Cell biomarker and drug screening for neuronal disorders

Abstract

There is provided a method of identifying a biomarker or group of biomarker for a disorder. The cells obtained from the individual are subjected to a perturbation that destabilizes the homeostasis of the cells. The response of the cells to the perturbation is analyzed by imaging. The biomarker or group of biomarkers for the disorder is identified by imaging the response of the cells to the perturbation.

Description

CELL BIOMARKER AND DRUG SCREENING FOR NEURONAL DISORDERSTECHNICAL FIELD

[0001] This disclosure relates to the field of cell biomarkers and methods of identifying cell biomarkers of disease and determining compounds for treating disease, particularly there is provided a cell biomarker for neuronal disorders and more particularly a cell biomarker for bipolar disorder.BACKGROUND OF THE ART

[0002] Currently, the first steps in identifying new therapeutic compounds involve two in vitro screening approaches: the first is based on known target (Target-Drug-Discovery, TDD) and the second is used without known target, it leverages the cellular phenotype defined as pathological (Phenotype Drug Discovery, PDD) to identify promising therapeutic compounds and subsequently biomarkers of the concerned disease.

[0003] In the TDD approach, since the cellular target is known (e.g. a receptor, transporter, etc.), therapeutics screening can be carried out on cells from the patient's diseased tissue or organ, or on cells into which the target has been transfected. This type of screening is used to identify molecules that are effective as agonists, antagonists or modulators of the known target. The promising molecules identified therefrom can then move on to the next stage in their validation pathway, which includes cell toxicity tests, etc. Nevertheless, the TDD approach requires a good knowledge of the cellular target, which is information that can be very difficult to obtain. Furthermore, this approach does not consider that several cellular targets may be concomitantly involved in a pathology.

[0004] In the second approach, i.e., PDD, where no specific cellular target is identified, the screening is carried out on a cellular phenotype defined as pathological, i.e., on cells originating from the part of the organ or tissue affected by the disease. In this case, the objective is to identify molecules which, when applied to these diseased cells, are capable of restoring them to a healthy phenotype. Such phenotypic restoration is validated by monitoring the evolution of various parameters or cellular behaviours (morphological or dynamic parameters, restoration of an altered cellular function, etc.), those which were used to define the phenotype as pathological versus healthy. The PDD approach therefore requires that the phenotype be well pronounced, and therefore that the patient's disease be in an advanced clinical condition.

[0005] These two methods, however, generally rely on having access to a tissue biopsy from the patient. As mentioned above, this is particularly problematic for neuropsychiatry disorders, where prevention and early identification (i.e. , early diagnosis) are key.

[0006] The aspect of prevention is particularly important for neuropsychiatric diseases with neurodevelopmental components (schizophrenia, bipolarity, major depression, autism...) or neurodegenerative components (Parkinson's, dementia...), since it is now well established that the pathophysiological processes that lead to disease begin more than 10 years before the onset of clinical symptoms, which in turn signal very advanced phases of the disease and therefore incurability.

[0007] Another important aspect to mention concerning neuropsychiatric diseases is the nonpossibility of performing brain biopsies, and therefore the unavailability of neural cells from patients to apply the PDD and / or TDD approaches.

[0008] Moreover, different neuropsychiatry disorders can share common clinical signs and symptoms (similar clinical phenotypes) in some cases, which can make it difficult to diagnose and differentiate them from each other. For example, depressive and bipolar disorders can have a similar clinical phenotype but require very different treatments. Concretely, if antidepressants are prescribed to bipolar individuals presenting an acute depressive episode, this can cause a manic episode. It is the occurrence of this antidepressant-induced manic episode that enables the psychiatrist to distinguish between an acute depressive episode constituting a depressive disorder and one constituting a bipolar disorder, whose treatments are very different (antidepressant versus thymoregulator and neuroleptics). However, a manic episode is traumatizing to the patient who may require intensive care and supervision over multiple months in some cases as treatment. The patient must be monitored by the psychiatrist until the situation has stabilized and a reliable diagnosis of bipolar is made. It is therefore desirable to identify biomarkers of neuropsychiatry diseases such as bipolar disorder so that the right diagnosis can be made quickly and the right treatment put in place without delay, thus avoiding the progression of an inadequately treated disease and the occurrence of complications linked to inappropriate drug treatments, such as the occurrence of an antidepressant-induced manic episode.

[0009] Accordingly, improvements in the identification of biomarkers particularly for neuropsychiatry disorders where a tissue biopsy is not available are highly desired.SUMMARY

[0010] In one aspect, there is provided a method of identifying a biomarker or group of biomarkers of a disorder, the method including optionally culturing cells obtained from an individual having the disorder; subjecting the cells to a perturbation that destabilizes the homeostasis of the cells; analyzing the response of the cells to the perturbation by live cell imaging and optionally determining a mechanism of action that drives the cell response; and identifying the biomarker or the group of biomarkers for the disorder with the imaging of the response of the cells to the perturbation or by the mechanism of action (i.e. , cell processes) or a subgroup of mechanisms that explain this response or by a combination of the specific response of the cells to the perturbation (or to a set of perturbations) and a subgroup of mechanisms that explains the response. In some cases, the method can further comprise identifying compounds to treat the disorder by screening a library of compounds and selecting compounds with the ability to normalize the response of the cells to the perturbation, i.e. to make the cell response similar or as close as possible to that measured in cells from healthy controls not having the disorder. The human cells can be obtained invasively or non-invasively. The cells are for example one of stem cells, blood cells, fibroblast cells, microglial cells, skin cells, urinary tract cells, hair follicle cells, olfactory bulb cells, oral cells or nasal mucosal cells. In some embodiments, the disorder is neuropsychiatric disorder and optionally the cells obtained are stem cells and they are differentiated to become neural cells (e.g., neuronal cells, astrocytes or microglial cells). In some embodiments, the microscopy is fluorescent microscopy or quantitative phase microscopy (such as digital holographic microscopy). In some cases, the step of analyzing the response of the cells further includes studying with different techniques (biochemical or molecular approaches as well as different -omics techniques such as genomics, proteomics, metabolomics, etc.) a mechanism (or subgroup of mechanisms) of the response of the cells to the perturbation which explains the response. In some embodiments, the step of subjecting the cells to a perturbation includes subjecting the cells to a list of perturbations. In some embodiments, the step of analyzing is supported by machine learning. In some embodiments, the perturbation is an oxidative disturbance, an osmotic disturbance, a mechanical disturbance, thermal disturbance, acid-base disturbance, metabolic disturbance and / or a chemical perturbation such as neurotransmitters. In some embodiments, the method further compries treating the individual identified as having the disorder with an appropriate treatment regimen specific for the disorder

[0011] In a further aspect, there is provided a method of diagnosing bipolar disorder in an individual suspected to have bipolar disorder or having a predisposition for bipolar disorder, the method includes: optionally culturing cells obtained from the individual; subjecting the cells to anoxidative disturbance; measuring a change in water efflux in the cells in response to the oxidative disturbance using microscopy, optionally identifying by pharmacological approaches the mechanisms, particularly in relation with chloride (Ch), underlying the homeostatic water efflux and determining that the individual has bipolar disorder if the water efflux and optionally the Cl’- related mechanisms are different from that of healthy control cells subjected to the same culture conditions and oxidative disturbance. In some embodiments, subjecting the cells to an oxidative disturbance includes providing in the culture media 50 pM of f-butyl hydroperoxide. The cells are for example fibroblast cells. In some cases, the method further comprises identifying compounds for treating bipolar disorder by screening a library of compounds and selecting compounds with the ability to normalize water efflux, i.e. to make the water efflux similar or as close as possible to that measured in cells from healthy controls not having the disorder.

[0012] In yet a further aspect, there is provided a method of differentiating bipolar disorder (BD) from major depressive disorder (MDD), the method includes: optionally culturing cells obtained from the individual suspected to have BD or MDD; subjecting the cells to an oxidative disturbance; measuring a change in water ■ efflux in the cells in response to the oxidative disturbance using microscopy; and determining that the individual has bipolar disorder if the water efflux and optionally Ck-related mechanisms are different from that of control cells obtained from an individual with MDD and subjected to the same culture conditions and oxidative disturbance. The cells are for example fibroblast cells, neuronal cells, neuronal-like cells, skin cells or urinary tract cells.

[0013] In still a further aspect, there is provided a method of treating bipolar disorder in an individual suspected to have bipolar disorder or having a predisposition for bipolar disorder, the method includes: optionally culturing cells obtained from the individual; subjecting the cells to an oxidative disturbance; measuring a change in water efflux and optionally Ch related mechanisms in the cells in response to the oxidative disturbance using microscopy; determining that the individual has bipolar disorder if the water efflux and optionally Ckrelated mechanisms are different that of healthy control cells subjected to the same culture conditions and oxidative disturbance; and treating the individual with a therapeutically effective amount of an agent that restores in the cells the efflux of water, possibly by acting on chloride-related mechanisms, close to those measured in the cells coming from healthy controls. The cells are for example fibroblast cells.

