Abstract
Early detection and intervention in schizophrenia requires mechanism-based biomarkers that capture neural circuitry dysfunction, allowing better patient stratification, monitoring of disease progression and treatment. In prefrontal cortex and blood of redox dysregulated mice (Gclm-KO ± GBR), oxidative stress induces miR-137 upregulation, leading to decreased COX6A2 and mitophagy markers (NIX, Fundc1, and LC3B) and to accumulation of damaged mitochondria, further exacerbating oxidative stress and parvalbumin interneurons (PVI) impairment. MitoQ, a mitochondria-targeted antioxidant, rescued all these processes. Translating to early psychosis patients (EPP), blood exosomal miR-137 increases and COX6A2 decreases, combined with mitophagy markers alterations, suggest that observations made centrally and peripherally in animal model were reflected in patients’ blood. Higher exosomal miR-137 and lower COX6A2 levels were associated with a reduction of ASSR gamma oscillations in EEG. As ASSR requires proper PVI-related networks, alterations in miR-137/COX6A2 plasma exosome levels may represent a proxy marker of PVI cortical microcircuit impairment. EPP can be stratified in two subgroups: (a) a patients’ group with mitochondrial dysfunction “Psy-D”, having high miR-137 and low COX6A2 levels in exosomes, and (b) a “Psy-ND” subgroup with no/low mitochondrial impairment, including patients having miR-137 and COX6A2 levels in the range of controls. Psy-D patients exhibited more impaired ASSR responses in association with worse psychopathological status, neurocognitive performance, and global and social functioning, suggesting that impairment of PVI mitochondria leads to more severe disease profiles. This stratification would allow, with high selectivity and specificity, the selection of patients for treatments targeting brain mitochondria dysregulation and capture the clinical and functional efficacy of future clinical trials.
Introduction
Among the most perdurable observations in schizophrenia (SZ) is a functional impairment of cortical parvalbumin interneurons (PVIs) [1]. This subset of GABAergic cells controls the precise synchronous activity of pyramidal neuron ensembles by virtue of their high firing rates within the cortical network and thereby modulates gamma-frequency rhythmicity and its cognitive correlates [1]. Defects in PVIs are reported to underlie the disruption of neural synchrony and subsequent fragmentation of cognitive-behavioral processes typically observed in SZ [2]. To sustain the large energy demand required to drive the high firing rate and rhythmicity of PVIs, these cells possess high mitochondrial content [3], which renders them more susceptible to oxidative stress (OxS) [4]. Mounting evidence suggests the presence of mitochondrial dysfunction in SZ, as indicated by the loss of electron transport chain activity and a shift toward anaerobic activity [5]. However, the precise molecular and cellular mechanisms that would allow a translation from animal models to patients are still missing. Moreover, mechanism-based blood biomarkers mediating specific behavioral and cognitive alterations are needed for patient stratification in terms of treatments and prevention.
Recent research points to OxS as one “central hub” in SZ pathophysiology, with converging evidence from environmental and genetic studies linking this process to PVI impairment [6]: indeed, in series of animal models carrying either genetic and/or environmental risks, we showed that PVI deficits were all accompanied by oxidative stress in prefrontal cortex. OxS may result from dysregulation of systems typically affected in SZ, including glutamatergic, dopaminergic, immune, and antioxidant signaling. As convergent end-point, redox dysregulation has successfully been targeted to protect PVIs with antioxidants/redox regulators across several animal models [6,7,8]. More importantly, the antioxidant and glutathione precursor NAC gave promising results in the improvement of cognition in early psychosis patients [8,9,10,11,12].
Notably, disturbances in endogenous antioxidants levels, particularly decreased glutathione (GSH) levels were reported in blood, CSF, and prefrontal cortex of SZ patients [8, 13,14,15]. Furthermore, variants of the genes for both modulatory (GCLM) and catalytic (GCLC) subunits of the key GSH-synthesizing enzyme glutamate-cysteine-ligase (GCL) were associated with SZ [16,17,18,19]. Accordingly, cultured skin fibroblasts of individuals expressing high-risk GAG-trinucleotide repeat (TNR) polymorphisms in the GCLC gene have decreased the GCLC protein expression, GCL activity, and GSH content. In addition, SZ associated polymorphisms and copy number variations of genes related directly to antioxidant system, lead to increased vulnerability to OxS [20,21,22,23]. However, these association studies based on relatively small number of subjects would deserve to be validated in larger cohorts.
To explore biological mechanisms underlying phenotypes such as cognitive deficits, transgenic Gclm-KO + GBR mice were developed as a model of a genetic risk (GSH displaying 70% GSH deficit) combined with environmental risk (insults, stress, trauma) at various timing during the development [12, 24, 25]. Indeed, beyond exhibiting cortical deficiencies in PVIs [26,27,28,29] and alterations of beta/gamma oscillations, Gclm-KO mice display elevated levels of the oxidative DNA damage marker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in the extranuclear compartment. This is exacerbated by additional insults in Gclm-KO mice challenged with GBR12909 (GBR), a specific dopamine reuptake inhibitor used to mimic the increased dopamine signaling known to generate reactive oxygen species (ROS) through its catabolism, as observed in SZ. The 8-oxo-dG extranuclear localization is suggestive of mitochondrial DNA oxidation [25], potentially related to impaired mitophagy inducing an accumulation of defective mitochondria [30]. Indeed, mitophagy, critical for basal mitochondrial turnover, is also known as a “cleaning system” and is induced as a stress-response mechanism to eliminate damaged mitochondria, thus avoiding cellular apoptosis [31]. Mitophagy processing is mediated by the autophagy receptors BCL2 Interacting Protein 3 Like (Nix/BNIP3L), BCL2 Interacting Protein3 (BNIP3), and FUN14 Domain Containing 1 (FUNDC1), which are responsible for delivery to autophagosomes by binding to Microtubule-associated protein1A/1B-light chain3 (LC3) [32]. In addition to its important role in the regulation of synaptic function, including synaptogenesis, spine development and morphology [33, 34],, miR-137, a 23-nt noncoding microRNA located on chromosome 1, has been reported to be critically involved in mitophagy [35]. Mitophagy is under the post-transcriptional control of miR-137. Indeed, miR-137 inhibits mitophagy by controlling Fundc1 and Nix expression [35]. MiR-137 expression is enriched in the mouse and human brain, with high expression in the cortex and hippocampus and low expression in the cerebellum and brain stem [34].
