WO2025247961A1

WO2025247961A1 - Lpa-lpar signaling as a therapeutic target or diagnostic tool for brain diseases

Info

Publication number: WO2025247961A1
Application number: PCT/EP2025/064764
Authority: WO
Inventors: Nicolas TONI; Thomas LARRIEU
Original assignee: Centre Hospitalier Universitaire Vaudois CHUV
Current assignee: Centre Hospitalier Universitaire Vaudois CHUV
Priority date: 2024-05-31
Filing date: 2025-05-28
Publication date: 2025-12-04
Anticipated expiration: 2026-11-30

Abstract

The present invention provides agents and methods of modulating the expression, abundance and/or activity of a Lysophosphatidic acid receptor LPAR or Lysophosphatidic acid (LPA) for the treatment and/or prevention of a neurodevelopmental, a neurological or a psychiatric disorder or disease.

Description

LPA-LPAR signalling as a therapeutic target or diagnostic tool for Brain diseases

FIELD OF THE INVENTION

The present invention provides agents and methods of modulating the expression and/or activity of a Lysophosphatidic acid receptor (LPAR) or Lysophosphatidic acid (LPA) for the treatment and/or prevention of a neuropsychiatric disorder or emotion dysregulation disease or disorder.

BACKGROUND OF THE INVENTION

Anxiety disorders represent the most prevalent forms of mental illnesses, affecting a significant portion of the population (Penninx, B.W., Pine, D.S., Holmes, E.A. & Reif, A. Anxiety disorders. Lancet 397, 914-927 (2021)). Their high chronicity and prevalence contribute to their substantial impact on global health, accounting for approximately 3% of the overall burden of disease (Gustavsson, A. et al. Cost of disorders of the brain in Europe 2010. Eur Neuropsychopharmacol 21, 718-779 (2011)). Moreover, anxiety frequently co-occurs with various psychiatric conditions, exacerbating the severity of these disorders and increasing the risk of recurrence or relapse (Goldstein-Piekarski, A.N., Williams, L.M. & Humphreys, K. A trans-diagnostic review of anxiety disorder comorbidity and the impact of multiple exclusion criteria on studying clinical outcomes in anxiety disorders. Transl Psychiatry 6, e847 (2016)). Unfortunately, anxiety often goes untreated, leading to prolonged suffering for individuals and a persistent burden on healthcare systems.

Thus, to gain a deeper understanding of the underlying mechanisms driving anxiety and its interplay with other psychiatric disorders, adopting a trans-diagnostic framework that incorporates anxiety as a fundamental dimension is crucial.

Implementing a trans-diagnostic perspective can also pave the way for personalized treatment approaches. SUMMARY OF THE INVENTION

The present invention provides an agent modulating the expression and/or activity of a Lysophosphatidic acid receptor LPAR or the concentration of circulating Lysophosphatidic acid (LPA16:0) for use in the treatment and/or prevention of a disease affecting, or linked to, the hippocampus, wherein the LPAR is selected from the group consisting of LPAR1 and LPAR3, or a combination thereof, and wherein the disease affecting, or linked to, the hippocampus is a neuropsychiatric disorder or emotional dysregulation disease or disorder.

The present invention also provides a pharmaceutical composition comprises a therapeutically effective amount of an agent of the invention, and a pharmaceutically acceptable carrier or diluent.

The present invention also provides methods of treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance of LPA16:0, i.e. a psychiatric disorder or emotional dysregulation disorder.

The present invention also provides a method for diagnosing a neurodevelopmental, a neurological or a neuropsychiatric disorder or disease characterized by an overexpression or an overabundance of LPA16:0 in a subject, the method comprising:

(a) detecting and measuring the level of LPA16:0 in a biological sample obtained from said subject;

(b) comparing said LPA16:0 level to a control biological sample for the same LPA16:0; wherein a differential of LPA16:0 level in said biological sample, relative to the level of corresponding said LPA16:0 in a control biological sample, is indicative of the subject having a neurodevelopmental, a neurological or a psychiatric disorder or disease characterized by an overexpression of LPA16:0, i.e. a psychiatric disorder or emotional dysregulation disorder.

Further provided is a kit for performing a method according to the invention or for use in the treatment and/or prevention of a psychiatric disorder or emotional dysregulation disorder, said kit comprising

(a) one or more agents and/or pharmaceutical compositions, and

(b) instructions for use. DESCRIPTION OF THE FIGURE

Figure 1. LPA16:0-LPARi signaling regulates stress-susceptibility and neurogenesis.

A. Experimental design for the anxiety trait assessment and LPA16:0 measurement. B. Histogram of the anxiety score of spontaneously low anxious (LA) and high anxious (HA) mice. C. Histogram of the concentration of LPA16:0 in the serum of LA vs HA mice. D. Correlation between LPA16:0 serum concentrations and individual anxiety score. E. Receiver Operating Characteristic (ROC) curve fort the discrimination of high anxious individuals by LPA16:0. F. Experimental design for in vivo cLPA16:0 administration. G. Histogram of the anxiety score of vehicle and cLPA16:0-treated mice under baseline condition post treatment (left). Anxiety score following a sub threshold 20minute-acute restraint stress (right). H. Quantification of Ki-67⁺ cells in the total dentate gyrus (left). aNPC proliferation in the BBA assay after treatment with serum from vehicle- and cLPA16:0-treated mice (right). I. Pearson correlation between individual serum cLPA16:0 concentration and aNPC proliferation in the BBA assay. J. Experimental design for in vivo KI16425 (LPARi inhibitor) administration under acute stress condition. K. Histograms of the anxiety score of mice injected with vehicle or KI16425 after 12 days of treatment under baseline condition (left) and after 6 hours of ARS (right). L. Quantification of Ki-67⁺ cells in the total dentate gyrus. M. Representative confocal micrographs of Ki-67 and DAPI staining in the DG in vehicle-, and cLPA-treated groups. N. Experimental design for in vivo KI16425 administration under chronic stress condition. O Histograms of the anxiety score (left) and marbles buried (middle) of control and stress mice injected with vehicle or KI16425 after 25 days of treatment under baseline condition and (right) after a 6 minute-FST challenge. P. Quantification of Ki-67⁺ cells in the total dentate gyrus. Q. Experimental design for in vivo KI16425 and TMZ (Temozolomide, a brainpermeant inhibitor of cell proliferation) administration under acute stress condition. R. Histograms of the anxiety score of mice injected with KI16425 and TMZ under baseline condition (left) and after 6 hours (right) of ARS. S. Quantification of Ki-67⁺ cells in the total dentate gyrus. T. Experimental design for in vivo KI16425 and TMZ administration under chronic stress condition. U-W. Histograms of (U) marbles buried, (V) latency to eat in a novelty suppress feeding test of control and stress mice injected with KI16425 and TMZ. W. Quantification of Ki-67⁺ cells in the total dentate gyrus. X. Number of newly-formed neurons. Histograms show average ± SEM. * p<0.05; ** p<0.01; ** p<0.01; ns: not significant. Figure 2 Platelet depletion reduces circulating LPA16:0 and increases stress resilience andadult neurogenesis.

A. Table showing platelets count and levels of LPA16:0 in activated platelet rich plasma from mice treated with an anti-platelet serum or control serum for 2 days. B. Timeline showing the experimental design. Mice received an intraperitoneal injection of anti-platelet serum or control serum every other day for 20 days, followed by assessment of resilience to a 6 hours ARS. C, D. Histograms of the anxiety score of mice injected with control (white) or anti-platelet serum ( grey) after 20 days of treatment under (C) baseline conditions (tl4 = 2.922, p = 0.0011, unpaired t-test, two-tailed; control: t = 1.651, p > 0.05, One sample t test and anti -platelet: t =2.610, p = 0.0349, One sample t test, n = 8 mice per group) and (D) after 6 hours of ARS (tl4 =5.005, p = 0.0002, unpaired t-test, two-tailed; control: t = 11.29, p < 0.0001, One sample t test and anti -platelet: t = 0.852, p > 0.05, One sample t test, n = 8 mice per group). E. Quantification of Ki-67+ cells in the DG (t 14 = 2,452, p = 0.0279, unpaired t-test, two-tailed, n = 8 per group). Histograms show average ± SEM. * p<0.05; ** p<0.01; *** p<0.001; ns: not significant. Comparison between the group mean and the hypothetical value of 0.5, to assess anxiety withing each group are shown within each histogram bar. One-sample t-test. #: p<0.05; ##: p<0.01

Figure 3. Platelet depletion reduced LPA16:0 but not LPA18:1

Effect of the anti- platelets on platelets count and LPA 16:0 levels. Table representing the concentrations of the different forms of LPA in platelet-depleted mice (top line) or vehicle- treated mice (bottom line).

DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise/comprising" is generally used in the sense of "include/including", that is to say permitting the presence of one or more features or components. This term also encompasses the more restricted term "consist/consisting".

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, "at least one" means "one or more", "two or more", "three or more", etc.

The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus ten (10) percent (%).

While conducting analysis with untargeted metabolomics, the Inventors surprisingly identified the lysophosphatidic acid (LPA)16:0 as one of the most significantly increased systemic lipids released in anxious human patients and spontaneously anxious mice. Also, elevated levels of serum LPA16:0 is associated with individual trait anxiety in both mice and humans strongly suggesting that LPA16:0 is involved in emotional regulation. Until now, the scientific community has mainly focused on the study of LPA18: 1.

As used herein, the terms "peptide", "protein", "polypeptide", "polypeptide chain", "polypeptidic" and "peptidic" are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

As used herein the terms "subject"/" subject in need thereof', or "patient"/"patient in need thereof " are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other aspects, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Preferably, the subject is a human, most preferably a human suffering from a neurodevelopmental, a neurological or a psychiatric disorder or disease or a human that might be at risk of suffering from a neurodevelopmental, a neurological or a psychiatric disorder or disease.