[0014] In an additional aspect, there is provided the use of digital holographic microscopy in the diagnosis of bipolar disorder in an individual by measuring the efflux of water and optionally Cl’ related mechanisms in cells of the individual in response to an oxidative disturbance.

[0015] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1A is a label-free microscopy image of healthy human skin fibroblasts (H-HSF).

[0017] FIG. 1B is a graph showing different quantitative phase signal (QPS) traces of the H-HSF of Fig. 1A, after a cellular disturbance induced by f-butyl hydroperoxide.

[0018] FIG. 1C is a label-free microscopy image of major depressive disorder human skin fibroblasts (MDD-HSF).

[0019] FIG. 1D is a graph of the QPS traces of MDD-HSF of Fig. 1C, after a cellular perturbation induced by f-butyl hydroperoxide.

[0020] FIG. 1E is a label-free microscopy image of bipolar disorder human skin fibroblasts (BD-HSF).

[0021] FIG. 1F is a graph of the QPS traces of the BD-HSF of Fig. 1E, after a cellular disturbance induced by t-butyl hydroperoxide.

[0022] FIG. 1G is a graph showing the Homeostatic Capacity (HC) responses of fibroblasts stressed by f-butyl hydroperoxide (tBHP). These responses are measured by the time courses of the quantitative phase signal for the three fibroblast populations from control (H-HSF), bipolar (BD-HSF) and depressive (MDD-HSF) individuals. HSF= Human Skin Fibroblasts.

[0023] FIG. 1 H is a bar graph of the different QPS responses induced by t- butyl hydroperoxide(tBHP) and measured at their maximum (peak amplitude), for the 3 groups of participants (H- HSF, MDD-HSF and BD-HSF).

[0024] FIG. 2A is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide (tBHP) with the application of 5-nitro-2-(3-phenylpropylamino)benzoic acid] (NPPB) and without (control)

[0025] FIG. 2B is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with NPPB and without (control).

[0026] FIG. 2C is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with NPPB and without (control).

[0027] FIG. 2D is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF with NPPB and without (control) the application of NPPB (*** p < 0.05, Wilcoxon signed-rank test).

[0028] FIG. 2E is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with the application of 4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on- 5-yl) oxobutyric acid (DCPIB) and without (control).

[0029] FIG. 2F is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with the application of DCPIB and without (control).

[0030] FIG. 2G is a graph showing the two QPS mean (+ SEM) traces of BP-HSF in response to f-butyl hydroperoxide with the application of DCPIB and without (control).

[0031] FIG. 2H is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF with DCIP and without (control) the application of DCPIB (*** p < 0.05, Wilcoxon signed-rank test).

[0032] FIG. 2I is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with (IAA-94) and without (control) the application of Indanyloxyacetic acid 94 (IAA-94).

[0033] FIG. 2J is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with Indanyloxyacetic acid 94 (IAA-94) and without (control).

[0034] FIG. 2K is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with the application of IAA-94 and without (control).

[0035] FIG. 2L is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of IAA-94 (*** p < 0.05, Wilcoxon signed-rank test).

[0036] FIG. 3A is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with furosemide (Furos.) and without (control) the application of furosemide.

[0037] FIG. 3B is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with furosemide (Furos.) and without (control) the application of furosemide.

[0038] FIG. 3C is a graph showing the two QPS mean (+ SEM) traces of BP-HSF in response to f-butyl hydroperoxide with furosemide (Furos.) and without (control) the application of furosemide.

[0039] FIG. 3D is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of furosemide (*** p < 0.05, Wilcoxon signed-rank test).

[0040] FIG. 3E is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with the application of [(dihydronindenyl)oxy] alkanoic acid (DIOA) and without (control).

[0041] FIG. 3F is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with the application of DIOA and without (control).

[0042] FIG. 3G is a graph showing the two QPS mean (+ SEM) traces of BP-HSF in response to f-butyl hydroperoxide with the application of DIOA and without (control).

[0043] FIG. 3H is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of DIOA (*** p < 0.05, Wilcoxon signed-rank test).

[0044] FIG. 4A is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with the application of 4,4'-diisothiocyanostilbene2,2'-disulphonic acid (DI DS) and without (control).

[0045] FIG. 4B is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with the application of DI DS and without (control).

[0046] FIG. 4C is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with the application of DI DS and without (control).

[0047] FIG. 4D is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of DIDS (*** p < 0.05, Wilcoxon signed-rank test).

[0048] FIG. 4E is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with Anthracene-9 carboxylic acid (A9C) and without the application of A9C (control).

[0049] FIG. 4F is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with A9C and without A9C (control).

[0050] FIG. 4G is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with A9C and without A9C (control).

[0051] FIG. 4H is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of A9C (*** p < 0.05, Wilcoxon signed-rank test).

[0052] FIG. 5A is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with NS398 and without NS398 (control).

[0053] FIG. 5B is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with NS398 and without NS398 (control).

[0054] FIG. 5C is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with NS398 and without NS398 (control).

[0055] FIG. 5D is a bar graph showing the peak amplitude of QPS (means + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of NS398 (*** p < 0.05, Wilcoxon signed-rank test).

[0056] FIG. 5E is a graph showing the two QPS mean (+ SEM) traces of H-HSF in response to f-butyl hydroperoxide with lithium and without lithium (control).

[0057] FIG. 5F is a graph showing the two QPS mean (+ SEM) traces of MDD-HSF in response to f-butyl hydroperoxide with lithium and without lithium (control).

[0058] FIG. 5G is a graph showing the two QPS mean (+ SEM) traces of BD-HSF in response to f-butyl hydroperoxide with lithium and without lithium (control).

[0059] FIG. 5H is a bar graph showing the peak amplitude of QPS (mean + SEM) in response to f-butyl hydroperoxide for H-HSF, MDD-HSF and BD-HSF without (control) and with the application of lithium (*** p < 0.05, Wilcoxon signed-rank test).

[0060] FIG. 6 is a graph showing the ratio in term of QPS amplitude between a tBHP application in presence of drug vs a same application of tBHP alone for H-HSF, MDD-HSF and BD-HSF, representing HSFs homeostatic capacity (HC) after tBHP stress in the presence of different pharmacological agents. Results are presented as means ± SEM (# p < 0.05, ## p < 0.01 , ### p < 0.005, One-way Anova / posttest « Tukey»).

[0061] FIG. 7 is a graph showing the ratio of the scoring (average value of the normalized efficiency of each pharmacological Cl' blocker in inhibiting the tBHP induced QPS responses) for 39 individuals (dots in group 1 are healthy individuals, dots in group 2 are MDD individuals and dots in group 33 are BD individuals).

[0062] FIG. 8 is a graph showing the measurement of the CHC of BD-HSF fibroblasts to tBHP in the presence of Lithium enables personalized medicine. This graph shows the Normalized QPS of fibroblasts from 13 individuals diagnosed with BD to tBHP in the presence of Lithium.

[0063] FIG. 9A is a graph showing the application of norepinephrine (NE) (100 pM for 5min) which triggered a transient increase of the QPS in the three cell lines. The average traces (means) obtained after NE application from H-HSF (nind = 4), MDD-HSF (nind = 4) and H-HSF (nind = 3) indicating a lower tBHP induced QPS for BD-HSF in comparison with H-HSF and MDD-HSF.

[0064] FIG. 9B is a bar graph showing the maximum values of peak amplitude of QPS induced by NE in presence of specific blockers such as Propranolol, Prazosine, or Reboxetine and also a cocktail of specific agonists with Isoproterenol + Phenylephrine for H-HSF, MDD-HSF and H-HSF from left to right.

[0065] FIG. 10A is a graph showing that the application of NE (10pM; 10min) triggered a transient increase of the QPS in the two cell lines of USCs. The average traces (means) clearlyshow that the NE-induced QPS was strongly higher for H-LISCs (nind = 6) in comparison with BD- USCs (nind = 3).

[0066] FIG. 10B is a graph showing the application of ACH (10pM; 10min) triggered a transient increase of the QPS in the two cell lines of USCs. The average traces (means) clearly show that the NE-induced QPS was strongly higher for H-LISCs (nind = 6) in comparison with BD- USCs (nind = 3). In contrast to NE, the average traces (means) ACH-induced QPS was higher for BD-USCs.

[0067] FIG. 10C is a bar graph showing the maximum values of peak amplitude of QPS (means + SEM) induced by NE or ACH. The observed amplitude difference between NE- and ACH-induced QPS for H-LISCs is well marked, compared to what was observed for BD-USCs, where the maximum amplitudes of NE- and ACH-induced QPS remain similar. (*** p < 0.005, unpaired t-test).

[0068] FIG. 10D is a bar graph showing that the ratio between NE and ACH response is higher for H-USCs in comparison with BD-USCs.

[0069] FIG. 11 is a graph showing the application of Glutamate (30 pM for 1min) triggered a transient decrease of the QPS in two neurons (1 and 2) shown in the image on the top left. After perfusion of MK801 (40 pM), the application of glutamate is strongly decreased indicating an involvement of the NMDA receptor.

