10.2 REDOX DYSREGULATION, OLIGODENDROCYTES AND WHITE MATTER ALTERATIONS IN SCHIZOPHRENIA

Abstract Background Widespread (Klauser et al., 2016) and progressive (Cropley et al., 2017) cerebral anomalies of white matter diffusion properties (i.e. fractional anisotropy, FA) have been observed in the Australian Schizophrenia Research Bank (ASRB), one of the largest samples of patients with schizophrenia. From a topological perspective, widespread alterations of white matter tend to concentrate into hub regions that interconnect brain areas over long-distances in a so-called “rich-club” (van den Heuvel et al., 2013; Klauser et al., 2016) in which the metabolic demand is high and thus are most likely to suffer from oxidative stress. Evidence from human and animal models suggests that redox dysregulation leading to oxidative stress during neurodevelopment is implicated in schizophrenia pathogenesis (Steullet et al., 2017). At the cellular level, the triad composed of NMDAR hypofunction, neuroinflammation and dopamine dysregulation interacts with redox imbalance and leads to oxidative stress, affecting oligodendrocytes precursor cells (OPC) and parvalbumine interneurons (Steullet et al., 2016). However, the links between redox imbalance, oligodendrocytes and gross alterations of white matter integrity are largely unexplored. Under oxidative stress induced in vitro by impairing the synthesis of glutathione (GSH), the key player in antioxidant defense, OPC showed a decreased proliferation mediated by an upregulation of Fyn kinase activity. In the prefrontal cortex of a mouse model with impaired GSH synthesis, mature oligodendrocyte numbers as well as myelin markers were decreased at peripuberty (Monin et al., 2014). FA was also reduced in fornix-fimbria and anterior commissure, a change accompanied by a reduced conduction velocity (Corcoba et al., 2015). Methods 49 patients with psychosis and 64 healthy controls were scanned with the same 3-Tesla scanner. The diffusion spectrum imaging (DSI) sequence included 128 diffusion-weighted images with a maximum b-value of 8000 s mm−2. White matter diffusion properties were estimated using generalized fractional anisotropy (gFA). Total blood cysteine (Cys, protein-bound form, free reduced and free oxidized form), the rate-limiting precursor of GSH, was measured by high performance liquid chromatography from plasma samples collected at the same time-point as MRI brain scans. Whole brain voxel-based analyses were performed using cluster-based non-parametric permutation testing on gFA maps. Cerebral levels of GSH were assessed by localized 1H-MRS measurements from a volume of interest in medial prefrontal cortex. Results As previously described in ASRB, we observed widespread abnormalities of white matter in patients. Interestingly, the degree of white matter alterations (i.e. decreased gFA) patients could be predicted by the levels of blood cysteine, a precursor of GSH, strongly suggesting the important role played by oxidative stress in the pathophysiological mechanism. Also, we found that white matter alterations could be reversed by 6 months of add-on treatment with the antioxidant and GSH precursor N-acetyl-cysteine (NAC). Most importantly, this improvement was positively correlated with an increase in prefrontal GSH levels. Discussion We propose that developmental redox imbalance inducing oxidative stress may lead to impairments of oligodendrocytes, myelin formation and eventually to the disruption of fibers integrity and conductivity, especially in brain regions having high metabolic demand. In patients, alterations of white matter are inversely correlated with blood levels of GSH precursor cysteine and could be prevented by the early administration of the antioxidant NAC.

of glia in normal brain functions and brain disorder pathology. A growing body of evidence shows that diversified populations of astrocytes, microglia, oligodendrocyte precursors and mature oligodendrocytes play a critical role in the regulation of synaptic functions, blood-brain barrier, immune response regulation, myelination and axonal conduction, and in the synthesis of the extracellular matrix, a key regulator of neural plasticity. Building on this evidence, exciting new findings are beginning to emerge, shedding light on glia abnormalities in schizophrenia and their impact these functions. This symposium aims to discuss and integrate the current state of knowledge on direct evidence for glial abnormalities in schizophrenia and their underlying mechanisms. Dr. Juliana Nascimiento will present novel findings on the effects of NMDAr antagonists and antipsychotics influence glial cell lines and 3D cultures as neurospheres and cerebral organoids. Results from these studies point to the central role of glycolysis, EIF2 signaling and translational machinery in oligodendrocytes and astrocytes. Dr. Paul Klauser will report on elegant investigations on the implication of developmental redox imbalance inducing oxidative stress leading to impairments of oligodendrocytes, myelin formation and eventually to the disruption of white fibers integrity and conductivity, especially in brain regions where the metabolic demand is high. In patients, alterations of white matter were found to be inversely correlated with blood levels of GSH precursor cysteine and could be prevented by the early administration of the antioxidant N-acetyl cysteine. Dr. Sabina Berretta will discuss recent findings on novel modalities of interaction between glial cells, extracellular matrix and neurons, postulated to affect synaptic structural plasticity and axonal conductance. A growing body of evidence from her group shows disruption of such interactions in schizophrenia, potentially contributing to synaptic pathology and impacting neural connectivity. Dr. Dost Ongur will build on previous work showing abnormal diffusion of neuron-specific metabolite NAA in frontal white matter in patients with chronic schizophrenia in the absence of abnormalities in the diffusion of non-specific metabolites Cr and Cho. State-of-theart recent studies on first episode psychosis patients and matched healthy controls show that NAA diffusion is normal in first episode patients but Cr and Cho diffusion is abnormal, suggesting that white matter abnormalities in non-neuronal elements in early phases of schizophrenia which are followed by neuronal damage in chronic disease. Background: A number of basic and translational studies have clearly indicated the vital role of glia in brain function and the pathophysiological mechanisms of neuropsychiatric disorders, including schizophrenia. The difficulty on studying the molecular basis of glial cells in vivo, led to the development of animal models, which are considered the gold standard to this type of understanding. However, the inherent difficulties in establishing these models for psychiatric disorders and the simplicity of in vitro models, especially given the recent advances in stem cell-based technologies have driven the development of sophisticated in vitro models, which may be attractive for studying the molecular basis of schizophrenia. Methods: Here, we report our investigations in terms of proteome while establishing protocols to generate human pluripotent stem cells-derived cerebral organoids as well as human cerebral organoids-derived astrocytes and oligodendrocytes.

