WO2017066434A1 - Brain-derived neurotrophic factor (bdnf)-tropomyosine kinase b (trkb) inhibition for improving cognitive deficits - Google Patents

Abstract

The present invention relates to methods and treatments for improving cognitive functioning in patients in need thereof. In certain aspects, the methods comprise administering at least one brain-derived neurotrophic factor (Bdnf)-tropomyosine kinase B (TrkB) inhibitor.

Description

BRAIN-DERIVED NEUROTROPHIC FACTOR (BDMF)~TROPOMYOSINE KINASE B (TRKB) INHIBITION FOR IMPROVING COGNITIVE DEFICITS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application Ser, No. 62/241,362 filed October 14, 2015, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created o» October 10, 2016, is named O1O01-OO4974-WOO_SL.TXT and is i .273 bytes in size,

GRANT INFORMATION

This invention was supported in part with government support under NIH grant number R21 MH09925.1 awarded by National institutes of Health. The United States Government may have certain rights in the invention,

FIELD OF THE INVENTION^'

The present invention relates to methods and treatments for improving cognitive functioning in patients in need thereof, in certain, aspects, the methods comprise administering at. least one brain-derived neurotrophic factor (Bdnfj-troporayosine kinase B (TrkB) inhibitor,

BACKGROUND OF THE INVENTION

Studies showed that histone deacetylase (H^'DAC) inhibitors can reverse cognitive deficits found in neurodegenerative disorders and age-related memory decline. However, the role of HDACs in stress-induced cognitive deficits has not been investigated. Deficits in executive cognitive functions occur in 20 to 30% of patients with major depression (Mclntyre et al., 2013; Miiiati et al., 2012) as welt as in a large subset of patients with unipolar depression (Trivedi and Greer, 2014). Such deficits include widely replicated findings of deficits in working memory (WM) and attention, i.e., deficits that -are principal mediators of psychosocial impairment, including impaired workforce performance (Mclntyre et af, 2013). The observations that patients in remission from depression still exhibit cognitive deficits, and that the presence of such deficits contributes to relapse, indicates that the cognitive deficits are core deficits rather than an epiphe.no.me.non of depression (Milan et al., 2012; Mcltttyre et al, 2013), The causes of these cognitive deficits are noi known and, at present, no mechanisms have been identified that could explain their emergence.

Thus, there is an urgent need for therapeutics and treatments tor stress-induced and related cognitive deficits.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method of treating cognitive defects in a mood disorder in a patient in need thereof, .comprising administering a therapeutically effective amount of at ieast one brain-deri ved neurotrophic factor (Bdni)- tropomyosme kinase B (TrkB) inhibitor to the patient.

la additional embodiments, the present invention relates to a method of improving cognitive functioning in a patient in need thereof comprising administering a therapeutically effective amount of at least one brain-derived neurotrophic factor (Bdrd)4:roporo.yosine kinase B (TrkB) inhibitor to the patient.

in certain embodiments, the treating comprises at least a 10% improvement of cognitive functioning, compared with a baseline cognitive functioning reference or control.

In additional embodiments, the cognitive functioning improvement is greater than, about 50%.

in additional embodiments, the TrkB inhibitor is a TkrB antagonist,

in further embodiments, the TrkB inhibitor is administered by mouth, topically, rectally, or intravenously.

in additional embodiments, the mood disorder is an anxiety disorder or stress- induced disorder including early life stress, or a combination thereof.

hi additional embodiments, the mood disorder is a panic disorder, an obsessive compulsive disorder, a post-traumatic stress disorder, or any combination thereof.

In additional embodiments, the patient is an adolescent or young adult.

In additional embodiments, the TkrB inhibitor comprises ANA- 12.