[0070] FIG. 12A is a graph showing QPS (main + / - SEM) recorded via DHM from cultured Mice astrocytes (MAs) after 21 days in culture. A perfusion of Noradrenaline (NE; 100pM) triggered a strong decrease of the QPS, this persists for the duration of the NE. It can be concluded that MAs expressed functional NE receptors. Additional application of glutamate (Glut; 10OpM for 2min) triggered a supplemental transient decrease of QPS concluding to an expression of functional glutamate receptors. After a washout of NE, a same application of glutamate triggered a same decrease of QPS, suggesting that the glutamate-induced QPS is independent to the NE-induced QPS through two distinct pathways.

[0071] FIG. 12B is a phase image of cultured mice astrocytes visualized in DHM.DETAILED DESCRIPTION

[0072] The method described in the present disclosure was motivated by the resolution of the problems identified in the above approaches, namely that: 1) in a very large number of diseases numerous cellular biomarkers are simultaneously involved and they are often only very partially known, or even unknown for certain diseases; 2) in many situations when a pathological cellular phenotype is expressed, the disease is already well established and is often at an advanced stage of progression. The present disclosure thus proposes a multi-step method for mapping cellular biomarkers specific to a disease or disorder, even at an early stage. Once the map of specific cellular biomarkers linked to a disease or disorder has been obtained, both the identification and thus the diagnosis of a pathology can be carried out. Likewise, the technology disclosed herein allows the design, identification, selection and / or testing of the best therapeutic agents for the said disease or disorder by measuring their effect on the mapped biomarkers.

[0073] Thanks to developments in the field of stem cells, it is now possible by collecting easily accessible cells from individuals (e.g., fibroblasts from a skin biopsy, blood cells from a blood sample, urothelial cells from urine samples) to reprogram them into stem cells (in particular induced pluripotent stem cells, iPSCs) and then to differentiate into any cells of the body, including neurons and astrocytes. This technique makes it possible to obtain different kinds of cells with the genetics of the individual concerned. This is key to obtain human cells and tissues to study the pathophysiology of major disorders and more specifically in identifying new biomarkers and discovering new therapeutic targets of various diseases. Indeed, this is particularly relevant in neuropsychiatry disorders because in these disorders it is not possible to obtain brain tissue biopsies on which to test biomarkers or therapeutic agents. Thus, providing living neuronal cells from the individual by producing them in vitro is definitely an attractive option.

[0074] There is provided herein a process of identifying a biomarker of a disease or disorder by first disturbing or perturbing cells obtained from an individual having the disease or disorder, and then analyzing the capacity of the cells to return to the pre-disruption equilibrium or to another equilibrium, or fail to regain an equilibrium, leading to cell death. This is referred to herein as the capacity of homeostasis, i.e., the propensity to restore or return to equilibrium. In concrete terms, when the equilibrium of a cell's internal environment, which enables it to function properly, is disturbed by changes in its environment, the cell reacts by activating a whole series of processes or mechanisms designed to re-establish the equilibrium of its internal environment, which is conducive to its proper functioning. The actual dynamics of this homeostatic capacity reflects aset of mechanisms or processes recruited. Hence, both the monitoring of the dynamics of this homeostatic capacity and the identification (nature) and characterization (intensity, duration, etc.) of the homeostatic mechanisms recruited or stimulated reveal both cellular biomarkers of disease as well as potential new targets for therapeutic compounds respectively. Concretely such biomarkers are discriminated on the basis of homeostatic capacity (the dynamics of the process i.e. , the efficiency of returning to equilibrium, of returning to another equilibrium, of not returning to an equilibrium) reflecting different recruited homeostatic mechanisms between the disease and the control case. On the other hand, dissecting the homeostatic mechanisms recruited by the cell can also reveal important mechanisms involved in the pathophysiology of the disease leading thus to the identification of new potential drug targets. Promising new therapeutic compounds are therefore identified globally by their ability to modify or restore a homeostatic capacity in cells close to that observed in healthy control cells (in particular the dynamic aspects) and specifically by their way of acting on one or other or several of the mechanisms underlying the homeostatic capacity, these mechanisms representing thus promising potential drug targets.

[0075] The present approach can be used to analyze cells that are still functioning normally in the early stages of disease, particularly slow-onset diseases, but which have become fragile as a result of physiopathological processes. This is sometimes referred to as the pathogenesis phase of the disease. During this phase, the cells do not present a pathological phenotype since they continue to function like healthy cells and thus the individual does not show any clinical symptoms. It has been presently found that inducing a perturbation by disrupting the homeostasis of the fragilized cells with a disturbing agent in the pathogenesis phase reveals their fragility which results in a deteriorated homeostatic capacity. This has been leveraged herein for the screening, identification and treatment of disease.

[0076] As will be described in further details below, cellular imaging is used to determine cell homeostasis and its response to a stressor. The preferred imaging methods are non-invasive and quantitative, and are performed without the use of imaging markers. The imaging method can determine a number of different cellular parameter which alone or in combination can be indicative of a return to equilibrium for the cell after being disrupted (i.e., disturbed). It is also contemplated herein that further steps can be taken to identify the mechanism of the cellular processes that were responsible or played a role in the return to equilibrium. This can be performed by using known drugs that inhibit or active certain cellular pathways (i.e, pharmacological agents) and then subject the cells to a stressor (i.e, a disturbing agent) and observe any change in the return to equilibrium. If they are unable to return or they take longer to return, then a specific homeostaticpathway can be identified. In turn, this information can be used to develop a therapeutic approach I regimen, or to identify new drugs for treating a disorder. The term “pharmacological agent” as used herein can be defined as including inhibitors of transmembrane ionic channels, chelators, inhibitors of cellular pathways, receptor agonists or antagonists, neurotransmitter uptake inhibitors, or pharmacological drugs or active agents (e.g. lithium). In some embodiments, the pharmacological agent is selected from among in one or combination of agents described in tables 1 to 3. The present methods can be applied to cell culture or to primary cells, such as cells derived from an individual. The cells may be in their pathogenesis phase which would show no difference in phenotype compared to healthy cells, but can be identified as being pathogenic using the present method. Early identification of disease improves significantly the capability to treat or alleviate the disease. This is particularly relevant for neuropsychiatric disorders, and more specifically neurodegenerative diseases.

[0077] Using cell reprogramming techniques developed some ten years ago, it is now possible to reprogram cells that are readily available in an individual (skin cells, urine cells, mouth cells, etc.) into so-called pluripotency-induced cells, which can then be differentiated, in vitro by choosing the right culture media, into any cell type in the body, including neural cells (astrocytes and neurons), the main cells that make up the brain.

[0078] In this context, the present methods can be successfully applied to neuronal and astrocytic cells derived from skin fibroblasts as well as from urinary cells taken from healthy and sick donors or donors suspected to have a psychiatric disorder or donors at risk of developing a psychiatric disorder, e.g. genetic predisposition (bipolar disorder, schizophrenia, major depression, autism, Alzheimer’s, Parkinson’s, addicts, etc.).

[0079] Importantly, the present methods also revealed altered homeostatic cell capacity (HCC) in urinary cells taken from urine samples of individuals, as well as in fibroblasts taken by skin biopsy from individuals with bipolar disorder (compared with HCC from healthy participants). Although urinary cells, fibroblasts and neurons are very different cell types, they share a number of common mechanisms involving a number of similar membrane elements (receptors, cotransporters and the like). In addition, the pharmacological experiments carried out on urinary cells and fibroblasts to dissect this altered HCC have identified a number of mechanisms underlying this alteration, and consequently a number of potential therapeutic targets linked to these mechanisms. This alteration in HCC and the identification of its underlying mechanisms in skin fibroblasts and in urinary cells from bipolar individuals have enabled to define a urine cell, aswell as a fibroblast cell biomarker specific to bipolar disease, which constitutes a diagnostic test for this illness.

[0080] The method of the present disclosure can thus identify biomarkers for neuropsychiatry disorders without requiring a neuronal tissue biopsy which is an undesirable invasive procedure. Neuronal cells can be obtained from an individual suspected of having a disorder, suspected of being at risk for the disorder, or having a predisposition to the disorder by collecting cells from easily accessible peripheral tissues (skin, buccal mucosa, etc.), from blood or secretions such as urine, which are then reprogrammed into stem cells (induced pluripotent cells, iPSCs) and differentiated in vitro to obtain neural cells. It is also possible to obtain directly stem cells that can then be differentiated into a neuronal cell lineage (umbilical cord stem cells, for example). The reprogramming and differentiation steps are, however, optional for the identification of biomarkers and novel drug targets related to the homeostatic capacity. Indeed, it was surprisingly discovered herein that observing the homeostatic capacity of human fibroblast cells and urinary cells (collected form skin biopsy) can be used for determining whether an individual has bipolar disorder. Indeed, from neurodevelopmental perspective, the differentiated and specialized cells that constitute the body's various tissues all originated from identical stem cells that have progressively differentiated from one another through various stages and phyla. As a result, and depending on the branching level at which they separated, differentiated and specialized, cells from very different tissues may still share many common features, notably in terms of transmembrane proteins. As a result, neurons, and in particular some of their highly specific structures including synapses share many of the same transmembrane proteins (such as ion channels) with other cell types, which have a priori nothing to do with neurons.

