Inhibition of Prolyl Oligopeptidase Restores Prohibitin 2 Levels in Psychosis Models: Relationship to Cognitive Deficits in Schizophrenia

Cognitive impairment represents one of the core features of schizophrenia. Prolyl Oligopeptidase (POP) inhibition is an emerging strategy for compensating cognitive deficits in hypoglutamatergic states such as schizophrenia, although little is known about how POP inhibitors exert their pharmacological activity. The mitochondrial and nuclear protein Prohibitin 2 (PHB2) could be dysregulated in schizophrenia. However, altered PHB2 levels in schizophrenia linked to N-methyl-D-aspartate receptor (NMDAR) activity and cognitive deficits are still unknown. To shed light on this, we measured the PHB2 levels by immunoblot in a postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects, in the frontal pole of mice treated with the NMDAR antagonists phencyclidine and dizocilpine, and in rat cortical astrocytes and neurons treated with dizocilpine. Mice and cells were treated in combination with the POP inhibitor IPR19. The PHB2 levels were also analyzed by immunocytochemistry in rat neurons. The PHB2 levels increased in DLPFC in cases of chronic schizophrenia and were associated with cognitive impairments. NMDAR antagonists increased PHB2 levels in the frontal pole of mice and in rat astrocytes and neurons. High levels of PHB2 were found in the nucleus and cytoplasm of neurons upon NMDAR inhibition. IPR19 restored PHB2 levels in the acute NMDAR inhibition. These results show that IPR19 restores the upregulation of PHB2 in an acute NMDAR hypoactivity stage suggesting that the modulation of PHB2 could compensate NMDAR-dependent cognitive impairments in schizophrenia.This research was funded by a Miguel Servet grant, MS16/00153-CP16/00153 to BR, financed and integrated into the National R+D+I and funded by the Instituto de Salud Carlos III (ISCIII, Spanish Ministry of Health)—General Branch Evaluation and Promotion of Health Research—and the European Regional Development Fund (ERDF). This work was also supported by ISCIII PI18/00213 to BR, the Predoctoral Fellowship Program from the ISCIII (PFIS) FI19/00080 to E.V, FPU fellowship from the Spanish Ministry of Education, Culture, and Sports FPU17/06000 to E.E., the CONICYT-Doctorado Becas Chile 2015, 72160426 to AV, and the CIBERSAM (Spanish Ministry of Economy, Industry, and Competitiveness, Institute of Health Carlos III). CIBERSAM will be encharged to fund open access publication fees

Analysis of Molecular Networks in the Cerebellum in Chronic Schizophrenia: Modulation by Early Postnatal Life Stressors in Murine Models

Despite the growing importance of the cerebellum as a region highly vulnerable to accumulating molecular errors in schizophrenia, limited information is available regarding altered molecular networks with potential therapeutic targets. To identify altered networks, we conducted one-shot liquid chromatography–tandem mass spectrometry in postmortem cerebellar cortex in schizophrenia and healthy individuals followed by bioinformatic analysis (PXD024937 identifier in ProteomeXchange repository). A total of 108 up-regulated proteins were enriched in stress-related proteins, half of which were also enriched in axonal cytoskeletal organization and vesicle-mediated transport. A total of 142 down-regulated proteins showed an enrichment in proteins involved in mitochondrial disease, most of which were also enriched in energy-related biological functions. Network analysis identified a mixed module of mainly axonal-related pathways for up-regulated proteins with a high number of interactions for stress-related proteins. Energy metabolism and neutrophil degranulation modules were found for down-regulated proteins. Further, two double-hit postnatal stress murine models based on maternal deprivation combined with social isolation or chronic restraint stress were used to investigate the most robust candidates of generated networks. CLASP1 from the axonal module in the model of maternal deprivation was combined with social isolation, while YWHAZ was not altered in either model. METTL7A from the degranulation pathway was reduced in both models and was identified as altered also in previous gene expression studies, while NDUFB9 from the energy network was reduced only in the model of maternal deprivation combined with social isolation. This work provides altered stress- and mitochondrial disease-related proteins involved in energy, immune and axonal networks in the cerebellum in schizophrenia as possible novel targets for therapeutic interventions and suggests that METTL7A is a possible relevant altered stress-related protein in this context