In additional embodiments, the method further comprises administering at least one selective serotonin reuptake inhibitor (SSRt) selected from the group consisting of

Citalopram (Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat);

Fluoxetine (Prozac); Fluvosamine (Luvox); Sertraline (Zoloft, Lusirai), and combinations thereof.

in certain embodiments, the patient exhibits a mood disorder comprising an anxiety disorder or stress-induced disorder including early life stress, or a combination thereof. la additional embodiments, die patient exMbits a mood disorder comprising_, a panic disorder, an obsessi ve compulsive disorder, a post-traumatic stress disorder, or any combination thereof.

la additional embodiments, the patient is a mammal. In additional embodiments, the patient is a human. In additional embodiments, the patient exhibits a cognitive deficit in one or more of the fo.Howi.ng features: working memory or attention set-shifting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs, 1A-C are diagram^'s showing the experimental design and dosing timeline for the model mice. Fig, LA is a timeline showing that mice exclusively used for HDACl and HDAC3 CMPs were exposed to the IMS from P2 and Pi 5, weaned at P28 and raised to adulthood (P60). At P60, their brains were removed, SFR mice raised in paraiiei served as controls. Fig, IB is a timeline showing how other groups of mice were chronically treated with either Ana.- 12 (IMS Balb/c mice) or with MS -275 (SFR Ba!b/c mice) from postnatal age P35 until P59. At the end of treatment, mice were tested in tire ASST followed by training and testing in the WM test. One hour post completion of the WM test at 20 s delay (typically around P84), brains were collected for real-time RT-PCR and CMP experiments. Age-arid sex-matched vehicle-treated mice served as controls. Fig, 1 C shows a timeline of the Forster IMS C57BI/6 or foster IMS Balb/c mice that were raised by Balb/c mothers during IMS exposure, weaned ai P2S, aid tested in the ASST in adulthood (at P66). One hour post completion of the IDS phase of the ASST, brain were collected for real-time PCR and ChJP experiments. Age- and sex-matched non-tested mice served as controls.

Figs. 2A-D are graphs showing reduced HDACl levels at promoter ill of the Bdnf gene in IMS Balb/c mice. Fig. 2 A is a graph illustrating comparison of the levels of HDACl and H.DAC3 at promoters ill and IV of the Bdnf gum and the promoters of the Bgr2 and fos genes in SFR and IMS Balb/c mice. Data are mean ± sent (n ~ 8/group for the HDAC3 Bdnf Pill ChIP (4 males and 4 females) and n ^::: 6/group for ail other groups (3 males and 3 females)) and were compared by Student's t-tesi. SFR and IMS Balb/c mice differ significantly in their levels of HDAC l associated with #dw/promotor ΪΪΪ. Fig, 2B shows Bdnf transcript variant IK expression in non-tested and WM-tested (20 s delay) SFR and IMS Balb/c mice. Data (mean ± sem; n ~ ⁽S/group) were compared by 2-way ANOVA (see Results) and statistical differences were resolved post hoc (Takey HSD test) as indicated. Fig. 2C shows a comparison of the levels of Pol II association at Mff/promotor III between non- tested and WM-tested IMS Balb/c mice. Data (mean * sem; n - 6/group) were compared using two-tailed Student's t test. Fig, 2D shows Egr2 mRNA expression levels in non-tested and WM tested SFR and IMS Bato/c mice. Data (mean ± sem; a = 6/group) were compared by 2-way ANOV A dot revealed ad flgptfkftittdil&raDces.

Rfk 3A-B are gnphs showing the effects of adokftcent ANA-12 treatment of IMS and SFRBalh/c imce on cognitive task perfonnauce in adnltibood. Fig.3 A is a graph showing perJfonnauce of SFR, IMS and Ana-12-treated SFR and IMS BaJb/c mice in the ASST. Dan are mean ± sem of 8 animab per group (4 males and 4 females) and woe coopered by 2-way ANOVA (lee Remits). Statistical dilfcrences were resolved post hoc (Tukey HSO tests) as indicated. Compared to SFR mice, IMS Balb/c mice exhibited significant deficits in the EDS phase of uta ASST, a defied that is abdiehedmAna-12-treeled IMS mice, m contnut, Ana- 12 treatment of SFR Balb/c mice led to the atme deficits found in IMS mke. Fig. 3B is a graph illustrating (he performance of me same groups of mice in the WM test Data were compared by 2-way ANOVA (see Results) and significant differences resolved post hoc (Tnkey HSD tests) are iixbcaied. When tested at 20 s inter-trial delays, the percentages of correct arm entries of IMS Balb/c and Ana-12-treated SFR mice were signiflcantiy lower conmaiiedtoiioii-treatedSFRan^