A disease affecting, or linked to, the hippocampus refers to any medical condition characterized by structural, functional, biochemical, or physiological abnormalities in the hippocampus region of the brain, which may result in, or contribute to, impairments in memory, learning, spatial navigation, emotional regulation, or other hippocampus-mediated processes.

Individuals diagnosed with anxiety disorders often exhibit heightened reactivity to stress. One of the cellular mechanisms underlying pathological stress response involves alterations of brain plasticity (McEwen, B.S. et al. Mechanisms of stress in the brain. Nat Neurosci 18, 1353-1363 (2015)), specifically affecting the most drastic form of it known as adult neurogenesis (Surget, A. & Belzung, C. Adult hippocampal neurogenesis shapes adaptation and improves stress response: a mechanistic and integrative perspective. Mol Psychiatry 2022 Jan;27(l):403-421. doi: 10.1038/s41380-021-01136-8. Epub 2021 May 14. 2022). Adult neurogenesis refers to the continuous generation of new neurons in the adult brain, a process primarily observed in the hippocampus (Altman, J. & Das, G.D. Post-natal origin of microneurones in the rat brain. Nature 207, 953-956 (1965)). In the hippocampus, adult neurogenesis is highly sensitive to changes in living conditions such as social isolation (Stranahan, A.M., Khalil, D. & Gould, E. Social isolation delays the positive effects of running on adult neurogenesis. Nat Neurosci 9, 526-533 (2006)), inflammation (Goshen, I. et al. Brain interleukin- 1 mediates chronic stress-induced depression in mice via adrenocortical activation and hippocampal neurogenesis suppression. Mol Psychiatry 13, 717-728 (2008)) or chronic stress (Zeh, B. et al. Chronic psychosocial stress and concomitant repetitive transcranial magnetic stimulation: effects on stress hormone levels and adult hippocampal neurogenesis. Biol Psychiatry 52, 1057-1065 (2002)), all of which are associated with emotional deficits (Lehmann, M.L., Brachman, R.A., Martinowich, K., Schloesser, R.J. & Herkenham, M. Glucocorticoids orchestrate divergent effects on mood through adult neurogenesis. J Neurosci 33, 2961-2972 (2013)). In such conditions, the generation of new neurons in the hippocampus is adversely affected. Inversely, the presence of new hippocampal neurons is crucial for regulating mood (Anacker, C. & Hen, R. Adult hippocampal neurogenesis and cognitive flexibility - linking memory and mood. Nat Rev Neurosci 18, 335-346 (2017)), promoting stress resilience (Anacker, C. et al. Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus. Nature 559, 98-102 (2018)), and mediating the effects of antidepressant treatments (Santarelli, L. et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301, 805-809 (2003)). Finally, experimental evidence demonstrates that increasing adult neurogenesis itself can produce antidepressant effects (Hill, A.S., Sahay, A. & Hen, R. Increasing Adult Hippocampal Neurogenesis is Sufficient to Reduce Anxiety and Depression-Like Behaviors. Neuropsychopharmacology 40, 2368-2378 (2015)). Thus, adult neurogenesis is both a key target of anxiety and stress-related mechanisms as well as a mediator of stress resilience. However, the underlying mechanisms driving anxiety / stress vulnerability and its impact on adult neurogenesis are not yet fully understood.

The Inventors identified LPA16:0, a circulating lysophosphatidic acid as necessary modulator of stress-induced anxiety and hippocampal aNPC activity. They showed that LPA16:O-LPAR1 signaling is both sufficient and necessary to mediate stress susceptibility/resilience. Using pharmacological strategy of neurogenesis ablation, they also showed that neurogenesis is necessary for LPAR1 antagonist-mediated stress resilience.

Furthermore, they demonstrated that systemic inhibition of platelet function reduced LPA16:0 and can mimic the beneficial effects of LPAR1 antagonist by enhancing adult hippocampal NPC proliferation and conferring stress resilience in mice.

The present invention thus provides an agent modulating the expression and/or activity of a Lysophosphatidic acid receptor LPAR or the concentration of circulating Lysophosphatidic acid (LPA16:0) for use in the treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance of LPA16:0.

In one aspect, the disease or disorder is affecting, or linked to, the hippocampus, and is selected from a neuropsychiatric disorder or an emotional dysregulation disease or disorder. In one aspect, the LPAR is selected from the group consisting of LPAR1 and LPAR3, or a combination thereof.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator primarily derived from membrane phospholipids. LPA initiates cellular effects upon binding to a family of G protein-coupled receptors, termed LPA receptors. LPA can be detected in many body fluids, although blood (platelets) is the major source of this lipid mediator in mammals [Yung YC, Stoddard NC, Chun J. LPA receptor signaling: pharmacology, physiology, and pathophysiology. J Lipid Res. 2014;55: 1192-214], LPA is synthetized through different metabolic pathways, being the autotaxin (ATX) the primary enzyme responsible of its production. The synthesis of LPA through the different metabolic routes results in the production of several LPA species, depending on the acyl group, that may differ in their biological actions. Among these different chemical species, the most abundant in human blood are the 16:0, 18:0,18: 1, 18:2 and 20:4 acyl LPA [Baker DL, Desiderio DM, Miller DD, Tolley B, Tigyi GJ. Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry. Anal Biochem. 2001;292:287-95],

There are six receptors that interact with lysophosphatidic acid (LPA): protein names LPA1 - LPA6 and italicized gene names LPAR1-LPAR6 (human) and Lparl-Lpar6 (non-human). In the present context, LPA receptors are referred as LPAR1-LPAR6. These receptors are essentially known for their implication in cancer.

According to one aspect, the agent of the invention modulates at least one LPAR which is selected from the group comprising LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6, or a combination thereof (such as e.g. LPAR1/LPAR3, ...). Preferably, the agent of the invention modulates at least one LPAR selected from LPAR1, LPAR3, or a combination thereof.

LPAR1, is known to enhance metastasis and tumor motility. Aberrant LPAR1 expressions were observed in many cancer cell lines and primary tumor.

LPAR2 activation has been shown to associate with cell survival because of its anti-apoptosis function.

LPAR3 is the predominant receptor subtype in colon, liver, and lung cancers. LPAR3- expressing cells significantly promote motility and invasiveness through Ras-, Rac-, Rho-, and PI3K-signaling pathways. Direct targeting of LPAR3 by miR-15b has been shown to repress cell proliferation and drive the senescence and apoptosis of ovarian cancer cells through the PI3K/Akt pathway, suggesting the potential mRNA treatment against ovarian cells (Li GC, Qin XL, Song HH, Li YN, Qiu YY, Cui SC, Wang YS, Wang H, Gong JL. Upregulated microRNA- 15b alleviates ovarian cancer through inhibition of the PI3K/Akt pathway by targeting LPAR3. J Cell Physiol. 2019 Dec;234(12):22331-22342).

In contrast to LPAR1-3, LPAR4 and LPAR5 have been shown to negatively affect cancer cell proliferation and motility. LPAR4 has been shown to attenuate tumor motility and colony formation in colon cancer cell lines.

LPAR5 was considered a negative regulator in cancer cell motility and survival. The inhibitory effect of LPAR5 on cell motility has been shown in pancreatic cancer and sarcoma.

Reports regarding LPAR6 in cancer are relatively limited compared with other LPARs. Several articles investigated the role of LPAR6 in liver, pancreatic, and colon cancers. LPAR6 expression in hepatocellular carcinoma correlated with poorer survival and increased microvascular invasion.

According to one aspect of the invention, the modulation of the expression and/or activity of a Lysophosphatidic acid receptor LPAR corresponds to a decrease equal or superior to about 5 %, preferably equal or superior to about 20 %, more preferably equal or superior to about 40 %, most preferably equal or superior to about 60 %, more preferably equal or superior to about 500%, even more preferably equal or superior to about 1000 %, in particular equal or superior to about 5000 % when compared to the level of corresponding expression and/or activity level of said LPAR determined previously (e.g. in a control biological sample or a reference sample or value).

According to one aspect of the invention, the modulation of the concentration of circulating Lysophosphatidic acid (LPA16:0) corresponds to a decrease equal or superior to about 5 %, preferably equal or superior to about 20 %, more preferably equal or superior to about 40 %, most preferably equal or superior to about 60 %, more preferably equal or superior to about 500%, even more preferably equal or superior to about 1000 %, in particular equal or superior to about 5000 % when compared to the level of corresponding expression and/or activity level of said LPAR determined previously, i.e. a normal biological sample (e.g. a control sample) or from a reference biological sample. The concentration of the circulating Lysophosphatidic acid (LPA16:0) is usually expressed in nM.

According to one aspect of the invention, the modulation comprises, or consist of, i) reducing the expression of LPA, ii) reducing the biological activity of LPA, iii) reducing the expression of a LPAR, and/or iv) impairing the binding of LPA to an LPAR.

In one aspect, the receptor (LPAR) is selected from the group comprising LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6, or a combination thereof (such as e.g. LPAR1/LPAR3, . . .). In a preferred aspect, the receptor is selected from the group comprising LPAR1, LPAR3, and a combination thereof (LPAR1/LPAR3).

The agent of the invention can be selected from the group comprising a nucleic acid, a chemical compound, a peptide or analog thereof, an antibody or an antigen-binding fragment thereof, and an antibody mimetic, or a combination of one or more thereof.

The terms "nucleic acid", "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any kind of deoxyribonucleotide (e.g. DNA, cDNA, ...) or ribonucleotide (e.g. RNA, mRNA, ...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g. DNA/RNA) polymer, in linear or circular conformation, and in either single - or double - stranded form. These terms are not to be construed as limiting with respect to the length of a polymer and can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g. phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity, i.e., an analogue of A will base-pair with T.