Here, we investigated the impact of redox dysregulation on mitophagy deficits in PVIs using the Gclm-KO model. The role of OxS was examined using a quantitative assessment of markers for OxS, PVIs, and mitophagy in the anterior cingulate cortex (ACC), a prefrontal area known to be affected and to display redox dysregulation in SZ [36]. We then investigated the effects of redox dysregulation on miR-137 expression given its involvement in mitophagy regulation [35] and robust evidence of genetic association with SZ [37]. We also investigated cytochrome c oxidase subunit VIa polypeptide2 (COX6A2), a subunit of cytochrome c oxidase complex IV (COX-IV), the terminal enzyme in the mitochondrial respiratory chain [38]. Indeed, altered function of COX, mainly related to dysregulation of mitochondrial gene expression, underlies mitochondrial dysfunction in brain tissue [39].
We next explored whether mitochondria-targeted antioxidant treatment with mitoquinone mesylate (MitoQ) can restore PVI integrity and mitophagy deficits. Finally, by adopting a reverse translational approach, we sought to validate our preclinical observations in a cohort of patients in the early phase of psychosis (EPP) to determine the potential mechanistic pathway through which disturbed mitophagy may affect PVI-mediated gamma oscillations as assessed by the auditory steady-state response (ASSR) in EEG, which are critically involved in cognitive performance [2, 40].
Materials, subjects, and methods
Preclinical, mice study
The tissue preparation, immunohistochemistry, imaging and image processing, microRNA in situ hybridization of transgenic mice lacking the glutamate-cysteine ligase modifier subunit (Gclm-KO; B6.129-Gclmtm1Tdal crossbred with C57BL/6J mice over more than 10 generations) [26] as well as the electron microscopy study and the quantification of plasmatic miR-137 were described in Supplementary materials. All animal procedures were conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Local Veterinary Office.
Reversal study with MitoQ
The selective mitochondrial antioxidant MitoQ has shown promise in preclinical models of disease related to mitochondrial dysfunction, such as the EAE model or multiple sclerosis, a condition in which some people may develop psychotic symptoms [41]. Half of the animals from each litter (4 females and 4 males/Group) received a single subcutaneous (s.c.) injection daily with GBR12909 (GBR) from P10 to P20. As a control, the other half of the animals received a single injection daily with phosphate buffered saline. Thereafter, from P21 to P40, half of the animals were treated with a MitoQ solution (500 nM, dissolved in water), and the other half were treated with water. The MitoQ solution was freshly prepared every other day and delivered through water bottles that were light-protected to prevent rapid oxidation. The mice were then sacrificed at P40.
Local reversal study with miR-137 inhibitor
miRNA solution
Two solutions of miRNA Inhibitor (miRCURY LNA miRNA custom Inhibitor in vivo large scale, Qiagen) were used in this study, one with our specific sentence of MIR-137 (CGTATTCTTAAGCAAT) and one with a scrambled sentence (as a sham). The final concentration of our solution was at 0.24 nmol/500 nl per injection.
miRNA injection
Mice 6–8 week old were anesthetized using Ketamine-Xylazine (83 and 3.5 mg/kg, respectively) and placed on a heating blanket to maintain the body temperature at 37 °C. The animal was head fixed on a stereotaxic apparatus (Kopf model 940, Tujunga, CA) to perform the surgery.
The bone was exposed at the desired position through a small skin incision and small craniotomies (<0.5-mm diameter) were performed above the site of injection at (anteroposterior (AP), mediolateral (ML), depth from cortical surface (DV), in stereotaxic coordinates from Bregma): 0.7; ±0.3; −1.2 to target the ACC. Each solution of miRNA were injected with a thin glass pipette (5-000-1001-X, Drummond Scientific, Broomall, PA) pulled on a vertical puller (Narishige PP-830, Tokyo, Japan).
At the end of the surgery, mice received a dose of analgesic (Buprenorphine, s.c. 0.1 mg/kg body weight). After 4 days, mice were injected i.p. with a lethal dose of penthobarbital and intracardial injection of about 50 ml of paraformaldehyde 4% was done to collect the organs. The brains were collected in PBS 0.1 M 30% sucrose overnight at 4 °C and sliced with a microtome (Microm HM440E, section thickness: 60 μm). The slices were disposed in 12-well plates filled with 0.1 M PB for immunohistochemistry.
Human clinical study
This study was carried out in accordance with the Declaration of Helsinki and was approved by the local Ethics Committee (Commission cantonale d’éthique de la recherche sur l’être humain (CER-VD)). Written documentation of informed consent for this study was obtained from each participant….