Figs.4A-D are graphs iBmtratmg the effect of adolescent MS-275 treatment of SFR BabVc mice on cognitive task perfixmance and expression of B

is a graph quantifying the performance of SFR Balb/c mice treated with MS-275 in the ASST. Data are mean ± sem (n - 8/groop). Sigmjfcaut differences between non-treated and MS-275-treated SFR mice were found only for the EDS phase of the ASST (Stadenft t test). Fig.4B is a graph Castrating the performance of die same group of mice shown in Fig.4A in the spatial WM test At 20 s delay, significant differences were found between the two groups (Sbjdenfs t test). Fig. 4C is a graph of the real-time RT-PCR measures of Bdnf transcript variant Dl expression. Date

one-way ANOVA (F (2,16) - 4.56; p - 0.02) were resolved post hoc (Tukey-Kramer multiple comparisons test) only for tested MS-275-treated mke that exhibited significantly increased expression of Bdnf mRNA. Fig.4D is a graph for the same group of mice shown in Fig. 4C, quantifying the real-time RT-PCR measures of Egr2 mRNA and revealed no significant differences between the three groups (ANOVA, F(2,16) - 0.5; p- 0.62).

Fare. SA-B avp graphs showing that fort* IMS C57BV6 mice have deficits in the

ASST and reduced HDAC1 levels at (he promoter of (he Egr2 gene. Fig. 5A β a graph iltastrating ASST performance of SFR and IMS CS7BI/6 mice raised by their biological mothers and foster IMS C37BU6mice raised by Balb/c mothers. Date are mean ± sem 0np7/gronp) and were compared by 2-way ANOVA (see Examples). Statistical differences were resolved poet boc (Tukey BSD lasts) as mdHaled, Fig. 5B is a graph coatwmsj the levels of HDACl aid HD AC3 et promoters 1Π and IV of the Bdnfgaa* and the pomotort of tibe £gr2 and For geoei in SFR and IMS C57B1/6 mice raised by their biological mothea and foster IMS C571/6 mice raised by Balb/c mothers. Data are mean ± sen (n-6/group). One- way ANOVA revealed sigmficant diflerences between groups only for the levels of HDAC1 at the -%r2 gene promoter (F(2,14)-9.1¾ p-0.005). Poet hoc Tokey-Kramer multiple compariaoos resolved these differences for foster IMS C57BI/5 mice that had significantly lower levels of HDAC1 cocnpared to SFR and IMS C57BI/6 mice.

Figs.4LA-D are graphs showing that Fooler IMS C57BI/6 mice exhibit increased Egr2 mRNA expression after ASST testing and increased histone H3 acetylation at the Egr2 promotor. Fig. 6A is a coixnarison of Bdnf transcript variant m and Fig. 6B shows Egr2 mRNA levels between non-tested and ASST-tested foster IMS CS7B1/6 and Balb/c mice. Data (mean ± sem; n=6/group) were compared by 2-way ANOVA (see Results). Significanl differences resolved post boc (Tukey HSD tests) are indicated Whereas foster IMS Balb/c mice exhibited increased Bdnf mRNA expression after ASST testing, ASST tested foster IMS C57B1/6 mice expressed significantly higher levels of Egr2 mRNA. *p < 0.05 in (Fig. 6A) and p < 0.01 in (Fig. 6B). Fig. 6C shows the consparison of the levels of Pol Π aaMciation with the Egr2 promotor between non-tested and ASST-tested foster IMS C57B1/5 mice. Data (mean ± sem; n - 6/group) were compared ttsiag two-tailed Stuoefifs t test Fig. 6D shows die ratio of acH3K9 over H3K9me3 levels associated with the Egr2 promoter of SFR and IMS C57BI/6 and Balb/c mice compared to foster IMS C57B1/6 mice. Data are mean ± sem (n - 5/group). One-way ANOVA revealed iigniftcant diflerences between groups (F(4,19) « 3.9; p - 0.02) that were resolved post hoc for foster IMS C57B1/6 mice that exhibited a significantly higher acH3KMDK9me3 ratio. *p < 0.05 compared to all other groups.

