METHODS FOR CLASSIFICATION AND TREATMENT OF PSYCHOTIC DISORDER SUBJECTS

Abstract

The present invention relates to the filed of diagnostic and/or prognostic and/or subject stratification biomarker assays for the prognosis and/or diagnosis and/or therapy of high risk early psychosis subjects, wherein psychotic disorder may include schizophrenia, bipolar disorder (manic depression), epilepsy, mood disorder, age-related disorders, or cognitive impairment, or another psychotic disorder. The expression markers used are miR-137 and COX6A2. The present invention also relates to the use of mitochondria-targeted antioxidant in the treatment of subjects classified as high-risk early psychosis subjects and to a kit comprising means for determining said markers.

Claims

1. A method for classifying a subject as high-risk psychotic disorder subject, the method comprising the steps of: (a) providing a biological sample obtained from a subject; (b) determining the expression level of miRNA-137, and the expression level of COX6A2; and (c) classifying the subject as high-risk psychotic disorder subject based on expression levels determined in step (b).

2. The method of claim 1, wherein the psychotic disorder is selected from the group containing schizophrenia; epilepsy; mood disorder; bipolar disorder; age-associated diseases; cognitive impairment; other psychiatric disorders.

3. The method of claim 2, wherein the psychotic disorder is schizophrenia, preferably the early stage schizophrenia, more preferably the early stage schizophrenia with cognitive impairment.

4. The method of claim 3, wherein the classification comprises differential classification between high-risk psychotic disorder subjects and low-risk psychotic disorder subjects.

5. The method of claim 1, wherein the biological sample comprises peripheral blood, plasma, serum, cerebrospinal fluid, blood-derived exosomes, plasma-derived exosomes, neural exosomes, cortical tissue, post-mortem brain tissue, fibroblast cell culture, induced pluripotent stem cell culture, derived neuronal precursor cell culture.

6. The method of claim 1, wherein the relative expression level of miRNA-137 normalised to reference genes is higher than a threshold value.

7. The method of claim 6, wherein the threshold value is 4.8 a.u, as normalized with respect to the average expression level of miR-16, snRNA-U1 and snRNA-U6.

8. The method of claim 6, wherein the threshold value corresponds to at least two-fold increase of miR-137 expression level as compared with the healthy subjects.

9. The method of claim 1, wherein the expression level of COX6A2 is lower than a threshold value.

10. The method of claim 9, wherein the threshold value is 1.2 ng/ml.

11. The method of claim 1, wherein expression level of miR-137 and expression level of COX6A2 are determined by an in vitro assay.

12. The method of claim 11, wherein the in vitro assay is selected from the group consisting of an immunoassay; an ELISA-based assay, an aptamer-based assay; an mRNA expression level assay; in situ hybridization assay; a proteomics-based assay; a PCR-based assay; a real time PCR-based assay, next generation sequencing; an electrochemistry-based assay; a lateral-flow assay; a nanobead-based assay; a microfluidics-based assay; and an oligonucleotide-templated reaction.

13. A method of treating a subject classified as high risk psychotic disorder subject, preferably a high-risk schizophrenia subject, wherein the method comprises administering a mitochondria-targeted antioxidant to the subject.

14. The method of claim 13, wherein the mitochondria-targeted antioxidant is selected from the group consisting of mitoquinone (MitoQ), 3-demethoxymitoquinone (DMMQ), 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1), SkQ3, coenzyme Q10 (CoQ10), methylene blue (MB).

15. A biomarker kit comprising reagents for determining miR-137 and COX6A2 expression levels as defined in claim 1, preferably wherein the COX6A2 expression level is determined as protein expression level.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0084] FIG. 1 represents the correlation between the expression level of miR137 and expression level of COX6A2 in differently classified groups of patients.

[0085] FIG. 2 shows performance of the use of miR-137 biomarker alone and in combination with COX6A2, NIX, LC3B, FUNDC1, MMP9 and sRAGE, presented as the Receiver Operating Characteristic (ROC) curves.

[0086] FIG. 3 shows a Table summarizing the data on early psychosis patients that have been included in the study to validate the biomarkers of this invention.

[0087] FIG. 4 illustrates the measurement of miR-137 expression level in subjects, and provides comparison data for early psychotic patient group and control group.

[0088] FIG. 5 shows a patient selection method using the two biomarkers miR-137 and COX6A2 in the population of patients classified based on the polymorphism of miR-137 gene.