In case the agent of the invention is a nucleic acid, then it will preferably be selected from the non-limiting group comprising a nucleic acid encoding a siRNA, a shRNA, a miRNA, a transfer RNA (tRNA), a piRNA, a heterogeneous nuclear RNA (hnRNA), an snRNA, an sgRNA used in a CRISPR-based loss- or gain-of-function system, an esiRNA, a single- stranded DNA, and an antisense oligonucleotide, or a fragment of one thereof or a combination of one or more thereof.

In one aspect, the nucleic acids described below will modulate the expression of a gene encoding an LPAR of the invention, preferably LPAR1 and/or LPAR3 , or a transcript of a gene encoding the LPAR1 or LPAR3 of the invention, or a regulatory sequence that controls the transcription of the gene encoding the LPAR1 or LPAR3 of the invention, or of an enzyme involved in the LPA16:0 synthesis (such e.g. acyltransferases, phospholipases, kinases) by acting on its mRNA.

The terms “microRNA,” “miRNA,” and “MiR” are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference, e.g. on a transcript of a gene encoding the LPAR1 or 3 of the invention such as on a transcript of a gene encoding the LPAR1 or 3 (see e.g. Li GC, Qin XL, Song HH, Li YN, Qiu YY, Cui SC, Wang YS, Wang H, Gong JL. Upregulated microRNA- 15b alleviates ovarian cancer through inhibition of the PI3K/Akt pathway by targeting LPAR3. J Cell Physiol. 2019 Dec;234(12):22331-22342).

The terms “siRNA” and “short interfering RNA” are interchangeable and refer to singlestranded or double-stranded RNA molecules that are capable of inducing RNA interference, e.g. on a transcript of a gene encoding the LPAR1 or 3 of the invention. siRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length (see e.g. Orosa B, Gonzalez A, Mera A, Gomez-Reino JJ, Conde C. Lysophosphatidic acid receptor 1 suppression sensitizes rheumatoid fibroblast-like synoviocytes to tumor necrosis factor-induced apoptosis. Arthritis Rheum. 2012 Aug;64(8):2460-70).

The terms “piRNA” and “Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between about 26 and about 31 nucleotides in length.

The terms “shRNA” as used herein refers to a nucleic acid molecule comprising at least two complementary portions hybridized or capable of specifically hybridizing to form a duplex structure sufficiently long to mediate RNAi (typically between about 15 to about 29 nucleotides in length), and at least one single-stranded portion, typically between approximately 1 and about 10 nucleotides in length that forms a loop connecting the ends of the two sequences that form the duplex. The mechanism of RNAi is based on the sequence-specific degradation of host mRNA (e.g. of a transcript of a gene encoding the LPAR1 or 3 of the invention) through the cytoplasmic delivery of double-stranded RNA (dsRNA) identical to the target sequence (see e.g. Doutt SW, Longo JF, Carroll SL. LPAR1 and aberrantly expressed LPAR3 differentially promote the migration and proliferation of malignant peripheral nerve sheath tumor cells. Glia. 2023 Mar;71(3):742-757).

The terms “snRNA” and “small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included. The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs.

According to one aspect of the invention, the expression of a gene encoding an LPAR 1 or LPAR3 of the invention, or a regulatory sequence that controls the transcription of the gene encoding the LPAR of the invention, or of an enzyme involved in the LPA16:0 synthesis (such e.g. acyltransferases, phospholipases, kinases) can be downregulated by using a gene editing system such as, e.g. the CRISPR-based gain/loss-of-function system. Usually, the CRISPR- based gain/loss-of-function system comprises at least one single guide RNA (sgRNA), or crRNA and tracrRNA, and a structure-guided endonuclease such as an RNA-guided endonuclease.

A non-limiting example of an enzyme involved in the LPA16:0 synthesis is autotaxin, which encoded by the ENPP2 human gene.

The terms “sgRNA” and “guideRNA” are interchangeable and refer to a specific RNA sequence that recognizes the target DNA region of interest and directs the endonuclease there for editing. The gRNA is usually made up of two parts: crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for a Cas nuclease. A specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene can also be present. Any suitable naturally occurring, or engineered, RNA-guided endonuclease can be employed as long as it is effective for binding a target DNA and it may be selected from the non-limiting group comprising Cas9, Casl2, Cpfl, and FEN-1. Preferably, the RNA-guided endonuclease is Cas9.

Within the context of this disclosure, the term "target DNA" refers, according to one aspect, to a gene encoding the LPAR of the invention (i.e. gene encoding LPAR1 and/or 3) as disclosed above, to a gene encoding an enzyme involved in the LPA synthesis, or to a regulatory sequence that controls the transcription of said genes.

Meng et al disclose a CRISPR-Cas) system inhibiting LPAR1 by silencing its gene (Meng F, Yin Z, Lu F, Wang W, Zhang H. Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts. Thorac Cancer. 2024 Feb;15(4):316-326).

The CRISPR/Cas9 system has become a remarkably flexible tool for genome manipulation over the years. A unique feature of Cas9 endonuclease is its ability to bind target DNA independently of its ability to cleave target DNA.

Within the context of this disclosure, the Cas9 endonuclease is preferably a modified Cas9 endonuclease such as, e.g. an enzymatically dead Cas9. Specifically, both RuvC- and/or HNH- nuclease domains can be rendered inactive by point mutations (e.g. D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 molecule that cannot cleave target DNA. However, the dead Cas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence, which sgRNA sequence is comprised in CRISPR-based gain/loss-of-function system.

In one aspect, the enzymatically dead Cas9 is tagged with one or more transcriptional repressors (see Andriy Didovyk, Bartlomiej Borek, Lev Tsimring, and Jeff Hasty. Curr Opin Biotechnol. 2016 Aug; 40: 177-184 which is incorporated herein by reference).

In another aspect, the enzymatically dead Cas9 is tagged with one or more epitope that is/are recognized by one or more antibody-activator/repressor effector. This enzymatically tagged dead Cas9 can then target the regulatory sequence resulting in robust transcription repression downstream target gene encoding an LPAR or an enzyme involved in the LPA synthesis of the invention. Designing and selecting a suitable siRNA, a shRNA, a miRNA, a tRNA, a piRNA, a hnRNA, a snRNA, a sgRNA used in a CRISPR-based loss- or gain-of-function system, an esiRNA, a single-stranded DNA, and an antisense oligonucleotide is well within the competences of one of ordinary skill in the art using routine experimentation, several commercial and noncommercial web sites available for nucleic acid design as well as the information provided herein (for a review e.g. Glen F. Deleavey, et al., Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing, Chemistry & Biology, Volume 19, Issue 8, 2012, Pages 937-954 as well as other references cited herein).

According to one aspect, the antisense oligonucleotide selectively targeting a gene encoding the LPAR 1 or LPAR3 of the invention, a transcript of a gene encoding the LPAR1 or LPAR3 of the invention, a gene encoding an enzyme involved in the LPA16:0 synthesis or any transcripts thereof, or a regulatory sequence that controls the transcription of said genes, is a modified antisense oligonucleotide. Preferably, the modified antisense oligonucleotide is a GapmeR or a GapmeR with fixed chemical modification architectures. More preferably, the GapmeR and/or GapmeR with fixed chemical modification selectively targets an exon of a gene encoding the LPAR of the invention, a gene encoding an enzyme involved in the LPA synthesis, or a regulatory sequence that controls the transcription of said genes.

A GapmeR with fixed chemical modification architectures is usually selected from the group comprising i) a gapmer with five 2'-O-methoxy ethyl (MOE) modifications in each flank, and a central gap of 10 unmodified dans (e.g. 5-10-5 MOE design), and ii) a gapmer employing three or four locked nucleic acid (LNA) modifications in each flank (e.g. 3-10-3 or 4-8-4 LNA designs), or a combination of one or more thereof (for a review e.g. Natalia Papargyri, et al., Chemical Diversity of Locked Nucleic Acid-Modified Antisense Oligonucleotides Allows Optimization of Pharmaceutical Properties, Molecular Therapy - Nucleic Acids, Volume 19, pages 706-717, 2020).

Non-limiting examples of a transcript of a gene encoding the LPAR1 or LPAR3 of the invention will be selected from the transcripts listed in Table 1. Table 1

*https://www.ensembl.org/

Examples of a human gene encoding the LPAR1 or 3 of the invention will be selected from ENSG00000198121.15 (LPAR1 gene) and ENSG00000171517.6 (LPAR3 gene).

As used herein, a “chemical agent” is a compound that produces change by virtue of its chemical composition and its effects on living tissues and organisms. The chemical agent of the invention may be a small molecule inhibitor (SMI), preferably a non-peptidyl molecule modulating the expression and/or activity of the LPA or LPAR of the invention.

The chemical agents of the invention can be tested using a number of techniques known to those of skill in the art.

According to one aspect of the invention, the chemical compound is preferably an antagonist inhibitor of aLPARl, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6, or a combination thereof. More preferably, the chemical compound is an antagonist inhibitor of a LPAR1 or LPAR3, or a combination thereof.