DETAILED DESCRIPTION

m die stress-susceptible mouse strain Balb/c, early fife stress triggers a persiatent decrease in HDAC expression in the fcrobrain neocortex, including reduced expression of class I HDACs. The same mice show pronounced cognitive deficits in adulthood, namely deficits, in -wciiing .memory and, attention set-shifflng. The present data i-tosteste mat these mice also exlubh reduced association of HDACl wim promotor ΙΠ of 4e brsMerived neurotiophic factor (βώφ gene, and mat cognitive testing leads to abnormally increased Bdnf mRNA expression. A pbarmacologica] reduction of Bdiif-tropotnyoeine kinase B receptor signaling efletfxveh/ reverses the cognitive deficits, indicating that enhanced transcriptional activation of the B&tf gene WMribi-tM to their enwgencc. In contrast to Balh/c mice, CS7B1/6 mice only dewlap attention set-s&fting deficits when raised by Balb/c foster modicn daring the time the pore are exposed to eeilyli&stre

promoter HI are unaltered in such C57B1/6 mice, although they exhibit decreased levels of HDAC1 at the nromotor of the earty-growtb response gene 2 (Egr2) and atmonnaOy increased Egi2 mRNA expression after cognitive testing. Hence, contrary to the beneficial effects of HDAC inhibition in nearodegeuerative diseases, the reduced HDAC1 levels at promoters of distinct plasticity-associated genes predispose animals exposed to eady lift stress to CTihimcfd expression of these genes upon cognitive challenge, an effect that

One promineni risk rector for depression is early Bfe stress (ELS) at described in Pechtel and Piragalti, 2011. Mouse models have been utilized as co-relating with human developmental learning, and models of human emotional behavior for many years (See MiOan, M. J. 2008 and Sdimtuss et al., 2014). For example, P1-P28 (moose pup day 1- mouse pup day 28) mice correspond developmentaDy to earty human childhood, with P21- P28 as the typical weaning time for ow mouse pups. Mice beyond P28 to P60 correspond to human adolescence (Set MiHan, M. J.2008). Mice mat are beyond P60(iiicwpi-p day 60), and m particular beyond P66 are coniidered to correspond to human adults.

Numerous studies on animal models of eariy life stress have denionstrated persistent changes in emotional behavior that are also found in human depression. For example, in die inbred mouse strain Balb/c a widely employed rodent paradigm of eariy life stress, infant maternal separation (IMS), chats not only iiicreased anxiety and depression-like behavior in adulthood, but also pronounced deficits in spatial WM and attention set-shifting (Mehta and Schmauss, 2011). Moreover, several of the gene expression changes found in IMS Bafo/c mice are found in the fcrebrain neocortex, and they are attributable to a lustone-based epigeDetic response to IMS exposure, namely increased acetylabon of histone H4 protein that is due to deuesaed activity of several dare Ι/Π HDACs, inclnding class I HDACs 1 and 3 (Levine et si, 2012). This epigenetic phenotype emerges during adolescent development and persists into adulthood. While it ameliorates the severity of foe emotional phenotype in adulthood (ticvine et aL. 2012) and if'^jTfif rasnonsiveness to antidernvseant it'tHR—il (Scnrnauw. 2015), only aphsimaco!ogical nwiimail that activated class I HDACs 1 and 3 owing adolescent development of IMS BanVc mice was able to abolish their cognitive deficits (Schmauss et al., 2014). This suggests that ledoced HDAC1 and/or HDAC3 activity contributes to foe emergence of cognitive deficits, a hypothesis tested in foe present study. Contrary to the pronounced deficits in WM and attention set shifting found^' in IMS Balb/c mice, these cognitive functions are onaffecied. after IMS exposure of the more resilient strain €5781/6 (Mehta and Schmauss, 2011). However, C57B1/6 foster pups raised by Balb/c mothers during IMS exposure also develop deficits in attention set-shifting, but no deficits in WM (Schmauss et ai,, 2014). Whether or riot the comparatively milder cognitive- deficits of foster IMS C57BI/6 mice are also functionally related to reduced HDACI and/or HDAC3 activity, is another hypothesis addressed in the present study.