[0089] FIG. 6 shows a patient selection method using the two biomarkers miR-137 and COX6A2 in the population of patients classified based on the clinical factors. Combined use of miR-137 and COX6A2 levels allows classification of subjects that are eligible for MitoQ treatment without miR-137 SNP rs1625579 genotype consideration. (A) Identification of high-risk psychosis subjects based on miR-137 expression levels. (B) Identification of high-risk psychosis subjects based on COX6A2 protein levels. (C) Identification of high-risk psychosis patients based on combined detection of miR-137 and COX6A2 levels.

[0090] FIG. 7 shows changes in expression levels of miR-137 as well as mitophagy markers NIX, FUNDC1 and LC3, upon inducing oxidative stress through knockout of Gclm gene (which mediates biosynthesis of glutathione) and treatment with compound GBR12909, and rescue of observed effect through treatment with MitoQ, a mitochondria-targeted antioxidant (as discussed in Examples 9 and 10).

[0091] FIG. 8 presents the comparison of clinical psychosis parameters between the group of subjects classified as high-risk psychotic disorder subject and healthy subjects, based on the methods disclosed herein. High risk psychotic disorder subjects exhibit more significant positive symptoms, lower functional outcome (GAF) and more severe cognitive impairment, which is measured by assessing processing speed, attention, vigilance, working memory and verbal learning).

[0092] The examples below present different embodiments of this invention and their application to practice.

[0093] Example 1 relates to the embodiments of this invention, wherein methods disclosed herein have been validated using a group of patients described below:

[0094] All the studies utilizing patient data or samples have been collected using the group of patient diagnosed as early psychotic disorder subjects based on clinical factors, whose population has been summarized in FIG. 3. The study population included early psychosis patients (EPP; n=138) and healthy controls (n=134), matched for gender and age. Patients were recruited from the Treatment and Early Intervention in Psychosis Program (TIPP, Lausanne University Hospital, Switzerland [1], which is a specialized 3-year program for the treatment of early psychosis patients. Inclusion criteria were: (i) individuals aged 18 to 35 years old; (ii) residence in Lausanne and surroundings areas; (iii) meeting threshold criteria for psychosis, as defined by the ‘Psychosis threshold’ subscale of the Comprehensive Assessment of At Risk Mental State (CAARMS) [2]; (iv) no more than 6 months of treatment with antipsychotic medication for psychosis; (v) no psychosis related to intoxication or organic brain disease; (vi) intelligence quotient ≥70; and (vii) ability to discern and to provide informed consent. The psychosis threshold and the diagnosis assessment resulted from an expert consensus including a senior psychiatrist, a psychologist and the case manager who closely followed-up the patient during the 3-year program. The duration of the illness considered the time elapsed from the psychosis threshold to the participation in the study and the diagnosis was based on DSM-IV criteria (American Psychiatric Association, 1994). Most of the patients (n=123) had antipsychotic medication (374.2±216.5 mg chlorpromazine equivalent dose (CPZ); It is noteworthy that patients who accepted to participate in the present study were representative of the entire clinical TIPP cohort. Healthy controls were recruited within same catchment area. They were assessed by the Diagnostic Interview for Genetic Studies (DIGS) in order to attest for the absence of any major psychiatric or substance use disorder. In addition, healthy controls who reported having a first-degree relative with psychotic disorder were excluded. Neurological disorders and severe head trauma were also exclusion criteria for all subjects.

[0095] Example 2 of this invention refers to an embodiment, wherein samples for determination of expression levels of biomarkers are isolated from exosomes, obtained from peripheral blood sample. For the isolation of exosomes from human plasma, 200-400 μl of plasma was separated by ultracentrifugation (Sorvall ultra Pro 80).

[0096] Example 3 relates to an embodiment, wherein expression level of circulating miR-137 is performed by using RT-PCR protocol, according to the following procedure:

[0097] miRNAs were extracted from exosome using miRNeasy Kit (Qiagen, Hilden, Germany). The protocol used was provided with the kit and described in “miRNeasy Handbook”. The quality of extracted miRNAs and their concentration were determined with a NanoDrop (ND-1000 spectrophotometer, Thermo Fisher Scientific, USA) by measuring the absorbance at 260 nm (A260) and 280 nm (A280). The A260/A280 ratio had to be −2.0 for pure miRNA (as for pure RNA).

[0098] For first-strand cDNA synthesis by reverse transcription, total purified miRNA samples were diluted to 5 ng/μ1. miRNA was reverse transcribed using the miRCURY LNA Universal cDNA Synthesis kit (Exiqon, Vedbaek, Denmark) according to the instructions enclosed. A mixture containing 4 μl of total miRNA, 2 μl of the Enzyme mix, and 4 μl of the 5×Reaction buffer concentrate was made up to 20 μl final volume with nuclease-free water. The cocktail was gently vortexed to thoroughly mix all reagents. Final solution was spun down and incubated for reverse transcription at 42° C. for 60 minutes, followed by 5 minutes at 95° C. The obtained cDNA templates were immediately cooled on ice and stored at 4° C.