Non-limiting examples of antagonist inhibitors of LPAR1 will be selected from the group comprising KH6425 (also known as Debio-0719, CAS No. 355025-24-0), KH6198 (CAS No. 147776-06-5), BMS-986020 (CAS No. 1257213-50-5), BMS-986278 (CAS

No. : 2170126-74-4), HY- 100619 (CAS No. : 1257213-50-5), AM095 (CAS No. : 1345614-59- 6), H2L5765834 (CAS No. : 420841-84-5), HY-16039 (CAS No. : 1345614-59-6), TAK-615 (CAS No. : 1664335-55-0), ONO-9780307 (CAS No. : 856691-44-6), ONO-0300302 (CAS No. : 856689-51-5), ML-161AZ-3451, ML-184 (CAS No. : 794572-10-4), Piperidine 18, Ro 6842262 (CAS No. : 1396006-71-5), AM966 (CAS No. : 1228690-19-4), AM095 (CAS No. : 1345614-59-6), CAS No. 1396006, -71-54-(4-(2-Isopropylphenyl)-4-((2-methoxy-4- methylphenyl)carbamoyl)piperidin-l-yl)-4-oxobutanoic Acidand SAR- 100842, or a combination thereof. Other antagonist inhibitors of LPAR1 can be found in Lescop C, et al. Discovery of a Novel Orally Active, Selective LPA Receptor Type 1 Antagonist, 4-(4-(2- Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin-l-yl)-4-oxobutanoic Acid, with a Distinct Molecular Scaffold. J Med Chem. 2024 Feb 22;67(4):2379-2396.

Non-limiting examples of antagonist inhibitors of LPAR2 will be selected from the group comprising KH6425, HY-18075, H2L5186303 as well as those disclosed in PCT/EP2011/003949 (WO2012028243), or a combination thereof.

Non-limiting examples of antagonist inhibitors of LPAR3 will be selected from the group comprising KH6425 (Debio-0719) and H2L5765834 (CAS No. : 420841-84-5), or a combination thereof.

Non-limiting examples of antagonist inhibitors of LPAR4 will be selected from the group comprising AM966 and BrP-LPA, or a combination thereof.

Non-limiting examples of antagonist inhibitors of LPAR5 will be selected from the group comprising H2L5765834, AS2717638 and TC LPA5 4, or a combination thereof.

Non-limiting examples of antagonist inhibitors of LPAR6 will be selected from the group comprising 4-methylene-2-octyl-5-oxotetra-hydrofuran-3-carboxylic acid (C75) and 9- xanthenyl acetic acid (XAA), or a combination thereof.

The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids. According to one aspect of the invention, the agent of the invention is an antibody, or antigen binding fragment thereof, that inhibits and/or impairs the binding of the LPA to at least one of its receptors (LPAR 1 to 6). Preferably, the LPA receptor is selected from LPAR1 or LPAR3, or a combination thereof (see e.g. Wasniewski T, Woclawek-Potocka I, Boruszewska D, Kowalczyk-Zieba I, Sinderewicz E, Grycmacher K. The significance of the altered expression of lysophosphatidic acid receptors, autotaxin and phospholipase A2 as the potential biomarkers in type 1 endometrial cancer biology. Oncol Rep. 2015 Nov;34(5):2760-7; and range of antibodies targeting LPARs provided by Alomone Labs https://www.alomone.com/).

As used herein, an “antibody” is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five distinct classes based on structural properties: IgA, IgD, IgE, IgG and IgM. The antibody may be a polyclonal (e.g. a polyclonal serum) or a monoclonal antibody, including but not limited to fully assembled antibody, single chain antibody, antibody fragment, and chimeric antibody, humanized antibody as long as these molecules are still biologically active and still bind to at least one peptide or protein of the invention. Preferably the antibody is a monoclonal antibody. Preferably also the monoclonal antibody will be selected from the group comprising the IgGl, IgG2, IgG2a, IgG2b, IgG3 and IgG4 or a combination thereof. Most preferably, the monoclonal antibody is selected from the group comprising the IgGl, IgG2, IgG2a, and IgG2b, or a combination thereof.

An “antigen binding fragment" comprises a portion of a full-length antibody. Examples of antigen binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

According to one aspect of the invention, the modulation comprises, or consist of, reducing the expression and/or the abundance of LPA16:0 by inhibiting platelet function.

In one aspect, the agent of the invention inhibiting platelet function can be selected from the group comprising a nucleic acid, a chemical compound, a peptide or analog thereof, an antibody or an antigen-binding fragment thereof, and an antibody mimetic, or a combination of one or more thereof. In one aspect, the agent reduces the amount of platelets i) by blocking the activity of the cyclooxygenase- 1 (COX-1) enzyme, ii) by inhibiting the P2Y12 receptor, iii) by inhibiting the ADP receptor, iv) by preventing the binding of fibrinogen or other proteins to the GPIIb/IIIa receptor, v) by inhibiting the PAR-1 receptor, vi) and/or by inhibiting a phosphodiesterase enzyme (PDE) selected from PDE2, PDE3 and PDE5.

According to one aspect of the invention, the modulation of the amount of platelets results in a decrease equal or superior to about 5 %, preferably equal or superior to about 20 %, more preferably equal or superior to about 40 %, most preferably equal or superior to about 60 %, more preferably equal or superior to about 500%, even more preferably equal or superior to about 1000 %, in particular equal or superior to about 5000 % when compared to the level of corresponding expression and/or activity level of said LPAR determined previously, i.e. a normal biological sample (e.g. a control sample) or from a reference biological sample. The amount of platelets is usually expressed as the number of platelets /pL of whole blood and is the result of a platelet count (PLT), a test that measures the number of platelets in blood.

In one aspect, the agent is an antibody, or antigen binding fragment thereof, that inhibits and/or impairs platelet aggregation. A non-limiting example of such agent comprises abciximab, an antiplatelet humanized chimeric Fab fragment of 7E3 that prevents blood clots by inhibiting platelet aggregation by specifically binding to the glycoprotein Ilb/IIIa (GP Ilb/IIIa) receptor on platelets.

In one aspect, the agent is a peptide or analog thereof, that inhibits and/or impairs platelet aggregation. Non-limiting examples of such agent comprise Eptifibatibe, a cyclic heptapeptide inhibiting glycoprotein Ilb/IIIa, or a prostacyclin analogue such as Ilpoprost.

In one aspect, the agent is a chemical compound, preferably a small molecule inhibitor belonging to a class of antiplatelets named glycoprotein Ilb/IIIa inhibitors. A non-limiting example of such agent comprises tirofiban, a small molecule inhibitor of the protein-protein interaction between fibrinogen and the platelet integrin receptor GP Ilb/IIIa.

In one aspect, the agent is a chemical compound, preferably a small molecule inhibiting a phosphodiesterase enzyme (PDE) selected from PDE2, PDE3 and PDE5. Non-limiting examples of such agent comprise those described in e.g. Gresele P, Momi S, Falcinelli E. Antiplatelet therapy: phosphodiesterase inhibitors. Br J Clin Pharmacol. 2011 Oct;72(4):634-46). In one aspect, the molecule inhibiting a phosphodiesterase enzyme (PDE) is selected from the group comprising cilostazol, dipyridamole, milrinone and anagrelide, or a combination thereof.

In one aspect, the agent is a chemical compound, preferably a small molecule inhibiting the protease-activated receptor 1 (PAR-1). Non-limiting examples of such agent comprise vorapaxar and atopaxar.

In one aspect, the agent is a chemical compound, preferably a small molecule inhibitor, more preferably an antagonist inhibitor of the P2Y12 receptor, a chemoreceptor for adenosine diphosphate (ADP) that belongs to the Gi class of a group of G protein-coupled (GPCR) purinergic receptors. Non-limiting examples of such agent comprise clopidogrel, ticlopidine, ticagrelor, prasugrel, and cangrelor.

In one aspect, the agent is a chemical compound, preferably a small molecule inhibitor, more preferably an inhibitor of the activity of the cyclooxygenase- 1 (COX-1) enzyme. Nonlimiting examples of such agent comprise acetylsalicylic acid (aspirin) as well as derivatives thereof (e.g. NCX-4016), or a pro-drug thereof.

According to an aspect, the agent of the invention is for use in the treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance of LPA16:0. According to an aspect of the invention, the disease characterized by an overexpression or overabundance of circulating LPA16:0 is reducing adult hippocampal neurogenesis and stress resilience, thereby leading to depression. In one aspect, the disease is affecting, or linked to, the hippocampus. The inventors have found that increased levels of LPA16:0 in the bloodstream are associated with a reduction in adult neurogenesis within the hippocampus. Moreover, inhibition of one of LPA's receptor (e.g. the LPAR1 receptor) was observed to counteract the adverse effects of chronic stress on adult hippocampal neurogenesis.

According to one aspect, the disease or disorder characterized by an overexpression or overabundance of LPA16:0 corresponds to a psychiatric disorder or emotional dysregulation disorder selected from the group comprising schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type, cognitive impairment associated with schizophrenia (CIAS), bipolar disorder, ADHD, anxiety, anxiety- related disorders, depression, cognitive dysfunction, borderline personality disorder (BPD), depression or cognitive impairment induced by cytokines or chemotherapies administration, inflammation-related cognitive impairment or mood impairment, Alzheimer’s disease, Parkinson's disease, Fragile X syndrome, post-traumatic stress disorder, or a combination of two or more thereof.

Preferably, the disease or disorder is selected from the group comprising schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type, cognitive impairment associated with schizophrenia (CIAS), autism spectrum disorder, bipolar disorder (BD), attention-deficit/hyperactivity disorder (ADHD), anxiety, anxiety- related disorders, depression, cognitive dysfunction, borderline personality disorder (BPD), depression or cognitive impairment induced by cytokines or chemotherapies administration, inflammation-related cognitive impairment or mood impairment, post- traumatic stress disorder (PTSD), frontal temporal dementia, or a combination of two or more thereof.

In one aspect, the modulation reduces the concentration of LPA16:0 in the blood and/or in the brain. The present invention further contemplates one or more pharmaceutical compositions. In one aspect, the pharmaceutical composition comprises a therapeutically effective amount of an agent of the invention, and a pharmaceutically acceptable carrier or diluent.