Results:
The proteome of cerebral organoids show major proteins from neuronal cells as expected, but also several glial markers, supporting the notion that glial cells may be obtained out of these organoids. Besides, the proteome of three schizophrenia and three control organoids have been investigated. Proteins found are broadly distributed on functional activities such as cell growth and maintenance, energy metabolism and cell communication and signaling, and are correlated to cortical brain tissue. We also succeeded in isolating astrocytes out of cerebral organoids. These cells are under investigation in terms of molecular differences associated to schizophrenia. Discussion: The generation of brain organoids and isolation of astrocytes and eventually oligodendrocytes hold great potential for the investigation of the role of glia in schizophrenia, providing an useful approach to drug screening and disease modeling, as our results showed in schizophreniaand control-derived cells. Additionally, proteomics adds knowledge about information and connections being formed into these models.  (Cropley et al., 2017) cerebral anomalies of white matter diffusion properties (i.e. fractional anisotropy, FA) have been observed in the Australian Schizophrenia Research Bank (ASRB), one of the largest samples of patients with schizophrenia. From a topological perspective, widespread alterations of white matter tend to concentrate into hub regions that interconnect brain areas over long-distances in a so-called "rich-club" (van den Heuvel et al., 2013;Klauser et al., 2016) in which the metabolic demand is high and thus are most likely to suffer from oxidative stress. Evidence from human and animal models suggests that redox dysregulation leading to oxidative stress during neurodevelopment is implicated in schizophrenia pathogenesis (Steullet et al., 2017). At the cellular level, the triad composed of NMDAR hypofunction, neuroinflammation and dopamine dysregulation interacts with redox imbalance and leads to oxidative stress, affecting oligodendrocytes precursor cells (OPC) and parvalbumine interneurons (Steullet et al., 2016). However, the links between redox imbalance, oligodendrocytes and gross alterations of white matter integrity are largely unexplored. Under oxidative stress induced in vitro by impairing the synthesis of glutathione (GSH), the key player in antioxidant defense, OPC showed a decreased proliferation mediated by an upregulation of Fyn kinase activity. In the prefrontal cortex of a mouse model with impaired GSH synthesis, mature oligodendrocyte numbers as well as myelin markers were decreased at peripuberty (Monin et al., 2014). FA was also reduced in fornix-fimbria and anterior commissure, a change accompanied by a reduced conduction velocity (Corcoba et al., 2015). Methods: 49 patients with psychosis and 64 healthy controls were scanned with the same 3-Tesla scanner. The diffusion spectrum imaging (DSI) sequence included 128 diffusion-weighted images with a maximum b-value of 8000 s mm−2. White matter diffusion properties were estimated using generalized fractional anisotropy (gFA). Total blood cysteine (Cys, proteinbound form, free reduced and free oxidized form), the rate-limiting precursor of GSH, was measured by high performance liquid chromatography from plasma samples collected at the same time-point as MRI brain scans. Whole brain voxel-based analyses were performed using cluster-based nonparametric permutation testing on gFA maps. Cerebral levels of GSH were assessed by localized 1H-MRS measurements from a volume of interest in medial prefrontal cortex. Results: As previously described in ASRB, we observed widespread abnormalities of white matter in patients. Interestingly, the degree of white matter alterations (i.e. decreased gFA) patients could be predicted by the levels of blood cysteine, a precursor of GSH, strongly suggesting the important role played by oxidative stress in the pathophysiological mechanism. Also, we found that white matter alterations could be reversed by 6 months of addon treatment with the antioxidant and GSH precursor N-acetyl-cysteine (NAC). Most importantly, this improvement was positively correlated with an increase in prefrontal GSH levels. Discussion: We propose that developmental redox imbalance inducing oxidative stress may lead to impairments of oligodendrocytes, myelin formation and eventually to the disruption of fibers integrity and conductivity, especially in brain regions having high metabolic demand. In patients, alterations of white matter are inversely correlated with blood levels of GSH precursor cysteine and could be prevented by the early administration of the antioxidant NAC.