[0081] Moreover, although certain diseases are classified as neuronal disorders, the cellular changes in an individual affect by the disorder can extend to cells other than the neurons. This can be explained in part by mechanism shared between neurons and other cells with transmembrane proteins or by the fact that the disease affects the body beyond just the neurons. Accordingly, any suitable cell type can be utilized in the present processes including but not limited to neurons, astrocytes, fibroblasts, epithelial cells such as skin cells or cells from the oral cavity, urinary tract cells, red blood cells, etc. These cells, in some cases, can be reprogrammed into neuron or neuronal-like cells in vitro to exhibit a certain characteristic of neuronal cells as explained below. It should be noted that cells obtainable non-invasively are preferred, such as are stem cells, blood cells, fibroblast cells, microglial cells, skin cells, urinary tract cells, hair follicle cells, olfactory bulb cells, oral cells and nasal mucosal cells.

[0082] The differentiated cells in culture are then subjected to an environmental perturbation to destabilize them which will disturb their equilibrium, particularly that of their internal milieu. Examples of environmental perturbations can include but are not limited to physico-chemical modifications of the environment caused by changes related to but not limited to temperature, neurotransmitters, osmotic pressure (addition of substances not internalized by the cell, such as mannose), acid / base balance, cell mechanical strain (e.g., applied by controlled fluid flows) and more specifically to the presence of oxidizing agents, energy substrate depletion / modification (e.g., glucose depletion). Any combination of these different perturbations can also be used. In some embodiments, the perturbation is an oxidative disturbance which could be performed by adding f-butyl hydroperoxide (tBHP) in the culture media. In certain neuropsychiatric disorders that have been well studied, there are known neurotransmitters that perturb the homeostasis of the cells and are responsible for the neuronal cells loosing at least a part of their normal function. Accordingly, neurotransmitter at non physiological concentrations can also be used as a disturbing agent particularly to target a known mechanism or response. It should be noted that neuropsychiatric disorders are complex and are rarely characterized by a single neurotransmitter. Accordingly, a combination or array of neurotransmitters specific for a given disorder can be used to more accurately perform a stress that is specific to that disorder. It should also be noted that the same biomarker can be indicative of different disorders based on the extent of the response to the disrupting agent. For example, a longer return to homeostasis could be indicative of a first disorder whereas a shorter return to homeostasis (still longer than for a healthy individual) could be indicative of a different, second disorder. It should also be noted that the level of disruption (e.g. pH, mechanical force, concentration of drug) applied on the cells must not kill the cell. Chemical concentrations as well as mechanical forces must be controlled to avoid inducing cell death. In the case of mechanical perturbations, different intensities or different types of mechanical stresses can be applied to the cells (e.g. compression, elongation, flow pressure etc.). Each disrupting condition can have a different intensity which could be indicative for different disorders.

[0083] The response of the cells to the perturbation reflecting the homeostatic capacity is then measured by microscopy, both the dynamics of the response and the mechanisms underlying this response. These underlying mechanisms are identified in particular by experiments combining the measurements of the homeostatic capacity with pharmacological investigations. These pharmacological investigations test compounds known for their specific actions on cellular mechanisms (i.e., pharmacological agent) while analyzing their effects on the resultinghomeostatic response. Once identified the underlying mechanisms can also be accurately deciphered using a variety of methods (such as biochemical or molecular approaches as well as different -omics techniques such as genomics, proteomics, metabolomics, etc).

[0084] Microscopy advantageously allows monitoring in real time the cell responses without affecting them. The microscopy techniques can for example be some fluorescent modalities or label free approached including digital holographic microscopy, a strictly non-invasive technique. In some embodiments, the optical signal obtained from microscopy is analyzed with algorithms which can be based on artificial intelligence, which has the ability to find specific links between a diagnosis and certain cellular characteristics (structural, functional, as well as resulting from responses to paradigms such as the homeostatic capacity test), which can result from the combination of several thousand parameters automatically extracted from images. Once the microscopy analysis has characterized the cell homeostatic capacity dynamics in response to the environmental perturbations, depending on the differences found between a cell population (a few hundred cells) derived from control subjects and those from individuals, a biomarker can already be identified as such. Secondly, the pharmacological experiments to analyze and determine the various cellular mechanisms underlying and explaining the specific homeostatic capacity of primary derived cells will enable both to better characterize the biomarker as well as to identify potential new therapeutic targets. This is for example the case for bipolar disorder, schizophrenia, major depression, autism, Alzheimer’s, and Parkinson’s as described in greater detail below in the Example section. Although bipolar disorder is used as an example of a neuropsychiatry disorder or even more generally as an exemplary disorder, the present disclosure is not limited to bipolar disorder and the methods described herein are also applicable to other disorders.

[0085] Although the present disclosure generally describes the identification of a biomarker (singular) it should be understood that the scope of the identification includes identifying an array of biomarkers. This is particularly relevant for neuropsychiatric disorder which are classified and identified based on a plurality of biomarkers or combination of biomarkers. This is referred to as mapping a disorder. In order to perform a mapping a plurality of different disturbing agents can be used to subject the cells to different disorders, an array of cells can be used and which cell can be subject to a different disturbing agent.

[0086] In some embodiments, the present disclosure leverages quantitative phase imaging in particular based on digital holographic microscopy (DHM) for the identification of biomarkersfor bipolar disorder (as well as other disorders). DHM is a microscopy technique which entails observing an specimen by interference between a reference electromagnetic wave and an object electromagnetic wave (the wave diffracted by the observed specimen) referred to as a hologram. The hologram numerical reconstruction is then used to calculate the object wave and thus obtain both the intensity images as well as the quantitative phase image of the observed specimen. Thanks to these images, this microscopy technique allows for the identification of morphological and dynamic aspects of cells as well as its contents with a sensitivity in the order of 10 nm. This method allows for quantification of different cell parameters including volume, shape (morphometry), dry mass, protein content membrane fluctuations and permeability, transmembrane water movements. Based on the cell refractive index which contributes to generating the measured quantitative phase signal, visualizing the inside of cells is made possible leading to the identification of different cell compartments and organelles in particular. DHM thus allows for the visualization of mechanisms of action in cells in real time. Additional technical description for DHM can be found in EP1451646 which is incorporated herein by reference in its entirety. Alternative microscopy techniques are also contemplated herein including but not limited to various fluorescence modalities such as but not restricted to epifluorescence, confocal, two photons, Stimulated Emission Depletion (STED) Photo-Activated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), Fluorescence Life Time Imaging (FLIM), Differential Interference Contrast (DIG) Microscopy, Forster Resonance Energy Transfer (FRET) Microscopy, Raman Microscopy, Polarized Light Microscopy, Surface Plasmon Resonance Microscopy (SPR), Total Internal Reflection Fluorescence Microscopy (TIRF), Scanning Probe Microscopy (SPM), Light-Sheet Fluorescence Microscopy (LSFM), Optical Coherence Tomography (OCT), and Optical Diffraction tomography.

[0087] Following the application of an oxidative perturbation (for example with f-butyl hydroperoxide (tBHP)) to fibroblasts obtained from the skin or urinary tract of control healthy individuals, depressive individuals and bipolar individuals, the experimental measurement performed with DHM reveals a dynamics of the homeostatic response which is specific to a given psychiatric disorder such as bipolar, schizophrenia, major depression, autism, Alzheimer’s, Parkinson’s. For example, a bipolar-specific homeostatic response can be characterized and it was found herein that it involves a higher water efflux through the fibroblast membrane. Then analysis, performed by combining DHM measurements with pharmacological approaches, reveals the cellular mechanisms underlying these particularly high transmembrane water efflux rates, and hence potential therapeutic targets. Concretely, the high water efflux specific to bipolardisorder involves ion movements in particular chloride ions, and the key elements responsible for this bipolar-specific water dynamics have been thus identified. As demonstrated in the Example section below, both a specific transmembrane water dynamics as well as the Ck-related underlying mechanisms were identified within fibroblasts and urinary cells as a cellular biomarker specific to bipolar, which in particular differentiates from major depressive disorder.