[0099] For qPCR amplification the cDNA templates and Exiqon Master mix (Exiqon, Vedbaek, Denmark) were used, following the product user instructions. This procedure suggests the use of three replicas for each plasma sample and for each miRNA. For the amplification, final reaction volumes of 10 μl were prepared in the following proportions: 5 μl of master mix, 1 μl of LNA primer set (0.5 μl of each forward and reverse primers) and 4 μl of 1.25% solution of the cDNA template in nuclease free water. qPCR amplification was performed with a qPCR Detection System with the thermal cycling parameters: 10 minutes at 95° C., 50 cycles (10 sec. each) at 95° C., 60° C. for one minute. qPCR values (quantification cycle “Cq”) for studied miRNAs were normalized to three reference miRNAs (miR-Ref) miR-16, snRNA-U1 and snRNA-U6

[0100] Example 4 relates to an embodiment, wherein the plasmatic level of COX6A2 is determined by using the Western Blot technique, according to the following protocol:

[0101] Total proteins were loaded on 12.5% acrylamide gels for SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in Tris Buffer Saline with 2% non-fat dried milk and hybridized overnight at 4° C. with a primary antibody diluted in blocking buffer (anti-COX6A2, rabbit, dilution 1:500). Proteins on immunoblots were revealed using appropriate secondary antibody conjugated to Horseradish peroxidase (HRP) corresponding to the primary antibody (anti-rabbit). Images were acquired and quantified using Fusion Imaging System (Fusion Solo-S). The comparison between the samples that originate from early psychotic disorder subjects and the control group is shown in FIG. 4.

[0102] Example 5 relates to an embodiment of present invention, wherein the plasmatic expression level of COX6A2 is determined by using the ELISA (Enzyme-Linked Immunosorbent Assay) technique, according to the following protocol:

[0103] ELISA Kit for COX6A2 detection was used according the provided instructions (Human Cytochrome c oxidase subunit 6A2, mitochondrial (COX6A2) ELISA Kit). Briefly, the assay sample and buffer are incubated in a pre-coated plate, together with COX6A2-HRP conjugate for one hour. After the incubation and several washes the HRP substrate [TMB (3,3%5,5′-tetramethylbenzidine)] is added. The intensity of color is measured using a spectrophotometer Tecan (Vitaris AG). A standard curve is plotted relating the intensity of the color to the concentration of standards. This standard curve is used to define COX6A2 level in each sample.

[0104] Example 6 relates to the embodiment of this invention, wherein selection of subjects is performed based on the expression levels of miR-137 and COX6A2, and the successful classification is checked against available genetic data that predisposes subjects to be high-risk schizophrenia subjects (FIG. 5). First, in the step (A) the subjects were selected based on expression level of miR-137, higher than the threshold of 4.8 a.u.. In the second step, the expression level of COX6A2 was examined, and subjects with expression level lower than 1.2 ng/mL have been excluded from the subject group (B). It should be noted that the correlation between the genetic biomarkers and molecular biomarkers further makes plausible the application of both biomarkers in the clinic.

[0105] Example 7 relates to an embodiment of this invention as in Example 6, with the difference that biomarker classification is checked against available clinical data, that is, classification of patients as high-risk psychotic disorder subjects according to the patient selection of Example 1. Example 7 further illustrates how combined use of both biomarkers miR-137 and can be used to exclude healthy subjects. FIG. 6 presents the details of performed classification. The left panel shows classification of two groups of subjects—one group containing early psychosis subjects (left) and the second group containing the healthy subjects (control group)—according to the expression level of miR-137. Most of the early psychosis subject group (68%) is included based on the expression level of miR-137 higher than the threshold (4.8 a.u. as normalized to miR-16, snRNA-U1 and snRNA-U6 expression levels). At the same time, only 17% of healthy subjects can be excluded based on the miR-137 expression level threshold of 4.8 a.u.. The right panel of FIG. 6 presents further classification of patients based on the expression level of COX6A2. While most of the subjects in early psychotic subject group that were selected based on miR-137 expression level also fulfilled the COX6A2 expression level requirements (set us lower than 1.2 ng/mL), there was significantly less correlation between the two parameters in the control group. Hence, 25% of control subjects that fulfill the criterion of miR-137 expression higher than threshold could be excluded based on the expression level of COX6A2.