GLIA-EXTRACELLULAR MATRIX INTERACTIONS IN THE PATHOPHYSIOLOGY OF SCHIZOPHRENIA AND BIPOLAR DISORDER Sabina Berretta* ,1 , Gabriele Chelini 1 , Harry Pantazopoulos 1 1 Harvard Medical School, McLean Hospital
Background: Growing evidence from our group and others indicates that key neural functions, including regulation of synaptic plasticity and axonal guidance and connectivity, arise from interactions between glial cells, neurons, and the extracellular matrix. Several distinct populations of glial cells critically contribute to the composition of main components of the extracellular matrix (ECM), synthesizing them and secreting them into the extracellular space, where they become incorporated in organized ECM structures. The brain ECM, and chondroitin sulfate proteoglycans (CSPGs) in particular, play a key role in brain development and adult life, in turn regulating glial functions as well as synaptic plasticity and neural connectivity. We have previously shown that glial cells expressing CSPGs are altered in the amygdala and entorhinal cortex of people with schizophrenia (SZ) and bipolar disorder (BD). These changes are accompanied by marked decreases of perineuronal nets (PNNs), organized ECM structures unsheathing distinct neuronal populations. Recent and ongoing studies are focused on novel CSPG-enriched ECM structures, related to synaptic complexes and myelinated axons, their relationship to glial populations and their involvement in the pathophysiology of SZ and BD. Methods: Postmortem tissue samples from the amygdala, entorhinal cortex and thalamus from a well characterized cohort of healthy control, SZ and BD subjects were included in these studies. Multiplex immunofluorescence combined with quantitative microscopy was used to quantify glial cells and CSPGs, while electron microscopy on human and mouse tissue were used to investigate ultrastructural morphology.
Step-wise ANOVA analyses included several potential confounds such as exposure to pharmacological agents and substance abuse. Results: Our results show that at least two novel ECM structures are present in the human brain. The first, enriched in CSPGs bearing chondroitin sulfation in position 6 (CS-6), and named here 'CS-6 clusters' was found to be markedly decreased in the amygdala of people with SZ and BD. Electron microscopy studies show that CS-6 clusters are composed of astrocytes synthesizing and secreting CS-6 CSPGs in the vicinity of adjacent groups of dendrites, where it is incorporated into postsynaptic densities of dendritic spines. The second CSPG-enriched ECM structure, i.e. axonal coats, has been observed in the human thalamus to envelope distinct populations of axons, interweaving with myelin sheets. Its main CSPG components appear to be synthesized and secreted by oligodendrocytes precursor cells located in the vicinity of axon bundles. Preliminary results show abnormalities affecting both oligodendrocyte precursors and axonal coats in SZ. Discussion: In summary, our results show complex interactions between glial cells, neurons and ECM, potentially affecting synaptic functions and axonal conductance. Results in SZ and BD point to a profound disruption of these interactions in several brain regions.

McLean Hospital
Background: In previous work we used diffusion tensor spectroscopy (DTS) to identify abnormal diffusion of the neuron-specific metabolite NAA in frontal white matter in patients with chronic schizophrenia in the absence of abnormalities in the diffusion of cell-type non-specific metabolites Cr and Cho. Methods: DTS relies on the same principles as DTI, but the diffusion characteristics of metabolites are probed, instead of those of water. Since brain metabolites are concentrated in specific cellular and sub-cellular compartments, their diffusion reflects the local geometry of these compartments. We have implemented a DTS approach at a 4 Tesla Varian MRI scanner (described in Du et al 2013).

Results:
We have now collected similar data from first episode psychosis patients and matched healthy controls. We find that NAA diffusion is normal in the frontal PFC in first episode patients, but Cr and Cho diffusion is abnormal. Discussion: Taken together, our studies suggest white matter abnormalities in non-neuronal elements in early phases of schizophrenia which are followed by neuronal damage in chronic disease.

University of California, Los Angeles
Overall Abstract: Recently aerobic exercise training has begun to be systematically examined in randomized controlled trials (RCTs) in schizophrenia. This symposium will report and discuss the results of RCTs that examined the impact of aerobic exercise on physical health, cognition, and everyday functioning across first-episode and established illness phases of schizophrenia. In addition, data on neurotrophic and brain structural changes will be examined as promising mechanisms of action. Dr. Amal Abdel-Baki of the University of Montreal has focused on the physical health benefits of interval training in her RCT with first episode and multi-episode schizophrenia outpatients. She is demonstrating improved waist circumference, diastolic blood pressure, HDL cholesterol, and social functioning in first episode and multi-episode patients. Dr. David Kimhy of Icahn School of Medicine at Mount Sinai in New York has focused on the impact of aerobic exercise training on cardiovascular fitness, Brain-Derived Neurotrophic Factor (BDNF), cognition, and functional outcome in individuals with an established schizophrenic illness. He has demonstrated beneficial effects at each of these levels. Furthermore, relationships between fitness improvements and BDNF increases and the cognitive and functional gains suggest potential mechanisms of action. Dr. Berend Malchow of Ludwig Maximilian University of