[0088] The methods contemplated herein can further comprise a treatment step or a treatment regimen determined based on the cell response to the homeostatic perturbance. For example, if a new drug is identified or if a known drug is identified to have a new therapeutic use, these drugs can be used to treat the individuals. There is thus provided, in some embodiments, a personalized medicine approach where a cell from the individual is extracted and subject to the present method in order to determine a suitable treatment step. The personalization in treatment is particularly useful in neuropsychiatric diseases which often have individuals respond differently to the same treatment due to complex underlying mechanisms. Determining whether a particularly treatment is appropriate to a particular individual is thus highly desirable and increases the chances of successfully treating or alleviating the symptoms for the individual.EXAMPLE 1

[0089] From a clinical point of view, it is extremely difficult to distinguish between an acute depressive episode related to a bipolar disorder (BP) and one related to depressive disorders including major depressive disorder (MDD). This is all the more problematic since the management of these two acute depressive episodes are radically different. Consequently, the identification of a reliable biomarker specific to bipolar disorder may be a crucial implement to achieve the differential diagnosis between these two types of depression, very similar clinically, which could then be treated quickly and appropriately. In the present example, the monitoring of the fibroblastic transmembrane water fluxes, in response to a perturbation triggered by a transient oxidative disturbance applied in cultured Human Skin Fibroblast (HSF) coming from Healthy (H- HSF), Bipolar Disorder (BD-HSF) and Major Depressive Disorder (MDD-HSF) individuals, is performed with Digital Holographic Microscopy (DHM), through the recording of its quantitative phase signal (QPS).

[0090] In the present example, Human Skin Fibroblast (HSF) cultures obtained from skin biopsies of Healthy (H ; n^d = 14), Major Depressive Disorder (MDD ; n^d = 12) and Bipolar Disorder (BD ; n^d = 13) individuals were cultured in HAM-F10 cell culture medium containing20% fetal calf serum and penicillin I streptomycin I Hygromycin solution. After a minimum of 5 passages, each HSF culture (H-HSF; MDD-HSF; BD-HSF) was placed on a Poly-Ornithine coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KOI, 5 mM D-glucose, 10 mM HEPES, 3 mM CaCI2, and 2 mM MgCI2(pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time was used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1-1 Hz). While DHM measurements were maintained, 50 pM of tBHP, the oxidizing agent used to disrupt cell equilibrium, was added to the perfusion medium for 10 min. Then, the initial perfusion medium (i.e. , without oxidizing agent) was replaced for at least 10 min under constant monitoring by DHM. This perfusion sequence (i.e., 1 - Perfusion medium for at least 15 min; 2 - Perfusion medium + oxidizing agent for 10 min; 3 - Perfusion medium for at least 10 min) provided the reference DHM signal for a given cell line.

[0091] In the second step, all 3 HSFs culture types were pre-incubated with different pharmacological agents (PA ; Table 1) known to inhibit specific intracellular pathways prior to tBHP application. Separate experiments respecting the perfusion sequence above were performed for each of the selected PA, during which the HSFs homeostatic capacity (HC) response was measured by DHM. The HC responses in the presence of each pharmacological agents was then be compared to the HC response in the absence of the PA, which was chosen as the reference value and therefore considered to be the 100% HSFs HC response.Table 1. Tested biomarker Pharmacological agents on H-HSF, MDD-HSF, and BD-HSF when disturbed with tBHP.

[0092] When cultured H-HSF, BD-HSF and MDD-HSF were exposed to terf-Butyl- HydroPeroxide (flBHP), an oxidation agent, all cell lines displayed a homeostatic response characterized in particular by transmembrane water efflux. The fBHP-induced homeostatic water efflux in BD-HSF is higher than that of H-HSF and MDD-HSF, which translates into a larger increase of the fBHP-induced QPS in BD-HSF. The pharmacological exploration of this difference in water efflux related to oxidative disturbance allows thus to identify the underlying mechanisms which involve in particular Ck ions. Specifically, this exploration leads to a pharmacological profile of the cultured fibroblasts which makes it possible to distinguish a bipolar individual from either a MDD individual or a healthy person with a high degree of sensitivity and specificity. It therefore represents an extremely valuable cellular biomarker not only to promote the adequate management of depressive episodes, but also, in the longer term, to better understand the pathophysiology of bipolar disorder. Indeed, the identification of the mechanisms underlying fBHP-induced homeostatic water efflux (in particular the Ck-related mechanisms) in BD-HSF reveals promising new druggable targets. Specifically, the targets here are related to elements that act on Ck homeostasis, and the outcome for testing promising compounds is their efficacy in restoring fBHP -induced water effluxes as close as possible to those measured to those measured in the fibroblasts coming from healthy controls.

[0093] Quantitative phase images of cultured H-HSF, MDD-HSF and BD-HSF are shown in Figs. 1A, 1 C, and 1 E respectively. The QPS recorded from H-HSF, MDD-HSF and BD-HSF are shown in Figs. 1 B, 1 D, and 1 F respectively. The application of tBHP (50 pM for 10 mins) triggereda transient increase of the QPS in the three cultures. Fig. 1G shows typical responses of fibroblasts from control (H-HSF), bipolar (BD-HSF), and depressed (MDD-HSF) individuals attempting to restore homeostasis after being perturbed by tBHP application. The HCC curves show cellular responses as measured by the quantitative phase signal of tBHP (means ± SEM) and processed as described in the example herein. Fig. 1G indicates a higher tBHP induced QPS for BD-HSF in comparison with H-HSF and MDD-HSF, meaning that the HCC curve under tBHP of fibroblasts from BP individuals is clearly different from those of fibroblasts from H and MDD individuals. However, at this stage, the difference between H and MDD individuals is clearly not significant. Fig. 1 H shows the graph of the maximum values of peak amplitude of QPS induced by tBHP for H-HSF (n^d = 14), MDD-HSF (n^d = 12) and BD-HSF (nind = 13), this last one being significantly larger than those of H-HSF and MDD-HSF.

[0094] The results of Figs. 2A-2L show that the tBHP-induced QPS is associated to a synergic opening of VRAC and CLIC-4 conductances. The cultured H-HSF (nind = 14), MDD-HSF (nind = 12) and BD-HSF (n^d = 13) were subjected to 100 pM [5-nitro-2-(3-phenylpropylamino)benzoic acid] (NPPB, a broad-spectrum blocker of Ch transporters). NPPB decreased the tBHP-induced QPS in all cell lines meaning that tBHP triggered a water efflux accompanied by a Ch efflux (Figs. 2A-2C). Fig. 2D confirms that NPPB significantly decreases the QPS induced by tBHP for H-HSF, MDD-HSF and BD-HSF. Accordingly, the return to homeostasis mechanism involves Ch transporters and can be modified by NPPB or other blockers of Ch transporters, as presented below.

[0095] H-HSF (nind = 14), MDD-HSF (nind = 12) and BD-HSF (n^d = 13) were cultured alone (i.e. only tBHP) or in the presence of 4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB, a selective blocker of VRAC) (100 pM). Even if all cell lines are sensitive to DCPIB, the decrease by this blocker of the tBHP-induced QPS is stronger in BD-HSF compared to the other two (Figs. 2E-2G). Indeed, Fig. 2H shows that DCPIB significantly decreases the QPS induced by tBHP for H-HSF, MDD-HSF and BD-HSF, but most particularly for BD-HSF.

[0096] H-HSF (nind = 14), MDD-HSF (nind = 12) and BD-HSF (n^d = 13) were cultured alone (i.e. only tBHP) or in the presence of 50 pM of Indanyloxyacetic acid 94 (IAA-94, a specific blocker of the CLICs-4). Similarly to DCPIB, IAA-94 decreases the tBHP-induced QPS in all cell line but in a stronger manner for BD-HSF (Figs. 2I-2K). Indeed, Fig. 2L shows that IAA-94 significantly decreases the QPS induced by tBHP for H-HSF, MDD-HSF, and BD-HSF. Nevertheless, the inhibitory effect of IAA-94 is significantly stronger in BD-HSF.

[0097] H-HSF (riind = 14) were cultured alone (i.e. only tBHP) or in the presence of furosemide (a broad-spectrum blocker of ionic co-transporters) (100 pM) or [(dihydronindenyl)oxy] alkanoic acid (DIOA, a specific blocker of Potassium Chloride Cotransporters (KCC)) (100 pM). It was found that neither furosemide or DIOA were able to strongly decrease the tBHP induced QPS for H-HSF (Figs. 3A and 3E). The same was done for MDD-HSF (nind = 12) and an inhibitory effect of furosemide was clearly observed in contrast to DIOA (Figs. 3B and 3F). Finally, the same was done again for BD-HSF and it was found that both blockers decreased the tBHP induced QPS (Figs. 3C and 3G). In summary, Figs. 3D and 3H show that furosemide significantly decreased the QPS induced by tBHP for MDD-HSF and BD-HSF and DIOA was efficient at blocking the same optical response for BD-HSF only, confirming that there is a specific involvement of KCC3 in the genesis of the tBHP induced QPS in the BD-HSF cell line.