[0106] Example 8 relates to the study of Examples 6 and 7, wherein the correlation between the expression level of miR-137 and the expression level of COX6A2 has been studied for different groups of subject. FIG. 1 clearly shows the correlation between both biomarker expression level for the group of high risk early psychotic disorder subjects, in both classification cases, disregarding genetic factors (p=0.0062 and R=0.532) and taking them into account (p=0.0014, R=0.547). At the same time, no significant correlation was observed for the control subject group, in both classification cases, disregarding genetic factors (p=0.51, R=0.090) and taking them into account (p=0.72, R=0.049).

[0107] Example 9 relates to an animal model of oxidative stress. Oxidative stress elicits mitophagy in the anterior cingulated cortex (ACC) of Gclm-KO mice (transgenic mice carrying permanent defect in glutathione GSH synthesis). Pharmacological challenge of Gclm-KO mice with GBR12909 (Gclm-KO+GBR) is known to induce elevated levels of the oxidative DNA damage marker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in the ACC of these animals. Because extranuclear localization of 8-oxo-dG labeling was observed, it could be surmised that mitochondrial DNA (mtDNA) was affected. Developmental oxidative stress resulted in abnormal mitochondrial morphology in the ACC of Gclm-KO+GBR (P20) as opposed to wild type (WT) animals. Specifically, qualitative assessment of electron micrographs revealed increased numbers of mitochondria with darker and a globular shape suggestive of impaired mitophagy. Principal markers of mitophagy including NIX, Fundc1 and LC3B were found to be significantly decreased in the Gclm-KO+GBR compared to WT mice (see FIG. 7 for details). In comparison with WT mice (Gclm-WT+PBS, wherein PBS refers to phosphate-buffered saline, a common buffer used in these experiments as vehicle), levels of the mitophagy receptors NIX and FUNDC1 and of the autophagosome LC3B were reduced in the ACC of Gclm-KO mice (Gclm-KO+PBS, at P40). Additional oxidative insult with GBR12909 (administered between P10-P20) further decreased NIX, FUNDC1 and LC3B staining intensity in Gclm-KO+GBR mice). Taken together, these observations are consistent with an oxidative stress-induced mitophagy deficit in prefrontal parvalbumin interneurons.

[0108] It has been shown that miR-137 plays a functional role in modulating synaptic function and in regulating the expression of the NIX and FUNDC1 mitophagy receptors. Combined in situ hybridization and immunohistochemistry was therefore applied to quantify the expression levels of miR-137 within the ACC, showing that it was significantly increased in Gclm-KO mice (FIG. 7). Additional oxidative challenge with GBR (administered between P10-P20) in Gclm-KO mice induced a marked increase in the co-localization of miR-137 staining in PVIs within the ACC at P40, suggesting that the elevated expression of miR-137 observed in the ACC was localized to parvalbumin interneurons. Consistent with previous results, parvalbumin immunoreactive cell count and puncta intensity were decreased in Gclm-KO and were further depleted in Gclm-KO+GBR animals. These findings suggest that in animals with genetically induced redox dysregulation, which are challenged with an additional environmental insult (i.e., Gclm-KO+GBR) there is upregulation of miR-137, which leads to decreased mitophagy and a subsequent accumulation of damaged mitochondria that further exacerbates oxidative stress and parvalbumin impairment.

[0109] Example 10 relates to mouse model experiment which demonstrates that treatment with MitoQ rescues oxidative stress induced elevated level of miR-137 expression, mitophagy, and parvalbumin interneurons alterations. In view of the detrimental effects of oxidative stress on integrity of parvalbumin interneurons, miR-137 expression level as well as mitophagy during preweaning and pubertal stages of development, it was evaluated whether MitoQ treatment, a selective mitochondria targeted-antioxidant, could rescue these deficits in young KO mice. Treatment with MitoQ (P21-P40) in Gclm-KO+GBR mice normalized NIX, FUNDC1 and LC3B expression levels as well as miR-137 expression intensity, when compared to WT values (FIG. 7). Additionally, abnormalities in cell body numbers and staining intensity of parvalbumin interneuron processes in the ACC of Gclm-KO+GBR animals were similarly restored to WT levels following treatment with MitoQ. Thus, MitoQ can reverse the oxidative stress induced miR-137 overexpression, mitophagy and parvalbumin interneuron impairment, highlighting that the upregulation of miR-137 and subsequent mitophagy defect constitutes the molecular mechanism underlying the oxidative stress-induced parvalbumin interneuron impairment.

[0110] Example 11 relates to the study of correlation between the high risk psychotic disorder classification based on the measurement of the expression level of miR-137 and COX6A2, and clinical indicators of psychosis. As shown in FIG. 8, high risk psychotic disorder subjects exhibit more significant positive symptoms, lower functional outcome (GAF) and more severe cognitive impairment, which is measured by assessing processing speed, attention, vigilance, working memory and verbal learning).