des souris adultes par contre, le stress n'avait pas d'effet dommageable. Idem chez des souriceaux auxquels les scientifiques avaient administré de l'antioxydant N-acétylcystéine avant et après la naissance. Leurs interneurones PV étaient entièrement protégés. Selon les chercheurs, l'administration de N-acétylcystéine à de jeunes personnes à risque pourrait prévenir la schizophrénie. «Ces données soulignent la nécessité de développer des médicaments sur la base d'antioxydants qui pourraient être remis à des enfants et adolescents à risque», estime le Dr Do dans un communiqué de l'éditeur Elsevier. (afp) 16