[0098] H-HSF (nind = 14), MDD-HSF (n^d = 12) and BD-HSF (n^d = 13) were cultured alone (i.e. only tBHP) or in the presence of 100 pM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DI DS, a blocker of HCOs' / CI' anionic exchangers). DI DS was found to decrease the tBHP induced QPS only in BD-HSF (Figs. 4A-4D). In addition, H-HSF (nind= 14), MDD-HSF (nind= 12) and BD- HSF (nind = 13) were cultured alone (i.e. only tBHP) or in the presence of 1 mM Anthracene-9 carboxylic acid (A9C, the specific blocker of calcium-activated chloride channel, CaCC). Similarly to DIDS, A9C decreased the tBHP induced QPS in BD-HSF only suggesting a specific involvement of both, the anionic exchanger and the CaCC, (Figs. 4E-4H).

[0099] H-HSF (nind = 14), MDD-HSF (nind = 12) and BD-HSF (n^d = 13) were cultured alone (i.e. only tBHP) or in the presence of 100 pM NS-398 (a specific blocker of the COX-2). A specific decrease of the tBHP-induced QPS was observe only in BD-HSF, (Figs. 5A-5D). Similarly, H- HSF (nind = 14), MDD-HSF (nind = 12) and BD-HSF (nind = 13) were cultured alone (i.e. only tBHP) or in the presence of 2mM lithium. An inhibitory effect of lithium was observed only on the tBHP induced QPS in BD-HSF (Figs. 5E-G). Fig. 5H confirms the significant blockade of the tBHP induced QPS by lithium for BD-HSF. Fig. 6 shows that each pharmacological agent (PA) is likely to modify the homeostatic capacity (HC) response of HSFs. Indeed, except for NPPB, DCPIB, and IAA-94, it was observed that the reducing effects of PA are significantly higher for BD-HSF compared to H-HSF and / or MDD-HSF. In other words, if a PA reduces the HC response compared to 100% (HC without PA), it means that the PA-blocked intracellular pathways are involved in the HC response. It is clear from the use of these different pharmacological agents that the profile of BD individuals is significantly different from that of H and MDD individuals. Specifically, this shows that different cellular pathways are more involved in the HC response inBD individuals (i.e., PAs for which the decrease from 100% is greatest). In other words, some intracellular pathways are over-activated in BD, and certain key elements are known about them (receptors, cotransporters, etc. on which the PA used to reveal the involvement of these pathways act), allowing them to be modulated. These key elements not only inform about the pathophysiology of BD, but also constitute a set of new therapeutic targets in the context of drug discovery. Indeed, the identification of molecules capable of modulating the over-activation of these cellular pathways present in BD is extremely promising. Specifically, the present methodology allows the screening of such molecules, since it can measure the HC response with great precision and in a non-invasive manner. It should be also noted that the differences between H and MDD individuals are more subtle, but exist for certain PA. This suggests that the presented methodology has an application in the diagnosis of MDD as well as in the diagnosis of BP.

[0100] By perturbing skin fibroblasts from control, depressed and bipolar individuals with oxidative disturbance using tBHP, it was possible to identify the fibroblastic homeostatic response aiming to re-establish the equilibrium of the intracellular milieu. This homeostatic response is characterized by a transmembrane efflux of water involving at some extent chloride mechanisms. BD individuals showed a larger water efflux compared to MDD individuals and healthy controls. These fibroblasts were then subjected to a series of signaling assays to block specific pharmacological pathways, which reveal, associated to this water efflux, the involvement of mechanisms that act on the Ch intracellular concentration. Fig. 7 shows the scoring (average value of the normalized efficiency of each Ch blocker) obtained from the HSF pharmacological profile related to Ch for 39 individuals. The two groups (depressed or bipolar) were differentiated based on a cut-off scoring of 60 (Fig. 7, see solid line identifying the cut off of 60). The discrimination of the two groups was performed with a specificity of 100% and a sensibility of 87%. Moreover, among these compounds that act on Ch, those that ultimately normalize water efflux the most, i.e., make it as close as possible to the water efflux measured in cells from healthy volunteers, represent promising compounds for the treatment of bipolar disorder, especially in the individual from whom the cells originate.EXAMPLE 2

[0101] Knowing the difficulties involved in finding an effective medication for an individual diagnosed with a neuropsychiatric disease, it would be ideal to be able to test the efficacy of certain commercial drugs on the individual's own cells, paving the way for personalized medicine. Thus, the method of determining therapeutic targets by studying the homeostatic capacity (HC)of a individual's cells described in Example 1 can also be used to test several therapeutic solutions (i.e. drugs, treatments, etc.), existing or to come, on the cells of said individual.

[0102] In Example 1 , lithium (Li) was used as a pharmacological agent. Indeed, Li is a first- line treatment, as a mood stabilizer, in the management of bipolar individuals. However, in clinical practice, it is known that not all bipolar individuals respond to Li. The aim of the present example was to measure the homeostatic capacity of Example 1 FSH-BDs, taken individually, following disruption with tBHP and in the presence of Li. Fig. 8 shows that the degree to which lithium blocks the tBHP response varies from one individual to another. By setting the threshold for response to Li at 80%, 9 individuals are classified as “responders” whereas 4 would be “nonresponders” to Li. For the latter, it is expected that Li will not be effective and that other therapeutic solutions will have to be considered.EXAMPLE 3

[0103] It is known that mood disorders, and more specifically BD, may be associated with dysfunction of several neurotransmitter systems. As with Example 1 above (i.e., study of tBHP- induced homeostatic water efflux), it was relevant to have another assessment of the optical responses to different neurotransmitters to see also their ability to modify or restore a homeostatic capacity in cells close to that observed in healthy control cells (especially dynamic aspects), and finally to be used as biomarkers to aid in early diagnosis. For this purpose, HSF cells were used, since these cells are known to express receptors for neurotransmitters such as acetylcholine, dopamine, noradrenaline, ATP and even glutamate.

[0104] In the present example, Human Skin Fibroblast (HSF) cultures obtained from skin biopsies of Healthy (H ; nind = 4), Major Depressive Disorder (MDD ; n^d = 4) and Bipolar Disorder (BD ; nind = 3) individuals were cultured in HAM-F10 cell culture medium containing 20% fetal calf serum and penicillin I streptomycin I Hygromycin solution. After a minimum of 5 passages, each HSF culture (H-HSF; MDD-HSF; BD-HSF) was placed on a Poly-Ornithine coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KOI, 5 mM D-glucose±10 mM HEPES, 3 mM CaCh, and 2 mM MgCh (pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time was used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1-1 Hz). While DHM measurements were maintained, 100 pM of norepinephrine (NE; disturbing agent) wasadded to the perfusion medium for 5 min. Then, the initial perfusion medium (i.e., without NE) was replaced for at least 10 min under constant monitoring by DHM.

[0105] In the second step, all 3 HSFs culture types were pre-incubated with different pharmacological agents (PA ; Table 2) known to inhibit specific intracellular pathways prior to NE application. Separate experiments respecting the perfusion sequence above were performed for each of the selected PA, during which the HSFs homeostatic capacity (HC) response was measured by DHM. The HC responses in the presence of each pharmacological agents was then be compared to the HC response in the absence of the PA, which was chosen as the reference value and therefore considered to be the 100% HSFs HC response.Table 2. Tested Pharmacological agent on H-HSF, MDD-HSF, and BD-HSF when disturbed with NE.

[0106] The present experiment aimed to assess the effects of norepinephrine (NE) on fibroblasts. Indeed, much evidence suggests that the noradrenergic system plays a critical role in mood episode switches, where depression might result from attenuation of NE transmission, whereas manic hyperactivity might result from an increase in NE tone. Therefore, when cultured H-HSF, BD-HSF, and MDD-HSF were exposed to NE, all cell lines exhibited a homeostatic response characterized by transmembrane water efflux. However, this NE-induced homeostatic water efflux was lower in BD-HSF than in H-HSF and MDD-HSF (Fig. 9A). Thus, such a difference in water efflux in the context of NE perturbation allowed the identification of the underlyingmechanisms involving several transmembrane proteins (NE receptors and / or NE transporters). Thus, it was found that propranolol or prazosine were less efficient in blocking the NE-induced quantitative phase signal (QPS) for BD-HSF (Fig. 9B). Similarly, the QPS induced by an NE agonist cocktail consisting of isoproterenol and phenylephrine was also weaker for BD-HSF (Fig. 8B), confirming a lower NE activity. More interestingly, reboxetine (norepinephrine transporter (NET) blocker) increased NE-induced QPS for BD-HSF (Fig. 9B), suggesting that the low NE activity may be related to excessive NE transporter activity for BD-HSF. It means that BD-HSF normally have a higher NE re-uptake activity, thus reducing the extracellular concentration of NE and, by consequence, reducing the effectiveness of this neurotransmitter.EXAMPLE 4

[0107] Some evidence suggests that BD has a genetic and neurodevelopmental component. Offspring of affected individuals are therefore at risk of developing one of these psychiatric disorders in their late teens. Thus, there is an urgent need to identify biomarkers in these at-risk children and their parents. Unfortunately, invasive medical procedures (such as skin biopsies or blood tests) cannot be performed on children because they cannot consent. Therefore, in order to perform such experiments on children, urine stem cells (USCs) were used as they can be easily collected from urine samples. These collected USCs were then cultured and challenged in the same way as the HSF in previous examples.