In one aspect, the pharmaceutical composition of the invention is for use in the treatment and/or prevention of a disease or disorder characterized by an overexpression of LPA 16:0, i.e. a psychiatric disorder or an emotional dysregulation disease.

The term "therapeutically effective amount" as used herein means an amount of an agent of the invention high enough to significantly positively modify the symptoms and/or condition to be treated, but low enough to avoid serious side effects (at a reasonable risk/benefit ratio), within the scope of sound medical judgment. The therapeutically effective amount of the agent of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the agent required to prevent, counter or arrest the progress of the disease or disorder characterized by an overexpression of LPA16:0.

“Pharmaceutically acceptable carrier or diluent” means a carrier or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes carriers or diluents that are acceptable for human pharmaceutical use.

Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The pharmaceutical compositions may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include macrocrystalline cellulose, carboxymethyf cellulose sodium, polysorbate 80, phenyletbyl alcohol, chiorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein.

The present invention further contemplates methods of treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance (e.g. originating from an external source) of LPA 16:0, i.e. a psychiatric disorder or emotional dysregulation disease.

In one aspect, the method of treatment and/or prevention comprises (a) providing an agent of the invention and (b) administering said agent to a subject in need thereof.

Preferably, said agent is in the form of a pharmaceutical composition comprising a therapeutically effective amount of the agent as described herein.

Administration of the agents and/or pharmaceutical compositions described herein may be accomplished by any acceptable method which allows the agents modulating the expression and/or activity the expression and/or activity of a Lysophosphatidic acid receptor LPAR1 or LPAR3 or Lysophosphatidic acid (LPA16:0) to reach its target. The particular mode selected will depend of course, upon factors such as the particular formulation, the specific agent, the severity of the state of the subject being treated, and the dosage required for therapeutic efficacy. The actual effective amounts of agent (the "drug") can vary according to the specific drug or combination thereof being utilized, the particular composition formulated, the mode of administration, and the age, weight, condition of the patient, and severity of the symptoms or condition being treated.

Any acceptable method known to one of ordinary skill in the art may be used to administer the agents and/or pharmaceutical compositions to the subject. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated.

Suitable routes of administration of the pharmaceutical composition of the invention include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), intradermal (ID) or infusion, oral and pulmonary, nasal, topical, transdermal, and suppositories.

In one aspect the administration is done by injections. Injections can be given at multiple locations. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets. Inhalation includes administering the composition with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the agents and/or pharmaceutical compositions are encapsulated in liposomes.

Preferably, the agents and/or pharmaceutical compositions delivery systems are provided in a manner which enables tissue-specific uptake of the agents and/or pharmaceutical compositions delivery systems. Techniques include using tissue or organ localizing devices, such as wound dressings or transdermal delivery systems, using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capability and configured as expansive devices or stent grafts.

The agents and/or pharmaceutical compositions may be delivered using a bio-erodible implant by way of diffusion or by degradation of the polymeric matrix. The administration of the agents and/or pharmaceutical compositions may be designed so as to result in sequential exposures to the agents and/or pharmaceutical compositions over a certain time period, for example, hours, days, weeks, months or years. This may be accomplished, for example, by repeated administrations of a formulation or by a sustained or controlled release delivery system in which the agents and/or pharmaceutical compositions is/are delivered over a prolonged period without repeated administrations. Administration of the formulations using such a delivery system may be, for example, by oral dosage forms, bolus injections, transdermal patches or subcutaneous implants. Maintaining a substantially constant concentration of the composition may be preferred in some cases. Other delivery systems suitable include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/or combinations of these. Microcapsules of the foregoing polymers containing nucleic acids are described in, for example, U.S. Patent No. 5,075,109. Other examples include nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the agent and/or pharmaceutical composition contained in a formulation within a matrix (for example, as described in U.S. Patent Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), or diffusional systems in which an active component controls the release rate (for example, as described in U.S. Patent Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. The system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the agent and/or pharmaceutical composition. In addition, a pump-based hardware delivery system may be used for delivery.

In one aspect, the pharmaceutical composition is administered in combination with an additional pharmaceutical composition or therapy. The pharmaceutical composition and the additional pharmaceutical composition or therapy are administered concomitantly, separately or staggered in time.

In one aspect, the pharmaceutical composition is administered in combination with antiplatelet drugs.

Further contemplated is a method of diagnosing a neurodevelopmental, a neurological or a psychiatric disorder or disease of the invention in a subject comprising:

(a) detecting, directly or indirectly, and measuring the level of LPA16:0 in a sample obtained from said subject;

(b) comparing said LPA16:0 level to a control biological sample for the same LPA; wherein a differential of LPA16:0 level in said biological sample, relative to the level of corresponding said LPA16:0 in a control biological sample, is indicative of the subject having a disease or disorder characterized by an overexpression of LPA16:0, i.e. a neurodevelopmental, a neurological or a psychiatric disorder or disease.

The control biological sample may be obtained from a subject, such as a normal or healthy subject, i.e. a subject who does not suffer from a disease or disorder described herein or from a reference biological sample.

Usually, the upregulated expression of LPA in a biological sample corresponds to an increase equal or superior to about 5 %, preferably equal or superior to about 20 %, more preferably equal or superior to about 40 %, most preferably equal or superior to about 60 %, more preferably equal or superior to about 500%, even more preferably equal or superior to about 1000 %, in particular equal or superior to about 5000 % when compared to the level of corresponding said LPA in a control biological sample of a disease-free subject.

In one aspect of the invention, the expression of LPA16:0 corresponds to, or is reflected by, the concentration of circulating LPA16:0 and is usually expressed in nM. The biological sample is selected, in the context of the present application, from the group comprising whole blood, serum, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, hair(s), cerebrospinal fluid, and brain cells (neuronal or glial cells), or a combination of one or more of these biological samples. Preferably, the biological sample is whole blood or a fraction thereof, more preferably the biological sample is serum.

One of ordinary skill in the art will appreciate that any suitable technique for detecting and quantifying lysophosphatidic acid (LPA) in biological samples may be employed in the present invention.

Non-limiting examples of techniques for detecting and quantifying LPA16:0 in biological samples are selected from the group comprising Liquid Chromatography-Mass Spectrometry (LC-MS, LC-MS/MS), High-Performance Liquid Chromatography (HPLC, with UV or Fluorescence detection), Enzyme-Linked Immunosorbent Assay (ELISA), Thin Layer Chromatography (TLC), Nuclear Magnetic Resonance (NMR) Spectroscopy, and Capillary Electrophoresis (CE).

In one aspect, the neurodevelopmental, neurological or psychiatric disorder or disease of the invention is a disease affecting or linked to the hippocampus, such as e.g. a neuropsychiatric disorder or an emotional dysregulation disease or disorder.

In one aspect, the neuropsychiatric disorder or emotional dysregulation disease or disorder is selected from the group comprising schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type, cognitive impairment associated with schizophrenia (CIAS), autism spectrum disorder, bipolar disorder (BD), attention-deficit/hyperactivity disorder (ADHD), anxiety, anxiety- related disorders, depression, cognitive dysfunction, borderline personality disorder (BPD), depression or cognitive impairment induced by cytokines or chemotherapies administration, inflammation- related cognitive impairment or mood impairment, post-traumatic stress disorder (PTSD), frontal temporal dementia, or a combination of two or more thereof. Also provided is a kit for performing a method according to the invention or for use in the treatment and/or prevention of a neurodevelopmental, a neurological or a psychiatric disorder or disease, said kit comprising

(a) one or more agents and/or pharmaceutical compositions, and

(b) instructions for use.

The present invention also contemplates a gene delivery vector, preferably in the form of a plasmid or a vector, that comprises one or more nucleic acid(s) encoding an agent of the invention. Preferably, said agent is a nucleic acid selected from the group comprising an siRNA, an shRNA, an snRNA, a piRNA, an siRNA capable of interfering the expression of short hairpin (sh), a guide RNA, and a nucleic acid including an antisense oligonucleotide, a modified antisense oligonucleotide (e.g. a GapmeR) or a combination of one or more thereof.

As used herein, a "vector" is capable of transferring nucleic acid sequences to target cells (e.g. monocytes or CTL) and may be selected from the group comprising e.g., viral vectors, non- viral vectors, particulate carriers, nano-delivery systems (such as LNP) and liposomes.

Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e. g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2p plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Various viral vectors are used for delivering nucleic acid to cells in vitro or in vivo. Nonlimiting examples are vectors based on Herpes Viruses, Pox- viruses, Adeno-associated virus, Lentivirus, and others. In principle, all of them are suited to deliver an expression cassette comprising an expressible nucleic acid molecule that codes for an agent of the invention. In a preferred aspect, said viral vector is an adenoviral vector, preferably a replication competent adenovirus.

It will be appreciated that in the present method the modification following the introduction of the gene delivery vector (plasmid or vector), or the one or more nucleic acid(s) encoding the agent of the invention, to a host cell may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other aspects, it may occur in vivo.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following Examples. These examples are non-limiting and are merely representative of various aspects of the disclosure.