Proteomic profile in postmortem brain in chronic schizopfrenia

[eng] Schizophrenia is a complex psychiatric disorder involving dysregulation of multiple pathways. In the last decades, the cortico-cerebellar-thalamo-cortical circuit has been proposed to play a key role in cognitive impairments in schizophrenia. The cerebellum is a brain area that forms part of this circuit that modulates synaptic responses of cortical regions, it has been proposed to play an important role in schizophrenia pathophysiology. Furthermore, the cerebellum, in connection with the dorsolateral prefrontal cortex, is involved in executive and working memory function. Thus, we hypothesized that the altered proteomic profile in the brain participates in the molecular network dysfunction in chronic schizophrenia. In this doctoral thesis we aimed to compare the proteomic profile in the postmortem cerebellar cortex and the prefrontal cortex of individuals with chronic schizophrenia by using (i) mass spectrometry, (ii) bioinformatic analyses to identify altered molecular networks, (iii) two double-hit schizophrenia murine models induced by maternal deprivation combined with an additional stressor for the hit proteins from the altered network in the cerebellum, (iv) immunohistochemistry techniques and (v) 3D projection analysis for unexplored candidate hits in human cerebellum. First, we performed a pilot proteomic analysis on postmortem human cerebellar tissue from patients with schizophrenia (n=4) and control (n=4) subjects in a pool design using differential isotope peptide labelling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Our results showed 1412 quantified proteins and 99 significantly altered proteins. 11 candidate proteins were selected as the most robust candidates from the enriched biological functions comprising cell communication/signal transduction in schizophrenia. In this study we report 68 new proteins altered in schizophrenia. In our individual proteomic study of the cerebellum, we analysed 12 cerebellar samples of subjects with schizophrenia and 14 healthy control individuals using one-shot liquid- tandem mass spectrometry. We identified a proteomic signature composed of 2578 identified proteins and 1474 quantified proteins with 250 significantly dysregulated proteins. This study reported 167 new proteins that are altered in the cerebellum in schizophrenia. Moreover, our study allowed us to identify the proteome profile and molecular networks altered in the cerebellum in chronic schizophrenia. Hierarchical clustering allowed schizophrenia subjects to be segregated from controls. Furthermore, our analyses showed that the 250 altered proteins could be under the transcriptional control of only11 transcription factors. The pathways regulated by these 11 transcription factors were related to transport, signalling, inflammation, and apoptosis. Our network generation analysis in the cerebellum showed two well-defined networks. The network generated from the up-regulated proteins showed a mixed module with enriched pathway interactions. This module consisted of proteins from vesicle-mediated transport and axon guidance pathways. In the axon guidance pathway, CLASP1 was a hit protein that we further studied in two independent double-hit murine models for schizophrenia. Our results showed CLASP1 to be down-regulated in the murine model of maternal deprivation combined with social isolation, while our proteomic study showed it to be up- regulated. The second network was formed by two modules generated from proteins that were found to be down-regulated in the cerebellum: an energy module and a neutrophil degranulation module. The energy module was well defined in our analysis, with NDUFB9 found to be a down-regulated hit protein in our proteomic study and in the double-hit murine model of maternal deprivation combined with social isolation. The second module was neutrophil degranulation. METTL7A was found to be a hit protein in this pathway. In our study, we found . and METTL7A was found to be consistently down-regulated both in schizophrenia subjects and murine models for schizophrenia. We demonstrated for the first time the expression of METTL7A in Bergmann glia cells in the human cerebellum. We also detected METTL7A in one of its canonical localizations, in lipid droplets in white matter cells. The prefrontal cortex analysis revealed 4407 identified proteins, 1989 quantified proteins and 43 significantly altered proteins. The enriched pathways were mainly related to the immune system. The network generated from the enriched pathway showed a mixed module with interactions between MHC class II antigen presentation, membrane trafficking, Golgi-to-ER retrograde transport, Nef-mediated CD8 down-regulation and the immune system. Together, the results presented in this Thesis suggest an imbalance in the immune system in two brain regions, the cerebellum and the prefrontal cortex. This suggests that the nervous system could be susceptible to an imbalance in the immune system in schizophrenia subjects. Moreover, the vesicle-mediated transport pathway was found to be altered in both the cerebellum and the prefrontal cortex, two brain areas that participate in the cortico-cerebellar thalamo-cortical circuit. This result suggests a possible alteration in synaptic efficacy and communication between these areas in schizophrenia

Analysis of Molecular Networks in the Cerebellum in Chronic Schizophrenia: Modulation by Early Postnatal Life Stressors in Murine Models