[0108] In the present example, USCs cultures obtained from urine of Healthy (H ; nind = 6) and Bipolar Disorder (BD ; nind = 3) individuals were cultured in 22.5 mL DMEM, 2.5 pL FBS, 250 pL de Pen / strep, 250 pL Glutamax, 250 pL Non Essential amino acids, 25 pL REBM, 25 pL EGF, 25 pL Insulin, 25 pL hydrocortisone, 25 pL Gent / ampho, 25 pL Epinephrine, 25 pL T3, 25 pL Transferrine solution, as described in Zhou et al., Nat. Protoc., 2012. After a minimum of 4 passages, each USCs culture (H-USC; BD-USC) was placed on a 0.1 % Gelatine coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KCI, 5 mM D-glucose±10 mM HEPES, 3 mM CaCI2, and 2 mM MgCI2(pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time was used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1-1 Hz). While DHM measurements were maintained, 10 pM of norepinephrine (NE) or 10 pM Acetylcholine (ACH) was added to the perfusion medium for 10 min as disturbing agents. Then, the initial perfusion medium (i.e., without NE or ACH) was replaced for at least 10 min under constant monitoring by DHM.

[0109] Such pharmacological tests on USCs from healthy individuals (H-LISC) vs. bipolar individuals (BD-LISC) showed some quantitative phase signal (QPS) differences (Figs. 10A-10D), with transient application of NE-induced QPS corresponding to a homeostatic water efflux (Fig. 10A). Importantly, the NE-induced QPS was significantly lower in BD-LISC than in H-LISC (Fig. 10C), as also demonstrated with H-HSF vs. BD-HSF in Example 2 (Fig. 9A). In contrast, application of acetylcholine (ACH), another neurotransmitter involved in the physiopathology of BD, induced a QPS with a higher maximum peak for BD-LISC compared to H-LISC (Fig. 10B). Consequently, the NE / ACH ratio for H-LISCs was clearly higher than for BD-USCs in comparison with BD-USCs (Figs. 10C-10D) suggesting that the balance in term of neurotransmitter activity between NE and ACH is clearly in favor of NE for H-USCs compared to BD-USC where an equilibrium is reached. This last result is relevant in terms of a reliable biomarker specific for BD, since it is known that periods of bipolar depression are associated with hypercholinergic activity, strongly suggesting that USCs are also a relevant biological model for psychiatric research. Moreover, these results are of particular interest as such increased cholinergic function also underlies depression.EXAMPLE 5

[0110] The example herein aims at demonstrating that mechanical stress can be used as a disturbing agent for the methods of the present disclosure. This is of particular interest as blood cells structural parameters can be used to study and potentially diagnose some psychiatric illnesses such as schizophrenia. Then, a mechanical stress was applied on blood cells by increasing the flow rate inside a closed flow cell. In other words, flow rate was used herein as a disturbing agent. Through simulations and flow cytometry (data not shown), it was possible to determine the velocity and shear stress at different flow rates used to stress the cells. The stressing flow rates were increased by gradually increasing the flow rate until a clear, constant signal was obtained. When the cells in the flow cell were stable at a flow rate of 0 mL / min, the DHM acquisitions started as explained in the examples above. After 3 seconds at 0 mL / min, the flow rate was increased to the stress level determined above and kept constant for 15 seconds. The flow rate was then stopped and returned to 0 mL / min.

[0111] Blood cells mass center displacement was used as the main parameters used to follow the deformation and recovery of each cell within the flow. The blood cell mass center corresponds to the center of mass at each timepoint minus the initial center of mass. The initial blood cell mass center position was determined during the first 3 seconds at 0 mL / min. Mechanical properties ofcells were modeled based on the homeostasis response to stress and recovery and it was confirmed that they are related to the type of cell state tested (i.e., healthy or disease state). Same measurements can be preformed in the presence and in the absence of pharmacological agents known to affect the cell membrane and cytoskeleton mechanical properties.EXAMPLE 6

[0112] A new powerful tool for neuropsychiatric investigation is the induced pluripotent stem cells (iPSCs) providing an opportunity to study astrocytes from individuals and therapeutic response profiles in vitro. Briefly, iPSCs can be generated from various somatic cells such as skin fibroblasts or exfoliated urothelial cells by transient overexpression of the reprogramming transcription factors. In other words, iPSCs clones can be differentiated to any other cell type by exposure to an appropriate combination of patterning molecules. Thus, iPSCs-derived astrocytes provide insight into disease pathogenesis, identifying altered signaling pathways for neuropsychiatric illnesses with a neurodevelopmental component (schizophrenia, bipolar disorder, major depression, autism, addiction, etc.) or neurodegenerative components (Parkinson's, Alzheimer’s, dementia, etc.).

[0113] In the present example, Human-derived astrocytes (HDAs) cultures were obtained from reprogramming HSFs or USCs of Healthy individuals and individuals suffering from neuropsychiatric illnesses. They were cultured in a propagation medium for 14 days, and then for 14 more days in CNTF medium (Neurobasal, 1% Glutamax, 1 % PenStrep, 1% NEAA, 0.2% B27 supplement, 10 ng / mL CNTF (Prospec Cyt-272). After a minimum of 6 weeks of culture, each reprogrammed HDA culture (healthy or ill) is placed on Geltrex Matrigel coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KCI, 5 mM D-glucose±10 mM HEPES, 1.8 mM CaCh, and 0.8 mM MgCh (pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time is used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1-1 Hz). While DHM measurements are maintained, disturbing agents such as 10 to 100 pM tert-butyl hydroperoxide (tBHP), or 10 to 100 pM Noradrenaline, or 10 to 100 pM Acetycholine, or 10 to 100 pM Serotonine, or 10 to 100 pM Dopamine, or 10 to 100 pM Glutamate, or 10 to 100 pM GABA are added to the perfusion medium for 10 min. Then, the initial perfusion medium (i.e., without disturbing agent) is replaced for at least 10 min under constant monitoring by DHM.

[0114] In a second step, all reprogrammed HDAs culture types were pre-incubated with different pharmacological agents (PA; Table 3) known to inhibit specific intracellular pathways prior to the disturbing agent application. Separate experiments respecting the perfusion sequence above were performed for each of the selected PA, during which the HDAs homeostatic capacity (HC) response is measured by DHM. The HC responses in the presence of each pharmacological agents were then be compared to the HC response in the absence of the PA, which was chosen as the reference value and is therefore considered to be the 100% HDAs HC response. These comparative experiments provide an example of screening that can be performed in the identification and mapping of HDAs biomarkers involved in neuropsychiatric illnesses. The present example is only one possible screening, a combination of different biomarkers for different diseases can be used in the same or similar manner as described herein.Table 3. Pharmacological agents screening

[0115] The mapping can be combined with the generation and characterization of induced pluripotent stem cell-derived neurons (Human-Like Neurons, HLNs) stemming from psychiatric individuals: toward cell biomarker identification. For example, HLNs cultures are obtained from reprogramming HSFs or USCs of Healthy individuals and individuals suffering from neuropsychiatric illnesses such as schizophrenia, bipolar disorder, major depression, autism, addiction, Parkinson's, Alzheimer’s, dementia, etc. Reprogrammed HLNs are cultured in DMEM / F12, 1XN2; KSR 10%, 1x B27, 20 ng / ml BDNF, 20 ng / ml BDNF, 20 ng / ml CTNF, 20 ng / ml GDNF, 500 pg / ml Dibutyryl cyclic-AMP, 1 pg / ml doxycycline. After a minimum of 3 weeks of culture, each HLNs culture (healthy or ill) is placed on a Poly-L-Ornithine + Matrigel coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KCI, 5 mM D-glucose±10 mM HEPES, 1.8 mM CaCI2, and 0.8 mM MgCI2(pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time is used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1- 1 Hz). While DHM measurements are maintained, disturbing agents such as 10 to 100 pM tertbutyl hydroperoxide (tBHP), or 10 to 100 pM Noradrenaline, or 10 to 100 pM Acetycholine, or 10 to 100 pM Serotonine, or 10 to 100 pM Dopamine, or 10 to 100 pM Glutamate, or 10 to 100 pM GABA are added to the perfusion medium for 10 min. Then, the initial perfusion medium (i.e., without disturbing agent) is replaced for at least 10 min under constant monitoring by DHM.