EXAMPLES

Materials and Methods

The blood-brain axis (BBA) assay

Cell culture: aNPC were isolated from the dentate gyrus (DG) of adult Fisher 344 rats and cultured in medium Dulbecco's Modified Eagle Medium (DMEM/F12) supplemented with N2 and FGF-2 (20ng/ml). They were plated at a density of 60,000 cells per well (total volume = 500pl) in a 24-well-plate coated with Poly-D-lysine hydrobromide at 0. Img/ml (P6407 sigma- aldrich) and Laminin at 20pg/ml (23017015 Life technologies). Twenty-four hours after plating, the medium was removed and replaced with fresh medium containing 0.2% serum for 24h. The medium was then supplemented with 5 pM BrdU for 30 min and washed twice with pre-heated fresh medium and fixed with 4% paraformaldehyde for 30 min. The plate was briefly washed with PBS 0.1M and stored at 4°C until used. Immunocytochemistry against BrdU and/or Ki-67 was processed as follows: BrdU immunohistochemistry was preceded by DNA denaturation with 2 M HC1 for 15 min at 37°C, and rinsed in 0.1 M borate buffer pH 8.5 for 15 min. Then, aNPC were incubated in blocking solution containing 0.3% Triton-XlOO and 10% deactivated horse serum (Gibco, 16050122) for one hour. Plates were incubated at 4°C with mouse monoclonal anti-BrdU (24 h, 1 :250, Abeam) or Rabbit anti-Ki-67 (1 :300, Abeam, abl5580) followed by goat anti-mouse 488 (1 :300, Invitrogen) or goat anti-rabbit 488 (1 :300, Invitrogen), respectively for 1 h at room temperature. After immunostaining, 10 minutes incubation into 4,6 diamidino-2-phenylindole (DAPI, 1 :500) was used to reveal nuclei.

Image acquisition and analysis: Images were acquired using an Eclipse Ti2 inverted microscope (Nikon). The number of BrdU- and Ki-67-labeled aNPC was counted in one randomly selected large field (tiles 4x4) in each well of the plate. For the validation of the BBA assay, we used 2-3 wells per condition in 3 independent replicates. For the rest of the experiments, each well stands for an individual and was replicated at least twice using 2 independent cohorts of mice for each condition (trait and state conditions). The number of BrdU- and Ki-67-labeled aNPC was compared with the total number of aNPC in each selected field to obtain a proliferation ratio, which was then normalized to the control group values to enable comparisons across conditions. Mice

All experiments were performed with the approval of the Cantonal Veterinary Authorities (Vaud, Switzerland) and carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609EEC). All experiments were performed on C57B1/6J mice obtained from Charles River Laboratories. After arrival, animals were housed four per cage and allowed to acclimate to the vivarium for one week. All animals were subsequently handled for 1 min. per day for a minimum of 3 days. Animals were weighted upon arrival as well as weekly to monitor health. Mice were maintained under standard housing conditions on corncob litter in a temperature- (23 ± 1°C) and humidity- (40%) controlled animal room with a 12h. light/dark cycle (0800-2000 hr), with unlimited access to food and water.

Chronic and acute restraint stress

This protocol involved 21 days of chronic retrain stress (CRS) as previously described (Lau, T., Bigio, B., Zelli, D., McEwen, B.S. & Nasca, C. Stress-induced structural plasticity of medial amygdala stellate neurons and rapid prevention by a candidate antidepressant. Mol Psychiatry 22, 227-234 (2017)). Animals were randomly assigned to either the control or CRS group. Animals were introduced head first into 50 ml Falcon tubes (11.5 cm in length; diameter of 3 cm) from which the cap was removed and the bottom was perforated with four 0.4 cm holes to enable breathing. Tissue was added at the caudal extremity to adjust the physical constraint to the mouse size and to allow the tail to expand out of the tube. The mice were subjected to this restrained environment for two consecutive hours every day for a period of 21 days. Control mice were left undisturbed in their home cage except for handling and body weighting each day for 21 days. The acute restraint stress protocol is based on the protocol as previously described (Zimprich, A. et al. A robust and reliable non-invasive test for stress responsivity in mice. Front Behav Neurosci 8, 125 (2014)). Animals of the stress group were restrained once, using the protocol described for the chronic stress. After one 20-min-restraining or 6-hours- restraining period, mice were transferred into their home-cage for an additional 20 min interval followed by an open-field test to assess anxiety -like behavior.

Behavioral tests

Open-field test (OFT): The test was performed as previously described (Larrieu, T. et al. Hierarchical Status Predicts Behavioral Vulnerability and Nucleus Accumbens Metabolic Profile Following Chronic Social Defeat Stress. Curr Biol 27, 2202-2210 e2204 (2017)). The apparatus consisted of a square Plexiglas arena (40 x 40 x 40 cm) that was illuminated with dimmed lights (25 lx). The floor was cleaned between each trial to avoid olfactory cues. Mice were introduced facing the wall of the arena and allowed to freely explore the arena for 10 min. A virtual centre zone (15 x 15 cm), thigmotaxis zone (30 x 30 cm) and an intermediate zone were included for the behavioral analysis as indicator for anxiety-like behavior. A video tracking system (Anymaze) recorded the path of each mouse as well as the total distance travelled, and the time spent exploring each zone.

Elevated plus maze test (EPM): The test was performed as previously described (Larrieu, T. et al. Hierarchical Status Predicts Behavioral Vulnerability and Nucleus Accumbens Metabolic Profile Following Chronic Social Defeat Stress. Curr Biol 27, 2202-2210 e2204 (2017)). The apparatus was made from black PVC with a white floor. The apparatus consisted of a central platform (5 x 5 cm) elevated from the ground (65 cm) with two opposing open (30 x 5 cm) and two opposing (30 x 5 x 14 cm) closed arms. Light conditions were maintained at 14-15 lx in the open arms, and 3-4 lx in the closed arms. Animals were placed at the end of the closed arms facing the wall, after which the animals were allowed to freely explore the apparatus for 5 min. Mice were tracked (Anymaze) to measure the time spent in each arm and in the risk zones (edge of the open arms).

Light Dark Test (LDT): The LDT was performed as previously described (Nasca, C., Bigio, B., Zelli, D., Nicoletti, F. & McEwen, B.S. Mind the gap: glucocorticoids modulate hippocampal glutamate tone underlying individual differences in stress susceptibility. Mol Psychiatry 20, 755-763 (2015)). A 60 x 40 x 21 cm high Plexiglas box was divided into a dark (20 x 40 x 21 cm) and a light (40 x 40 x 21 cm; 400 lux illuminated) compartments separated by an open door (5 x 5 cm) located in the centre of the partition at floor level. Each mouse was placed into the light chamber facing the door. Mice were allowed to freely explore the apparatus for 6 minutes. The Anymaze software was used to analyse anxiety-like behavior by calculating the time spent in each zone.

Marble burying test (MBT): The apparatus consists of an open transparent plastic box (40 x 25 x 20 cm) filled around 6 cm deep with bedding material across the whole cage. Twenty dark marbles (diameter: 16 mm) are spaced evenly in a 4 x 5 grid on the bedding. Mice were given 20 minutes to freely explore the cage (300 lx). At the end, the mice are removed, and every buried marble (more than 2/3 covered by litter) is counted. The number of buried marbles is used as an indicator of anxiety. Novelty suppressed feeding test (NSFT): The apparatus consists of a square Plexiglas arena (40 x 40 x 40 cm; > 400 lx). The floor was layered with approximately 2 cm of wood bedding. In the center of the square, a single pellet of food was placed on a white paper circular taped to a tissue culture disk (20 x 100 mm). Twenty-four hours before the test, all the food was removed from the home cage to food restricted the mice. The mice were introduced into the arena, facing the wall, and given 3 minutes for free exploration. Anymaze video tracking system recorded the movement trajectory of each mouse, and the videos were analyzed to assess the latency for the first bite. After the test, the food pellet was removed, and the mice were then returned to their cage with food and the amount of food consumed in 20 minutes was measured (in-cage consumption). A latency for the first bit in the pellet is used as an indicator of depressive- related behavior.

Anxiety scores: The anxiety score was calculated as previously described (Larrieu, T. et al. Hierarchical Status Predicts Behavioral Vulnerability and Nucleus Accumbens Metabolic Profile Following Chronic Social Defeat Stress. Curr Biol 27, 2202-2210 e2204 (2017)) with the average of standardized scores of each anxiety -related behavior tests. Standardization consisted in subtracting the minimum value of the whole population to the value of each animal and dividing the result by the maximum value of the whole population minus the minimum value of the whole population: (x - min value) / (max value - min value). This procedure yields scores which are distributed along a scale from 0 to 1, 1 reflecting high anxiety.

Mouse blood collection and preparation

Blood was collected (Multivette® 600 pl, Clotting Activator/Serum) by intracardiac punction using a 1ml syringe with a 21G7/8 needle after pentobarbital (10 ml/kg, Sigma-Aldrich, Buchs, Switzerland) anaesthesia before mouse perfusion. After sampling, the blood was left undisturbed at room temperature for 15 minutes to enable clotting. The clot was removed by centrifuging at 1,500 x g for 10 minutes in a refrigerated centrifuge. Following centrifugation, the resulting supernatant (i.e., serum) was transferred into a clean polypropylene tube using a Pasteur pipette. The samples were apportioned into 0.5 ml aliquots and stored at - 80°C.

Tissue collection and preparation

Mice received a lethal dose of pentobarbital (10 ml/kg, Sigma-Aldrich, Buchs, Switzerland) and were perfusion-fixed with 50 ml of 0.9% saline followed by 50 ml of 4% paraformaldehyde (Sigma-Aldrich, Switzerland) dissolved in phosphate buffer saline (PBS 0.1 M, pH 7.4). Brains were then collected, post-fixed overnight at 4 °C, cryoprotected 72h in 30% sucrose and slowly frozen on dry ice. Coronal frozen sections of a thickness of 40 pm. were cut with a microtomecryostat (Leica MC 3050S) and slices were kept in cryoprotectant (30% ethylene glycol and 25% glycerin in l x PBS) at -20 °C until being processed for immunohistochemistry.