The data provided herein illustrate mat both IMS Balb/c mice and foster IMS C57B1/6 mice exhibit reduced HDACI association with promoters of distinct plasticity-associated genes, and that cognitive test exposure triggers an. abnormally increased, expression of these genes. However, the affected genes differed between the two strains; In IMS Baib/c mice, reduced HDAC I levels axe found on promoter HI of the Aft/gene, and increased expression Bdnf and hence, increased Bdnf-iropomyosine kinase B (TrkB) receptor signaling can explain the cognitive phenotype of IMS Balb/c mice. Moreover, a pharmacological reduction of HDACI activity during adolescent development of non-stressed Balb/c mice precisely recapitulates this effect. In contrast, in IMS C57BI/6 mice fostered by Balb/c mothers, reduced HDAC i levels were detected at the promoter of the early growth response gene 2 (Egr2), and cognitive task exposure also enhanced the expression of the Egr2 gene above normal levels. Hence, while reduced HDACI association at promoters of distinct plasticity- associated genes is a common denominator contributing to early-life stress-triggered cognitive deficits, the genes affected are determined by the genetic backgrounds.

The present data using validated mouse models for these early stress conditions and corresponding cognitive deficits illustrate that in certain embodiments pharmacological treatment of a patient with a TrkB inhibitor such as ANA- 12 will lead to improvement of the cognitive deficits and improvement in cognitive functioning (executive cognitive functions such as working memory and attention set-shifting)., which may also serve to benefit certain emotional phenotypes or conditions such as MDD (major depressive disorder) without any additional pharmacological intervention, in certain alternative instances, it is expected, that in addition to treating a patient with a TrkB inhibitor such as ANA- 12, thai treating with one or more SS.RI. (including any combination of: Cita.lop.rara (Celexa); Escitalopraru (Lexapro, Cipratex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxamme (Luvox); Sertraline (Zoloft, Lustra!)) would result in improvement to both the cognitive deficits and emotional phenotype e.g., depressi ve condition/s as described herein. ASS- attrition MBMhiftiBg

ASST" attention set-shifting test;

Cii"* compound duciiminitKin;

ELS- early life stress (for example everts during childhood that exceed 0» child's coping resources inchiding physical, sexual. emotional tad vertMl abuse, lfttghm. deprivation, disaster, household dysAmctions such at witnessing violence, criminal activity, parental separation parental death or illness, poverty, and gubitancc abuse. See, Pechtri and Pizzagalli 2011);

IDS- iiaradimensional shift of attention;

IMS" infint maternal separation;

MOD- major depressive disorder,

Sr>½imple discrimination, for example between odor (scented tern, cotta pots) of texture (difieremmggiiigniedia);

WM » working memory.

TrUIaAMton

ANA-12 it a selective, smaU-molecule ncn-coiijpetiUve antegonist of TrkB (Kd■»· 10 nM and 12 uM for the high- and km-aflmity sties, respectively), the tnrin receptor of breixh derived neurotrophic factor (BDNF/Bdnf) (Cazorla 2011). The compound crosses the Wood- braiit-berrier and exerts central TrkB blockade, producing effects as early at 30 ramattt (-400 nM) and at long at 6 hours (-10 nM) fofowing hffltperitoneal injection in mice (Cazorla 2011). ft blocks the neurotrophic actions of BDNF without compromising neoron survival (Cauda 2011). ANA-12 produces rapid antidepressant- and anxwrytie-like eflects in animal models, the former of which have been ducidtted to be mediated by blockade of BI^ signahng m Ae nticleu

MS-275 is also known as Entinostat, or SNDX-275 and is a benzamide bjstonc deacetylaae inhibitor undergoing clinical trials for treatment of various cancers. Fjirmottat inhibits class I HDACl and HDAC3 win IC_M of 0.51 uM and 1.7 μΜ, respectively . It has the formula; Q»H*N-*¼.

Hwamptos of additional exemplary TrkB ioJnbiior conspounds include LOXO-lOl; N- T04; AZ623 and Enlrectinib. It it expected (hat any TrkB nrtbitor mat can penetrate the blood brain barrier will be suitable for die methods described herein. For example, LOXO- 101 is expected to be a suitable inhibitor and is described in: Cancer Discovery 5:1049-1057, 2015. Robert C. Doebefe el al. An Oncogenic NTRK Fusion in a Patient with SoA-Tissue Sarcoma with RMpoue to the TroponiyoeiR-Rilettxi Kinase Fusion tahmiior LOXQ-101.

