Continuous home cage monitoring of activity and sleep in mice during repeated paroxetine treatment and discontinuation

Rationale: Non-invasive home cage monitoring is emerging as a valuable tool to assess the effects of experimental interventions on mouse behaviour. A field in which these techniques may prove useful is the study of repeated selective serotonin reuptake inhibitor (SSRI) treatment and discontinuation. SSRI discontinuation syndrome is an under-researched condition that includes the emergence of sleep disturbances following treatment cessation. Objectives: We used passive infrared (PIR) monitoring to investigate changes in activity, sleep, and circadian rhythms during repeated treatment with the SSRI paroxetine and its discontinuation in mice. Methods: Male mice received paroxetine (10 mg/kg/day, s.c.) for 12 days, then were swapped to saline injections for a 13 day discontinuation period and compared to mice that received saline injections throughout. Mice were continuously tracked using the Continuous Open Mouse Phenotyping of Activity and Sleep Status (COMPASS) system. Results Repeated paroxetine treatment reduced activity and increased behaviourally-defined sleep in the dark phase. These effects recovered to saline-control levels within 24 h of paroxetine cessation, yet there was also evidence of a lengthening of sleep bouts in the dark phase for up to a week following discontinuation. Conclusions: This study provides the first example of how continuous non-invasive home cage monitoring can be used to detect objective behavioural changes in activity and sleep during and after drug treatment in mice. These data suggest that effects of paroxetine administration reversed soon after its discontinuation but identified an emergent change in sleep bout duration, which could be used as a biomarker in future preclinical studies to prevent or minimise SSRI discontinuation symptoms

Neuroinflammation in the dorsolateral prefrontal cortex in elderly chronic schizophrenia

Cognitive deterioration and symptom progression occur in schizophrenia over the course of the disorder. A dysfunction of the immune system/neuroinflammatory pathways has been linked to schizophrenia (SZ). These altered processes in the dorsolateral prefrontal cortex (DLPFC) could contribute to the worsening of the deficits. However, limited studies are available in this brain region in elderly population with long-term treatments. In this study, we explore the possible deregulation of 21 key genes involved in immune homeostasis, including pro- and anti- inflammatory cytokines, cytokine modulators (toll-like receptors, colony-stimulating factors, and members of the complement system) and microglial and astroglial markers in the DLPFC in elderly chronic schizophrenia. We used quantitative real-time reverse transcriptase poly- merase chain reaction (RT-PCR) on extracts from postmortem DLPFC of elderly subjects with chronic SZ ( n = 14) compared to healthy control individuals ( n = 14). We report that CSF1R, TLR4, IL6, TNF α, TNFRSF1A, IL10, IL10RA, IL10RB, and CD68 were down-regulated in elderly SZ subjects. Moreover, we found that the expression levels of all the altered inflammatory genes in SZ correlated with the microglial marker CD68 . However, no associations were found with the astroglial marker GFAP . This study reveals a decrease in the gene expression of cytokines and immune response/inflammation mediators in the DLPFC of elderly subjects with chronic schizophrenia, supporting the idea of a dysfunction of these processes in aged patients and its possible relationship with active microglia abundance. These findings include elements that might contribute to the cognitive decline and symptom progression linked to DLPFC functioning at advanced stages of the disease

Rebound activation of 5-HT neurons following SSRI discontinuation

Cessation of therapy with a selective serotonin (5-HT) reuptake inhibitor (SSRI) is often associated with an early onset and disabling discontinuation syndrome, the mechanism of which is surprisingly little investigated. Here we determined the effect on 5-HT neurochemistry of discontinuation from the SSRI paroxetine. Paroxetine was administered repeatedly to mice (once daily, 12 days versus saline controls) and then either continued or discontinued for up to 5 days. Whereas brain tissue levels of 5-HT and/or its metabolite 5-HIAA tended to decrease during continuous paroxetine, levels increased above controls after discontinuation, notably in hippocampus. In microdialysis experiments continuous paroxetine elevated hippocampal extracellular 5-HT and this effect fell to saline control levels on discontinuation. However, depolarisation (high potassium)-evoked 5-HT release was reduced by continuous paroxetine but increased above controls post-discontinuation. Extracellular hippocampal 5-HIAA also decreased during continuous paroxetine and increased above controls post-discontinuation. Next, immunohistochemistry experiments found that paroxetine discontinuation increased c-Fos expression in midbrain 5-HT (TPH2 positive) neurons, adding further evidence for a hyperexcitable 5-HT system. The latter effect was recapitulated by 5-HT1A receptor antagonist administration although gene expression analysis could not confirm altered expression of 5-HT1A autoreceptors following paroxetine discontinuation. Finally, in behavioural experiments paroxetine discontinuation increased anxiety-like behaviour, which partially correlated in time with the measures of increased 5-HT function. In summary, this study reports evidence that, across a range of experiments, SSRI discontinuation triggers a rebound activation of 5-HT neurons. This effect is reminiscent of neural changes associated with various psychotropic drug withdrawal states, suggesting a common unifying mechanism