Immunohistochemistry

Immunochemistry was performed as previously described (Gebara, E., Sultan, S., Kocher- Braissant, J. & Toni, N. Adult hippocampal neurogenesis inversely correlates with microglia in conditions of voluntary running and aging. Front Neurosci 7, 145 (2013)). Briefly, sections were washed 3 times in PBS 0.1M. BrdU detection required formic acid pre-treatment (formamide 50% in 2x SSC buffer; 2x SSC is 0.3 M NaCl and 0.03 M sodium citrate, pH 7.0) at 65 °C for 2 h followed by DNA denaturation for 30 min in 2 M HC1 for 30 min at 37°C and rinsed in 0.1 M borate buffer pH 8.5 for 10 min. Then, slices were incubated in blocking solution containing 0.3% Triton-XlOO and 10% deactivated horse serum (Gibco, 16050122) for 1 h. After blocking, samples were incubated with appropriate primary antibodies at 4°C overnight and then incubated with secondary antibodies for 1 h. After immunostaining, slices were incubated for 10 min into DAPI (1 :500) to reveal nuclei. One out of 6 section for the total DG were analysed. For in vivo proliferation experiments, an average of 10 sections was analysed per mouse for Ki-67 positive cells. The area of the DG was also measured using Zen blue edition software to assess whether any differences between groups could be due to changes in dentate gyrus area.

Reagents and antibodies

For primary antibodies: rabbit anti-Ki-67 (1 :300, Abeam, abl5580). For secondary antibodies: goat anti-rabbit 594 (1 :300, Invitrogen). Cyclic LPA16:0 (0.01 mg/ml in PBS 0.1M and 0.1% BSA fatty acid free, Polar Avanti, 7999268-68-1), KI16425 (0.5 mg/ml in ddH20 and 10% ethanol, Cayman Chemical, 10012659), temozolomide (TMZ, from Sigma-Aldrich, T2577, (25 mg/kg; 2.5 mg/ml in 0.9% NaCl i.p.).

Metabolomics: Targeted LPA16:0 quantifications: Mouse sera (15 pL) were extracted with 75 pL of ice-cold isopropanol (IP A) spiked with C17 cLPA as the internal standard (IS). To promote the lipid extraction and protein precipitation this solution was vortexed and centrifuged (at 4°C for 15 minutes at 14000rpm). The resulting supernatant was analyzed by reversed phase Liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS) in negative ionization mode operating in selective reaction monitoring mode (SRM, TSQ Altis triple quadrupole system interfaced with a Vanquish UHPLC system (Thermo Fisher Scientific)). Chromatographic separation was carried out on a Zorbax Eclipse Plus Cl 8 (1.8 pm, 100 mm * 2.1 mm I D.) column (Agilent technologies, USA). Mobile phase was composed of A = 60:40 (v/v Acetonitrile: water solution) with 10 mM ammonium acetate and 0.1% acetic acid and B = 88: 10:2 (Isopropanol: acetonitrile: water solution) with 10 mM ammonium acetate and 0.1% acetic acid. The linear gradient elution from 15% to 30% B was applied for 2 minutes, then from 30% to 48% B for 0.5 minutes, from 48% to 72% B and last gradient step from 72% to 99% B followed by 0.5 minutes isocratic conditions and a 3 min re-equilibration to the initial chromatographic conditions. The flow rate was 600 pL/min, column temperature 60 °C and sample injection volume 2pl. Data were processed using XCalibur 4.3 (Thermo Fisher Scientific) and concentrations were calculated to IS using a calibration curve.

Statistical analysis

All values are given as mean ± S.E.M. The normal distribution and homogeneity of variances were assessed using Shapiro-Wilk and Bartlett’s tests, respectively. The sample size was determined to obtain a power of at least 0.8 using Gpower Analysis Software (v3.1.9.2 Dusseldorf University, Germany). For all experiments, unpaired Student’s t-test, one sample t- test or one-way ANOVA followed by Tukey post-hoc test was performed. Although Tukey post-hoc test is less conservative, it allows pairwise comparisons between groups with different sample sizes and represents a good compromise for intra-group analysis. All statistical tests were performed with GraphPad Prism (GraphPad 9 software, San Diego, CA, USA) using a critical probability of p < 0.05.

Results

The regulation of stress resilience by LPA16:O-LPA1 signaling requires adult neurogenesis

To explore the role of LPA16:0-LPAi signaling in anxiety and stress response, we started by assessing LPA16:0 in mouse serum. To this aim, we used EPM and LDT to test trait anxiety in a new cohort of naive mice and divided them into two groups based on their anxiety scores, i.e., LA and HA groups (Fig. 1 A, B). We then collected blood samples and performed targeted metabolomics analysis of LPAs. HA mice displayed significantly higher serum concentrations ofLPA16:0, LPA20:4, and LP Al 8:2 than LA mice, but not in LPA18:0 and LPA18: l (Fig. 1C and data not shown). Notably, there was a positive correlation between LPA16:0 concentration and individual anxiety score (Fig. ID) and LPA16:0 concentration showed an accuracy of 80% at identifying HA from LA mice (Fig. IE), suggesting that LPA16:0 had a similar role in mice anxiety as in humans.

Next, we examined the effect of exogenous LPA16:0 on anxiety and neurogenesis. To this aim, mice were first assessed for anxiety using an EPM in order to distribute them in anxiety- matched groups. Mice were then treated with either cLPA16:0 or vehicle daily for 21 days, after which we assessed their anxiety -like behavior, sampled their serum, and processed their brains for histology (Fig. IF). To investigate the impact of cLPA16:0 treatment on anxiety- related behavior, we conducted an open-field test. Surprisingly, mice treated with cLPA16:0 did not exhibit any significant changes in baseline anxiety-like behavior compared to the vehicle-treated mice (Fig. 1G, left panel). Considering our previous findings in the human cohort, where LPA16:0 levels were elevated only in individuals at high risk and susceptible to anxiety disorders, we postulated that LPA16:0 might trigger an exaggerated stress response. To test this hypothesis, we subjected both cLPA16:0- and vehicle-treated mice to a subthreshold, 20 minutes acute restraint stress (ARS), a paradigm known to unveil a susceptible phenotype by lowering the animals' stress reactivity threshold. Mice treated with cLPA16:0 displayed increased anxiety-like behavior following 20 min. ARS compared to the control group (Fig. 1G, right panel), indicating that cLPA16:0 treatment exacerbated stress reactivity. Furthermore, cell proliferation in the dentate gyrus, assessed by immunohistochemistry against the endogenous cell proliferation marker Ki-67, was reduced in mice treated with cLPA16:0 (Fig. 1H, left panel). Similarly, serum from cLPA16:0-treated mice decreased aNPC proliferation in the BBA assay as compared to serum from vehicle-treated mice (Fig. 1H, right panel) and serum cLPA16:0 concentration negatively correlated with aNPC proliferation in the BBA assay (Fig. II, M). Thus, increased circulating LPA16:0 impaired hippocampal neural stem/progenitor cell proliferation and reduced stress resilience.

We then tested whether antagonizing the LPAi receptor might confer stress resilience. To this aim, we tested the resilience of mice to 6 hours of ARS, which is a protocol that induces anxiety-like behavior in naive mice. Anxiety-matched mice were injected daily for 12 days with either Kil6425 (i.p. 5 mg/Kg body weight) or vehicle and then assessed for their basal anxiety using an open-field test. One day later, they were subjected to 6 hours of ARS followed by an open-field test (OFT) 20 minutes afterwards to assess state anxiety (Fig. 1 J). The anxiety score before ARS did not differ between Ki 16425- or vehicle-treated mice (Fig. IK, left panel). After ARS however, vehicle-treated mice showed an expected increase in anxiety score, whereas mice treated with Ki 16425 exhibited no anxiety-like behavior (Fig. IK, right panel). The resilience profile exhibited by the Ki 16425 -treated mice was associated with an increase in aNPC proliferation (Fig. IL). Thus, antagonizing LPAi increased adult neurogenesis and resilience to an acute stress.

Next, we tested the effect of Ki-16425 on resilience to chronic restraint stress (CRS). To this aim, anxiety-matched mice were treated with either Ki 16425 or vehicle for 25 days and exposed to a 21 days CRS starting 4 days after Ki- 16425 exposure. These mice were compared to control, non-stressed mice (Fig. IN). After this, mice were assessed for basal anxiety using an open-field test, marble burying test, and response to a 6h-ARS. Before ARS, we found no group difference in anxiety in an open field test (Fig. 10, left panel). However, mice exposed to CRS displayed increased marble burying behavior as compared to control mice and to mice exposed to RCS after Ki 16425 injection (Fig. 10, right panel). All three groups showed similar anxiety levels after a 6h-ARS (Fig. IP, left panel). In addition, CRS reduced cell proliferation in the dentate gyrus as compared to control animals, an effect that was ablated with Ki- 16425 administration (Fig. IP, right panel).