The tenn "e-flective aaaousT of a coinpound b ft quantity sufficient to achieve · desixed tfaerspecitic and/or prophylactic effect fiV example, an anovnt which results in the alleviation, prevention of, or a decrease in the symptoms associated with a disease that is being treated.

"Activation," ''stimulation/* and "treatment,'* as it applies to ceils or to receptors, may have the same meaning, e.g., activation, sthmnation, or treatment of acefl or receptor with a tigand, unless indicated otherwise by me context or expboitjy. "Ligand*' encompasses natnml and synthetic Kgm-fa cytokines, cytokine variants, analogues, »** sod binding compounds derived from amibodies. "Ligand" also encc¾npasses small molecules, e^., peptide mimetic* of cytokines and peptide nmnetics of antibodies, "Activation" can reifar to ccfl activation as regulated by internal mechanisms as well as by external or environmental factors. Response,'^* eg., of a cell, tissue, organ, or otganitni, encompasses a change in biochemical or physiological behavior, e.g., concentration, density, adhftion, or migration within a biological conyartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms sixdh as genetic programming.

"Activity" of a molecule may describe or refer to the landing of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stmailatn gene expression or cefl signaling, differentiation, or tnatnrstion; to antigenic activity, to the modulation of activities of other molecules, and the hike. "Activity'' of a molecule may also refer to activity in modulating or maintaining cetHo-ccfl mteractiora, e.g.. adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. "Activity" can also mean specific activity, e*, [catalytic actrvity]/[mg protein], or [immunological activity)/[mg protein], cot-ceotEstion in a biological ttxi-partment, or me like. "Activity" may refer to modulation of cemponerrtsofthemnateof t^

*¾omology" refers to sequence similarity between two rwrynucleotide seqoenoes or between two polypeptide snnjuffnr** when they are optimally aligned. When a position in both of die two compared sequences is occupied, by die seme base or amino acid monomer sobuntt, e^, if a position in each of two DNA n»lecules is occupied by adenine, men the molecules are homologous at (hat position. The percent of homology is the number of honiologoos positions shared by die two sequences divided by the total number of positions compared *100. For example, if 6 of 10 of die positions in two sequences are matched or homologous whet* the sequences are optimally aligned then the two .sequences are 60% homologous. Generally, the comparison is made when two sequences are aligned to give maximum percent homology.

"Isolated nucleic acid molecule" means a DNA or RNA of genomic, raRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that "a nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes, isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified, sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequeoces that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

The phrase "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences thai are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequenee or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates hi the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words " trans■formants*' and "transformed cells" include the primary subject ceil and cultures derived therefrom without regard for the number of transfers, it is also understood that not ail progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have fee same function or biological activity as screened for in the originally tr afisfoaned cell are included. Where disimct designations ate ratended, it will be clear from the context

As used haninv "porymerate chain reaction" or "PCR" refers to a procedure or technique in which specific nucleic acid sequences, RNA and/or DNA, are amplified as described in, e,g., U.S. Pal No.4,683,195. Generally, sequence inJbniietioa from the ends of the region of interest or beyond is used to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5" terminal nocleotides of me two primers can coincide with the ends of die amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Muliis et at. (1987) Cold Spring Harbor Symp. Quant. BtoL 51.263; Erich, etL, (1989) PCR TECHNOLOGY (Stockton Press, N. Y.) As used herein, PCR is comridered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample axqmsing the use of a known nuclek acid as a primer and a nucleic acid polymerase to amplify or generate a -φβ^

With respect to cells, the term "isolated" refers to a cell mat has been isolated from its natural environment (e.g., from a tissue or subject). The term "ceD line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are donal populations derived from a single progenitor cefJ. It is further known in the art mat spontaneous or induced changes can occur in karyotype during storage or transfer of such cionelpci»lalions.71)m

identical to the ancestral ceHs or ctdtures, and the ceD line

As used herein, the terns "recombmsjit c*U" refers tt DNA segment, such as DNA segment mat leads to the transcription of a otologicalfy-active polypeptide or production of a biologkalhr active nucleic acid such as an RNA, has been introduced.