[0116] In a second step, all reprogrammed HLNs culture types were pre-incubated with different pharmacological agents (PA ; Table 3) known to inhibit specific intracellular pathways prior to the disturbing agent application. Separate experiments respecting the perfusion sequence above were performed for each of the selected PA, during which the HLNs homeostatic capacity (HC) response is measured by DHM. The HC responses in the presence of each pharmacological agents was then be compared to the HC response in the absence of the PA, which is chosen as the reference value and is therefore considered to be the 100% HLNs HC response. A screening of multiple biomarkers is therefore achieved, however, the screening should not be limited to the biomarkers exemplified herein and can be modified to include more biomarkers and also different biomarkers when considering other disorders.EXAMPLE 7

[0117] In the present example, human-like neurons (HLNs) cultures obtained from human- derived neurons (HDNs) cultures obtained from reprogramming Healthy HSFs. Reprogrammed HDNs are cultured in DMEM / F12, 1XN2; KSR 10%, 1x B27, 20 ng / ml BDNF, 20 ng / ml BDNF, 20 ng / ml CTNF, 20 ng / ml GDNF, 500 pg / ml Dibutyryl cyclic- A MP, 1 pg / ml doxycycline. After a minimum of 3 weeks of culture, HDNs culture is placed on a Poly-L-Ornithine + Matrigel coated plate and then inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KCI, 5 mM D-glucose, 10 mM HEPES, 1.8 mM CaCI2, and 0.8 mM MgCI2(pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements.

[0118]

[0119] While DHM measurements were maintained, 30 pM of glutamate was added to the perfusion medium for 1 min. Then, the initial perfusion medium (i.e., without Glutamate) was replaced for at least 10 min under constant monitoring by DHM. As shown in Fig. 11 , such application of glutamate triggered a decrease of QPS on HDNs triggered a decrease of QPS corresponding to a homeostatic water influx.

[0120] In the second step, Glutamate-disturbed HDNs culture were treated with 40 pM MK801 , a specific antagonist (i.e., pharmacological agent) of NMDA receptors, a subtype of ionotropic glutamate receptor involved in numerous brain physiological processes such as memory and pathological processes such as ischemia. Then, the measured glutamate-induced quantitative phase signal (QPS) strongly decreased, indicating the involvement of functional NMDA receptors in human-like neurons. Therefore, this observation is of particular interest as glutamatergic neurotransmission (i.e., functional glutamate receptors such as NMDA receptors) is playing an important role in memory as well as in the development of many major psychiatric disorders.EXAMPLE 8

[0121] A certain number of evidence supports neuroimmune, metabolic, and homeostatic roles for astrocytes in shaping neuronal function and cognition but also a consequential role in the initiation, progression and symptoms of psychiatric disorders. Thus, astrocytes are crucial for glutamatergic synaptic transmission since they are responsible for the vast majority of glutamateclearance in the brain through glutamate uptake by the Na+-dependent glial glutamate transporter (GLT-1) and the glutamate-aspartate transporter (GLAST). Moreover, astrocytes play key roles in the regulation of brain energy metabolism, which has a major impact on brain functions, including memory, neuroprotection, resistance to oxidative stress and homeostatic tone. Besides, astrocyte energy metabolism is involved of in the pathophysiology of major psychiatric disorders, including schizophrenia, mood disorders, and addictions.

[0122] In the present example, Mice Astrocytes (Mas) were cultured to confluence on Poly- L-ornithine coated glass cover slips in Dulbecco’s modified Eagle’s medium (DM EM / D7777 sigma) containing anti biotics (100 lll / ml penicillin and 100 pg / ml) and 10% fetal calf serum (FCS). Cells were incubated at 37°C in 5% CO2 and the medium was changed twice a week. After two weeks, the medium was replaced by DM EM containing 10% FCS and 250 pM dibutyryl cAMP to induce differentiation. After 21 days in culture, Mas cultures were inserted within a flow cell with a circulating perfusion solution containing 140 mM NaCI, 3 mM KCI, 5 mM D-glucose., 10 mM HEPES, 1.8 mM CaCh, and 0.8 mM MgCh (pH 7.4, 290-295 mOsm, Room temperature, 1-2 mL / min) for at least 15 min prior to DHM measurements. This time was used to obtain the baseline of each cell type for all subsequent DHM measurements (0.1-1 Hz).

[0123] While DHM measurements were maintained, 100 pM of Noradreanaline (NE) was added to the perfusion medium for 40 min and triggered a strong decrease of the quantitative phase signal (QPS), which persisted for the duration of the NE perfusion (Fig. 12A). This observation indicates that MAs astrocytes express functional NE receptors. Additional application of glutamate (Glut; 100pM for 2min) triggered a supplemental transient decrease of QPS concluding to an expression of functional glutamate receptors (Fig. 12A). After a washout of NE, a same application of glutamate triggered a same decrease of QPS, suggesting that the glutamate-induced QPS is independent from the NE-induced QPS through two distinct pathways (Figs. 12A-12B).

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a biomarker or group of biomarkers of a disorder, the method comprising: subjecting cells obtained from an individual having the disorder, suspected of having the disorder, or having a predisposition for the disorder, to a perturbation that destabilizes the homeostasis of the cells; analyzing the response of the cells to the perturbation by imaging; and identifying the biomarker or the group of biomarkers for the disorder with the imaging of the response of the cells to the perturbation.

2. The method of claim 1 , further comprising identifying compounds to treat the disorder by screening a library of compounds and selecting compounds with the ability to normalize the response of the cells to the perturbation, by making the cell response similar or as close as possible to that measured in cells from healthy controls not having the disorder.

3. The method of claim 1 or 2, wherein the cells are stem cells, blood cells, fibroblast cells, microglial cells, skin cells, urinary tract cells, hair follicle cells, olfactory bulb cells, oral cells and nasal mucosal cells.

4. The method of any one of claims 1 to 3, wherein the cells are obtained non-invasively.

5. The method of claim 4, wherein the cells are urinary tract cells, hair follicle cells, oral cells and nasal mucosal cells.

6. The method of any one of claims 1 to 5, further comprising culturing the cells.

7. The method of any one of claims 1 to 6, wherein the disorder is neuropsychiatric disorder.

8. The method of claim 7, wherein the cells obtained are stem cells and they are differentiated to become neural cells.

9. The method of any one of claims 1 to 8, wherein the imaging is performed by fluorescent microscopy or quantitative phase imaging.

10. The method of any one of claims 1 to 9, wherein the step of analyzing the response of the cells further comprises studying a mechanism or underlying mechanism of the response of the cells to the perturbation which explains the response.

11. The method of claim 10, wherein the underlying mechanism includes a subgroup of mechanisms.

12. The method of any one of claims 1 to 11 , wherein the step of subjecting the cells to a perturbation includes subjecting the cells to a panel of disturbances.

13. The method of any one of claims 1 to 12, wherein the step of analyzing is supported by machine learning.

14. The method of any one of claims 1 to 13, wherein the step of analyzing includes performing a biochemical or molecular assay.

15. The method of any one of claims 1 to 14, wherein the perturbation is an oxidative disturbance, an osmotic disturbance, a mechanical strain, a thermal disturbance, an acidbase disturbance, a metabolic disturbance and / or a chemical disturbance.

16. The method of claim 15, wherein the chemical disturbance is performed with one or more neurotransmitters.

17. The method of any one of claims 1 to 16, further comprising treating the individual identified as having the disorder with an appropriate treatment regimen specific for the disorder.

18. A method of diagnosing bipolar disorder in an individual suspected to have bipolar disorder or having a predisposition for bipolar disorder, the method comprising: subjecting cells obtained from the individual to an oxidative disturbance; measuring a change in water efflux in the cells in response to the oxidative disturbance using microscopy; and determining that the individual has bipolar disorder if the water efflux is different than that of healthy control cells subjected to the same oxidative disturbance.

19. The method of claim 18, further comprising culturing the cells.

20. The method of claim 19, wherein subjecting the cells to an oxidative disturbance comprises providing a culture media with 50 pM of f-butyl hydroperoxide.

21. The method of any one of claims 18 to 20, further comprising identifying compounds for treating bipolar disorder by screening a library of compounds and selecting compounds with the ability to normalize water efflux, by making the water efflux similar or as close as possible to that measured in cells from healthy controls not having the disorder.

22. The method of claim 21, further comprising treating the individual with the compounds identified.

23. A method of differentiating bipolar disorder (BD) from major depressive disorder (MDD), the method comprising: subjecting cells obtained from an individual suspected to have BD or MDD to an oxidative disturbance; measuring a change in water efflux in the cells in response to the oxidative disturbance using microscopy; and determining that the individual has bipolar disorder if the water efflux is different than that of control cells obtained from an individual with MDD and subjected to the same culture conditions and oxidative disturbance.

24. A method of treating bipolar disorder in an individual in need thereof, the method comprising: subjecting cells obtained from the individual to an oxidative disturbance; measuring a change in water efflux in the cells in response to the oxidative disturbance using microscopy; determining that the individual has bipolar disorder if the water efflux is different than that of healthy control cells subjected to the same oxidative disturbance; andtreating the individual with a therapeutically effective amount of an agent that normalized the efflux of water of the cells.

25. The method of claim 24, wherein the cells are fibroblast cells, neuronal cells, neuronallike cells, skin cells or urinary tract cells.

26. Use of digital holographic microscopy in the diagnosis of bipolar disorder in an individual by measuring the efflux of water and / or identifying the Ck-related underlying mechanisms in cells of the individual in response to an oxidative disturbance.

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