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

Phosphorylation of transcription factor specificity protein 4 is increased in peripheral blood mononuclear cells of first-episode psychosis

BACKGROUND: Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation. METHODS: A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons. RESULTS: We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons. CONCLUSIONS: The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may be regulated by lithium treatment in first-episode psychosis

Altered CSNK1E, FABP4 and NEFH protein levels in the dorsolateral prefrontal cortex in schizophrenia

Schizophrenia constitutes a complex disease. Negative and cognitive symptoms are enduring and debilitating components of the disorder, highly associated to disability and burden. Disrupted neurotransmission circuits in dorsolateral prefrontal cortex (DLPFC) have been related to these symptoms. To identify candidates altered in schizophrenia, we performed a pilot proteomic analysis on postmortem human DLPFC 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). We quantified 1315 proteins with two or more unique peptides, 116 of which showed altered changes. Of these altered proteins, we selected four with potential roles on cell signaling, neuronal development and synapse functioning for further validation: casein kinase I isoform epsilon (CSNK1E), fatty acid-binding protein 4 (FABP4), neurofilament triplet H protein (NEFH), and retinal dehydrogenase 1 (ALDH1A1). Immunoblot validation confirmed our proteomic findings of these proteins being decreased in abundance in the schizophrenia samples. Additionally, we conducted immunoblot validation of these candidates on an independent sample cohort comprising 23 patients with chronic schizophrenia and 23 matched controls. In this second cohort, CSNK1E, FABP4 and NEFH were reduced in the schizophrenia group while ALDH1A1 did not significantly change. This study provides evidence indicating these proteins are decreased in schizophrenia: CSNK1E, involved in circadian molecular clock signaling, FABP4 with possible implication in synapse functioning, and NEFH, important for cytoarchitecture organization. Hence, these findings suggest the possible implication of these proteins in the cognitive and/or negative symptoms in schizophrenia

SP Transcription factors in psychotic disorders

[eng] Psychotic disorders including bipolar disorder and schizophrenia are a leading cause of disability across the world but the underlying pathophysiological mechanisms remain poorly understood. Available treatments are inadequate for some sets of symptoms as is the case for negative symptoms in schizophrenia. Alterations in brain connectivity, synaptic plasticity, N-methyl D aspartate receptor (NMDAR) signalling and calcium homeostasis have been suggested to contribute to these disorders. However, the particular transcriptional programmes altered in these disorders are not fully characterised. Previous data suggested that the transcription factors specificity protein 4 (SP4) and SP1 may be involved in the pathophysiology of psychotic disorders. We hypothesized that the expression and/or function of SP4 and SP1 may be altered in psychotic disorders through the regulation of transcriptional programmes involved in neuronal patterning, synaptic plasticity and glutamate signalling. In this doctoral Thesis we aimed to characterise the contribution of SP4 and SP1 transcription factors to the pathophysiology of psychotic disorders. By using real time quantitative RT-PCR and/or immunoblot techniques, we analysed the expression of SP factors, of SP4 S770 phosphorylation and/or of selected SP-regulated gene targets in at least one of the following substrates: (i) rat cerebellar granule neurons (CGNs), (ii) the postmortem brains of bipolar disorder, schizophrenia and control subjects, (iii) peripheral mononuclear blood cells (PMBC) of first-episode psychosis, and (iv) the rodent hippocampus after NMDAR blockade and antipsychotic treatment. We found that membrane depolarisation regulates SP4 protein levels in CGNs by preventing SP4 degradation via the ubiquitin-proteasoma pathway and that lithium prevents SP4 degradation and increases SP1 gene expression in non-depolarising conditions. In postmortem human tissue, we found a reduction in protein but not mRNA expression of SP4 and SP1 in the cerebellum in subjects with bipolar disorder and in subjects with more severe negative symptoms in schizophrenia. We have also found reduced expression of protein and mRNA levels of SP4 in the prefrontal cortex in bipolar disorder and of SP1 in the same region in schizophrenia, suggesting a disorder-specific regulation in this area. In contrast, both SP4 and SP1 protein and mRNA levels were increased in the hippocampus in schizophrenia. Consistent with this, we also observed an increase of SP1 and SP4 protein levels in the hippocampus of a mouse model of psychosis, but not in the hippocampus of a rat model of chronic antipsychotic treatment, suggesting that this upregulation may be present from the early stages of psychosis. We further characterised the phosphorylation of SP4 at serine 770 (S770), which is regulated by membrane depolarisation and NMDAR activity. We found an increase of SP4 S770 phosphorylation in conditions where SP4 protein levels are reduced, namely in the cerebellum of bipolar disorder and of schizophrenia patients with more severe negative symptoms, as well as in PMBC in first-episode psychotic patients. These results suggest that an imbalance in SP4 abundance may be regulated by NMDAR-dependent SP4 phosphorylation in the brain. Moreover, we found that reduced expression of NR2A and DRD2 in the cerebellum of schizophrenia patients correlated with more severe negative symptoms and SP protein levels. Additionally, we show here evidence for an imbalance in the SP4-NWK2-NR1 pathway in the cerebellum of patients with bipolar disorder. This pathway is involved in NR1 subunit availability on the cell surface, suggesting that SP4 could contribute to altered NR1 receptor trafficking in psychotic disorders. Together, the results presented in this Thesis suggest an imbalance in SP4 and SP1 transcription factors in the brains of patients with bipolar disorder and schizophrenia that may contribute to alterations in NMDAR receptor signalling and thereby to the impaired synaptic plasticity and altered brain connectivity observed in psychotic disorders