The term "vector* includes any genetic castnsnt, such as it plasrnsi, phage, uaiisposon, coemid, chromosome, artificial chiornosome, virus, virion, etc, which Is capable of replication when wwr iatrrrt with the proper control mff0mr^m and which can transfer gene. yfnnm^fff ¾p^^Y|ffft ffflf Thus, me term mcludes e kningg and firprpMioii nrhicltn. HI well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which me nucleic acid segment to be transcribed is positioned under the ttssacriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or. introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operativeiy positioned," "operativeiy linked," "under control," or "under transcriptional control" means that the promoter Is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shKNA, miRNA) from a transcribed gene.

"Inhibitors" and "antagonists," or "activators" and ''agonists," refer to inhibitory or activating molecules, respectively, e.g., for the activation of, e.g., a iigand, receptor, cofactor, a gene, cell, tissue, or organ. A modulator of, e.g., a gene, a receptor, a Iigand, or a cell, is a molecule that alters an activity of the gene, receptor, iigand, or cell, where activity can be activated, inhibited, or altered in its regulator}¹ properties. The modulator may act alone, or it may use a cofactor, e.g., a protein, metal ion, or small molecule. Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g., a gene, protein, Iigand, receptor, or cell. Activators are compounds that increase, activate, facilitate, enhance activation, sensitize, or up regulate, e.g., a gene, protein, Iigand, receptor, or cell. An inhibitor may also be defined as a compound that reduces, blocks, or inactivates a constitutive activity. An "agonist" is a compound that interacts with a target to cause or promote an increase in the activation of the target. An "antagonist" is a compound that opposes the actions of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist. An antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist.

To examine the extent of inhibition, for example, samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activator or inhibitor and are compared to control samples without the inhibitor. Control samples, i.e., samples not treated with antagonist, are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 25%. Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, iMro patently*

often at leoet2>lrjl4

10-fold, preferably at least 20-fold, mare preferably at least 40-fbki, and n»st preferably over 40-foU higher.

Bndpoints in actuation or iidubitiaa can be momtorcd as follows. Activation, inhibition, and response to treatment, &g., of a cell, physiological find, tisane, organ, and predetermined qiiantity or percentage of, e.g., indicia of inflammation, oncogenicity, or cell degnmularionorse The cndpoim may cop.p.ise, eg., a predetennined quantity of ion fan, or transport; ccfl migration; ceo adhrsion; cell proliffe-etion; potential for tnctattsttr, cell differentiation; and change in pbenotype, e.g., change in expression of gene relating to tnflammarion, apoptosia, transfonnation, cell cycle, or metastasis (see, e.g_^ Knight (2000) Ann. CUn. Lab. Scl 30:145- 158; Hood and Cberesh (2002) Nature Rev. Cancer 2:91-100; Timme, et ai (2003) Oar. Drug Targets 4251-261; Robbins and Itzkowitz (2002) Med. CUn. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev. Genomic* Hum. Genet 3:101-128; Bauer, et ai.

(2001) Gila 36235-243; Stanimirovic and Satoh (2000) Brum Pathol. 10:113-126).

An endpoint of inmuftkn is generally 75% of the control or lest, preferably 50% of the control or less, more preferably 25% of the control or less, and most preferably 10% of the control or leas. Generally, an endpoint of actrvation is at least 150% the control, preferably at least two times me control, more preferably at least four times me control, and most preferably at least ten times the controL

"Small molecule" is defined as a molecule with a molecular weight mat is km man 10 kOa, typically less than 2 kDe, preferably less than 1 kDa, and most preferably less than about 500 Da. Small molecules include, but are not timited to, inorganic molecule*, organic molecules, organic molecules containing an aorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide nrimetics, and antibody nmnerks. As a therapeutic, a small molecule may be mora permeable to cells, less susceptible to degradation, and less apt to elicit an immune response than large molecules SmallinoJecnlee. such ss MM-M* mimrtiflt of antibodies and cvaikines. as well aa small nmlfffflile toxins, have been described (see, Cassef, et at. (2003) Btocmm. Bkpkys. Res. Cbmmm. 307:198-205; Muytdennans (2001) J. Btofedmol 74277-302; Li (2000) Nat. BtotechnoL 18:1251-1256; Apostokwofdoa, et al. (2002) Oar. Med. Chan. 9:411-420; Moiuwmni, etal.