To test whether adult neurogenesis may mediate the stress resilience effects of KH6425, we used the anti-mitotic drug temozolomide (TMZ) to suppress adult neurogenesis. Anxiety- matched mice were first treated with either NaCl or TMZ for 4 weeks, 3 days/week followed by, afterthe third week of TMZ treatment, 12 days administration of Ki 16425 and then assessed for resilience to a 6h-ARS (Fig. IQ). Before ARS, we found no group difference in anxiety in an open field test (Fig. 1R, left panel). After ARS, Ki 16425 +NaCl -treated mice did not display increased anxiety, confirming the resilience profile observed previously (Fig. 1R, right panel). Strikingly, mice treated with both Ki 16425 and TMZ showed increased anxiety (Fig. 1R, right panel), indicating a decreased stress resilience in the TMZ-treated group. In addition, Kil6425+TMZ-treated mice showed less cell proliferation than Ki 16425 +NaCl -treated mice (Fig. IS), confirming the effect of TMZ on the reduction of adult neurogenesis. These results suggest that the effect of Ki 16425 on acute stress resilience required intact adult neurogenesis. The experiments described above tested the effect of LPAi antagonism on anxiety after an acute stress. To test the effect of KH6425 on depressive-like behavior after a chronic stress, we repeated these experiments with 21 days of CRS. To this aim, anxiety-matched mice were treated for 4 weeks with either NaCl or TMZ. Starting after the second cycle of TMZ (11 days after the first day of TMZ treatment), mice were injected daily with Ki 16425 for 25 days and, starting 4 days after treatment initiation, were all subjected to CRS for 21 days. Two days later, mice were injected with BrdU. One day before the end of the CRS, anxiety-like behavior was assessed on a marble burying test (MBT) and, one day later, depressive-like behavior was tested on a novelty-suppressed feeding test (NSFT) (Fig. IT). We found no difference between groups in the number of marbles buried, suggesting no difference in anxiety (Fig. 1U). However, in a Novelty-suppressed Feeding Test (NSFT), mice treated with KH6425 showed a decreased latency to feed as compared to vehicle-treated animals, an effect that was abolished by TMZ treatment (Fig. IV). Following 21 days of CRS, KH6425 increased cell proliferation (Fig. 1W) and the number of newly formed neurons (Fig. IX), as compared to NaCl, an effect that was abolished by TMZ. Together, these results indicate that the increase in adult neurogenesis was required for the antidepressant effect of Ki 16425 after stress.

Platelet depletion reduces circulating LPA16:0 and increases stress resilience and adult neurogenesis.

Serum LPA is mostly produced by platelets. Consistent with this primary source, thrombocytopenia is characterized by a significant reduction in circulating LPA. We therefore assessed whether LPA16:0 may be reduced by decreasing platelets. To this aim, we injected 3 mice with anti-platelet or control serum for 2 days and analyzed their plasma by pooling all animals from each condition. Anti-platelet treatment reduced platelets and LPA16:0 to undetectable levels (Fig. 2A). We then tested the possibility that anti -platelets serum may regulate adult neurogenesis and stress resilience. To this aim, anxiety-matched mice were administered with anti-platelet serum every 2 days for 20 days and tested for anxiety in an OFT, followed by a stress-resilience test using a 6h ARS (Fig. 2B). Platelet-depleted mice exhibited reduced anxiety-like behavior under baseline conditions whereas mice injected with control serum displayed a normal anxiety score (Fig. 2C). After ARS, control mice showed an expected increase in anxiety score, whereas mice treated with antiplatelets exhibited no anxiety-like behavior (Fig. 2D). This resilience profile of platelet-depleted mice was associated with an increase in the proliferation of aNPCs in the DG (Fig. 2E). Thus, depleting platelets eliminated circulating LPA16:0 and mimicked the stress-resilience effect of LPAi antagonism. Platelet depletion specifically abolishes LPA16:0 production

To assess the effect of platelet depletion on the different forms of circulating LPA, serum from mice treated with anti-platelet serum was assessed by mass spectrometry. Anti-platelet serum abolished the production ofLPA16:0, but not the production of cLPA16:0, LPA18:0, LPA18: 1, LPA18:2, LPA20:4, cl6 cyclic LPA, or C18: l cyclic LPA. Thus, platelet ablation specifically affects the production of LPA16:0 in the serum. These results show that LPA16:0-LPAi signaling in mice bidirectionally regulated stress resilience and adult neurogenesis and the increase in stress resilience produced by LPAi antagonism was mediated by adult neurogenesis. Finally, the depletion of platelets, the main source of LPA in blood, specifically reduced LPA16:0 and induced stress resilience along with increasing aNPC proliferation in the dentate gyrus. Together, these results highlight a crucial role of platelets in stress susceptibility in anxious individuals, mediated by the production of LPAI 6:0 and the inhibition of adult hippocampal neurogenesis.

Claims

1. An agent modulating the expression and/or activity of a Lysophosphatidic acid receptor LPAR or the concentration of circulating Lysophosphatidic acid (LPA16:0) for use in the treatment and/or prevention of a disease affecting, or linked to, the hippocampus, wherein the LPAR is selected from the group consisting of LPAR1 and LPAR3, or a combination thereof, and wherein the disease affecting, or linked to, the hippocampus is a neuropsychiatric disorder or emotional dysregulation disease or disorder

2. The agent for use of claim 1, wherein the disease affecting, or linked to, the hippocampus is a neuropsychiatric disorder or emotional dysregulation disease or disorder characterized by an overexpression or an overabundance of LPA16:0 in the blood and/or in the brain.

3. The agent for use of claim 1 or 2, wherein the disease affecting, or linked to, the hippocampus is selected from the group comprising schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type, cognitive impairment associated with schizophrenia (CIAS), autism spectrum disorder, bipolar disorder (BD), attention-deficit/hyperactivity disorder (ADHD), anxiety, anxiety- related disorders, depression, cognitive dysfunction, borderline personality disorder (BPD), depression or cognitive impairment induced by cytokines or chemotherapies administration, inflammation- related cognitive impairment or mood impairment, post-traumatic stress disorder (PTSD), frontal temporal dementia, or a combination of two or more thereof.

4. The agent for use of any one of the preceding claims, wherein said modulation comprises, or consist of, i) reducing the expression and/or the abundance of LPA16:0, ii) reducing the biological activity of LPA16:0, iii) reducing the expression of LPAR1 and/or LPAR3, iv) impairing the binding of LPA to the receptor LPAR1 and/or LPAR3, and/or v) reducing the expression and/or the abundance of LPA16:0 by inhibiting platelet function.

5. The agent for use of any one of the preceding claims, which is selected from the group comprising a nucleic acid, a chemical compound, a peptide or analog thereof, an antibody or an antigen-binding fragment thereof, and an antibody mimetic, or a combination of one or more thereof.

6. The agent for use of claim 5, wherein the nucleic acid is selected from the group comprising a nucleic acid encoding an siRNA, a miRNA, a piRNA, an hnRNA, an snRNA, an sg RNA, a CRISPR-based loss-of-function system, an esiRNA, an shRNA, and an antisense oligonucleotide, or a combination of one or more thereof.

7. The agent for use of claim 5, wherein the agent is an antibody, or antigen binding fragment thereof, that inhibits and/or impairs the binding of the LPA to the receptor LPAR1.

8. The agent for use of claim 5, wherein the chemical compound is a small molecule inhibitor, preferably an antagonist inhibitor of LPAR1 and/or LPAR3.

9. The agent for use of claim 8, wherein the antagonist inhibitor of LPAR1 is selected from the group comprising Ki 16425, Debio-0719, Ki 16198, BMS-986020, BMS-986278, HY- 100619, AM095, H2L5765834, HY-16039, TAK-615, ONO-9780307, ONO-0300302, ML- 161AZ-3451, ML-184, Piperidine 18, Ro 6842262, AM966, AM095, , CAS NO. 1396006, - 71 -54-(4-(2-Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin- 1 -yl)-4- oxobutanoic Acidand SAR- 100842, or a combination thereof.

10. The agent for use of claim 8, wherein the antagonist inhibitor of LPAR3 is selected from the group comprising KH6425 and H2L5765834, or a combination thereof.

11. The agent for use of any one of claims 3 to 10, wherein said modulation reduces

- the concentration of LPA in the blood and/or in the brain, or

- the amount of platelets.

12. The agent for use of claim 11, wherein the agent reduces the amount of platelets i) by blocking the activity of the cyclooxygenase- 1 (COX-1) enzyme, ii) by inhibiting the P2Y12 receptor, iii) by inhibiting the ADP receptor, iv) by preventing the binding of fibrinogen or other proteins to the GPIIb/IIIa receptor, v) by inhibiting a phosphodiesterase enzyme (PDE) selected from PDE2, PDE3 and PDE5.

13. The agent for use of claim 5 or 12, wherein the agent is an antibody, or antigen binding fragment thereof, that inhibits and/or impairs platelet aggregation.

14. The agent for use of claim 5 or 12, wherein the agent is a peptide or analog thereof, that inhibits and/or impairs platelet aggregation.

15. The agent for use of claim 5 or 12, wherein the chemical compound is a small molecule inhibitor, preferably an antagonist inhibitor of the P2Y12 receptor.

16. A pharmaceutical composition comprising a therapeutically effective amount of an agent of any one of claims 1 to 15, and a pharmaceutically acceptable carrier or diluent.

17. The pharmaceutical composition of claim 16, which is administered by intravenous, intradermal, subcutaneous, intramuscular, intranasal, or intraperitoneal routes.

18. The pharmaceutical composition of claim 16 or 17, which is administered in combination with an additional pharmaceutical composition or therapy.

19. The pharmaceutical composition of claim 18, wherein said pharmaceutical composition and the additional pharmaceutical composition or therapy are administered concomitantly, separately or staggered in time.

20. A method for diagnosing a neurodevelopmental, a neurological or a neuropsychiatric disorder or disease characterized by an overexpression or an overabundance of LPA16:0 in a subject, the method comprising:

(b) comparing said LPA16:0 level to a control biological sample for the same LPA; wherein a differential of LPA level in said biological sample, relative to the level of corresponding said LPA in a control biological sample, is indicative of the subject having a neurodevelopmental, a neurological or a psychiatric disorder or disease characterized by an overexpression of LPA.

21. A kit comprising (a) an agent of any one of claims 1 to 15 and/or a pharmaceutical composition of any one of claims 16 to 19, and (b) instructions for use.

22. Use of the kit of claim 21 for the treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance of LPA16:0 in a subject in need thereof.

23. A method of treatment and/or prevention of a disease or disorder characterized by an overexpression or overabundance of LPA16:0 in a subject in need thereof, the method comprising administering an agent of any one of claims 1 to 15 and/or a pharmaceutical composition of any one of claims 16 to 19.