Increased SP4 and SP1 transcription factor expression in the postmortem hippocampus of chronic schizophrenia

Altered levels of transcription factor specificity protein 4 (SP4) and 1 (SP1) in the cerebellum, prefrontal cortex and/or lymphocytes have been reported in severe psychiatric disorders, including early psychosis, bipolar disorder, and chronic schizophrenia subjects who have undergone long-term antipsychotic treatments. SP4 transgenic mice show altered hippocampal-dependent psychotic-like behaviours and altered development of hippocampal dentate gyrus. Moreover, NMDAR activity regulates SP4 function. The aim of this study was to investigate SP4 and SP1 expression levels in the hippocampus in schizophrenia, and the possible effect of antipsychotics and NMDAR blockade on SP protein levels in rodent hippocampus. We analysed SP4 and SP1 expression levels in the postmortem hippocampus of chronic schizophrenia (n=14) and control (n=11) subjects by immunoblot and quantitative RT-PCR. We tested the effect of NMDAR blockade on SP factors in the hippocampus of mouse treated with an acute dose ofMK801. We also investigated the effect of subacute treatments with haloperidol and clozapine on SP protein levels in the rat hippocampus. We report that SP4 protein and both SP4 and SP1 mRNA expression levels are significantly increased in the hippocampus in chronic schizophrenia. Likewise, acute treatment with MK801 increased both SP4 and SP1 protein levels in mouse hippocampus. In contrast, subacute treatment with haloperidol and clozapine did not significantly alter SP protein levels in rat hippocampus. These results suggest that SP4 and SP1 upregulation may be part of the mechanisms deregulated downstream of glutamate signalling pathways in schizophrenia and might be contributing to the hippocampal-dependent cognitive deficits of the disorder.Peer Reviewe

Phosphorylation of transcription factor specificity protein 4 is increased in peripheral blood mononuclear cells of first-episode psychosis

Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation.A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons.We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons.The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may be regulated by lithium treatment in first-episode psychosis

The glial phosphorylase of glycogen isoform is reduced in the dorsolateral prefrontal cortex in chronic schizophrenia

Reduced glutamatergic activity and energy metabolism in the dorsolateral prefrontal cortex (DLPFC) have been described in schizophrenia. Glycogenolysis in astrocytes is responsible for providing neurons with lactate as a transient energy supply helping to couple glutamatergic neurotransmission and glucose utilization in the brain. This mechanism could be disrupted in schizophrenia. The aim of this study was to explore whether the protein levels of the astrocyte isoform of glycogen phosphorylase (PYGM), key enzyme of glycogenolysis, and the isoform A of Ras-related C3 botulinum toxin substrate 1 (RAC1), a kinase that regulates PYGM activity, are altered in the postmortem DLPFC of chronic schizophrenia patients (n = 23) and matched controls (n = 23). We also aimed to test NMDAR blockade effect on these proteins in the mouse cortex and cortical astrocytes and antipsychotic treatments in rats. Here we report a reduction in PYGM and RAC1 protein levels in the DLPFC in schizophrenia. We found that treatment with the NMDAR antagonist dizocilpine in mice as a model of psychosis increased PYGM and reduced RAC1 protein levels. The same result was observed in rat cortical astroglial-enriched cultures. 21-day haloperidol treatment increased PYGM levels in rats. These results show that PYGM and RAC1 are altered in the DLPFC in chronic schizophrenia and are controlled by NMDA signalling in the rodent cortex and cortical astrocytes suggesting an altered NMDA-dependent glycogenolysis in astrocytes in schizophrenia. Together, this study provides evidence of a NMDA-dependent transient local energy deficit in neuron-glia crosstalk in schizophrenia, contributing to energy deficits of the disorder.These studies were supported by Predoctoral Fellowship Program from ISCIII (PFIS) FI10/00177 to R.P. and the Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) (CB/07/09/0028) to B.R. Financial support of this work comes from Marie Curie Program IRG RTD REG/T.2 (2007)D/530573 to B.R. and Plan Nacional de Investigación BFU2008-01103 (MCINN) to B.R.Peer Reviewe