Despite the growing importance of the cerebellum as a region highly vulnerable to accumulating molecular errors in schizophrenia, limited information is available regarding altered molecular networks with potential therapeutic targets. To identify altered networks, we conducted one-shot liquid chromatography–tandem mass spectrometry in postmortem cerebellar cortex in schizophrenia and healthy individuals followed by bioinformatic analysis (PXD024937 identifier in ProteomeXchange repository). A total of 108 up-regulated proteins were enriched in stress-related proteins, half of which were also enriched in axonal cytoskeletal organization and vesicle-mediated transport. A total of 142 down-regulated proteins showed an enrichment in proteins involved in mitochondrial disease, most of which were also enriched in energy-related biological functions. Network analysis identified a mixed module of mainly axonal-related pathways for up-regulated proteins with a high number of interactions for stress-related proteins. Energy metabolism and neutrophil degranulation modules were found for down-regulated proteins. Further, two double-hit postnatal stress murine models based on maternal deprivation combined with social isolation or chronic restraint stress were used to investigate the most robust candidates of generated networks. CLASP1 from the axonal module in the model of maternal deprivation was combined with social isolation, while YWHAZ was not altered in either model. METTL7A from the degranulation pathway was reduced in both models and was identified as altered also in previous gene expression studies, while NDUFB9 from the energy network was reduced only in the model of maternal deprivation combined with social isolation. This work provides altered stress- and mitochondrial disease-related proteins involved in energy, immune and axonal networks in the cerebellum in schizophrenia as possible novel targets for therapeutic interventions and suggests that METTL7A is a possible relevant altered stress-related protein in this context

Analysis of networks in the dorsolateral prefrontal cortex in chronic schizophrenia : Relevance of altered immune response

The dorsolateral prefrontal cortex (DLPFC) has a crucial role in cognitive functioning and negative symptoms in schizophrenia. However, limited information of altered protein networks is available in this region in schizophrenia. We performed a proteomic analysis using single-shot liquid chromatography-tandem mass spectrometry of grey matter of postmortem DLPFC in chronic schizophrenia subjects (n = 20) and unaffected subjects (n = 20) followed by bioinformatic analysis to identify altered protein networks in schizophrenia (PXD024939 identifier in ProteomeXchange repository). Our results displayed a proteome profile in the DLPFC of 1989 proteins. 43 proteins were found significantly altered in schizophrenia. Analysis of this panel showed an enrichment of biological processes implicated in vesicle-mediated transport, processing and antigen presentation via MHC class II, intracellular transport and selenium metabolism. The enriched identified pathways were MHC class II antigen presentation, vesicle-mediated transport, Golgi ER retrograde transport, Nef mediated CD8 downregulation and the immune system. All these enriched categories were found to be downregulated. Furthermore, our network analyses showed crosstalk between proteins involved in MHC class II antigen presentation, membrane trafficking, Golgi-to-ER retrograde transport, Nef-mediated CD8 downregulation and the immune system with only one module built by 13 proteins. RAB7A showed eight interactions with proteins of all these pathways. Our results provide an altered molecular network involved in immune response in the DLPFC in schizophrenia with a central role of RAB7A. These results suggest that RAB7A or other proteins of this network could be potential targets for novel pharmacological strategies in schizophrenia for improving cognitive and negative symptoms

A Novel Localization of METTL7A in Bergmann Glial Cells in Human Cerebellum

Methyltransferase-like protein 7A (METTL7A) is a member of the METTL family of methyltransferases.Little information is available regarding the cellular expression of METTL7A in the brain. METTL7A is commonly located in the endoplasmic reticulum and to a lesser extent, in the lipid droplets of some cells. Several studies have reported altered protein and RNA levels in different brain areas in schizophrenia. One of these studies found reduced protein levels of METTL7A in the cerebellar cortex in schizophrenia and stress murine models. Since there is limited information in the literature about METTL7A, we characterized its cellular and subcellular localizations in the human cerebellum using immunohistochemical analysis with laser confocal microscopy. Our study reveals a novel METTL7A localization in GFAP-positive cells, with higher expression in the end-feet of the Bergmann glia, which participate in the cerebrospinal fluid-brain parenchyma barrier. Further 3D reconstruction image analysis showed that METTL7A was expressed in the contacts between the Bergmann glia and Purkinje neurons. METTL7A was also detected in lipid droplets in some cells in the white matter. The localization of METTL7A in the human cerebellar glia limitans could suggest a putative role in maintaining the cerebellar parenchyma homeostasis and in the regulation of internal cerebellar circuits by modulating the synaptic activity of Purkinje neurons