(2002) Cur. PharwL Des. 82185-2199; Dcmmgues, et ai. (1999) Nat. Struct Bid 6:652- 656; Sato and Some (2003) Biockem, J. 371 ;603-608; U.S. Patent No. 6,326,482 issued to Stewart, et a!),

"Treat" or "treating" refers to administering a therapeutic agent, such as a composition containing a TrkB inhibitor (e.g., ANA- 12 ) or combinations with, one or more SSRI inhibitor (e.g., Citalopiam (Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil Seroxat); Fluoxetine (Prozac); Fluvoxamine (Luvox); Sertraline (Zoloft, Lustral), or similar compositions described herein, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease or being at elevated at risk of acquiring a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such sympiomt^'s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the "therapeutically effective amount") may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g., a treatment method or article of manufacture) may not be effective in alleviating the target disease sympiom(s) in every subject, it should alleviate the target disease symptom(s) in a statistically significant number of subjects as determined, by any statistical test known in the art such as the Student's t-test, the chr-test, the U-test according to Mann and Whitney, the Kmska!-Waliis test (B-test), Jonckheere-Terpstra-tesi and the Wilcoxon-test.

"Treatment," as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications. "Treatment" as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses combination treatments including: one or more TrkB inhibitors, optionally in. combination with one or more SSR.T inhibitor (described herein)., or related methods described herein as applied to a human or animal subject, a cell, tissue, physiological compartment, or physiological fluid. Pharmaceutical Compositions and Administration

Formulations of therapeutic and diagnostic -agents may be prepared by mixing with acceptable carriers, exctpients, or stabilizers in the form of, e.g., iyophjfeed powders, slurries, aqueous solutions or suspensions (see, e.g., Hardmaa, et al. (2001) Goodman and Oilman '$ The Pharmacological Bam of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincotl, Williams, and Wilkins, New York, NY; Avis, et al (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Liehennan, et al (eds.) ( 1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipi&tt Toxicity and Safety, Marcel Dekker, inc.. New York, NY).

Toxicity and therapeutic efficacy of the therapeutic compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures to cell cultures or experimental animals, e.g., for determining the LD_S0 (the dose lethal to 50% of the population) and the EDse (the dose therapeutically effective in. 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD₅0/ EDse). hi particular aspects, therapeutic compositions exhibiting high therapeutic indices are desirable. The data obtained from these ceil culture assays and animal studies can be used in formulating a range of dosage for use in human.. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In an embodiment of the invention, a composition of the invention is administered to a subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal., or Inira-arterial

In. particular embodiments, the composition or therapeutic can be administered by an invasive route such as by injection (see above). In further embodiments of the invention, the composition, therapeutic, or pharmaceutical composition thereof, is administered intravenously, subcuianeously, intramuscularly, mtraarterial!y, iritra-attieularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery. Administration by non- invasive routes (e,g ., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can. be administered by injection with a hypodermic needle, including, e.g., a prefil!ed syringe or autoinjector.

The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; S,312,335;_: 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556.

As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the sympioms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.

in certain embodiments, the present methods are suitable for treating adolescent or young adult patients (with typical age ranges from 30-22; but including those as young as 5- 10), in certain embodiments, patients in need of treatment will include those that have experienced ELS (early life stress, as described for example in PechteJ. and Piz/agalli 201. 1). In certain embodiments, it is expected that the patient will exhibit a cognitive deficit in one or more of the following features; working memory and attention set-shifting.

hi additional embodiments, it is expected that the patient will exhibit improvement in at least one of these features including working memory and attention set-shifting, during and following treatment with an effective amount of at least one TrkB inhibitor, when compared to normal baseline or a control patient value. Additionally, in certain instances and based on the present results in the model data, treatment should continue until improvements arc observed, at which time treatment can be stopped. The benefits of the treatment are expected to endure following treatment, as shown in the model, data provided herein.

In certain embodiments, patients in need of the present treatments will also benefit from receiving, an effective amount of one or more of a selective serotonin reuptake iiiiiibitor (SSRI's), such as one or more of the following;

● Citalopram (Ce!exa)

● Bscitalopram {Lexapro. Cipralex) * Paroxetine- (Paxil, Seroxat)

_* f luoxetine (Prozac)

* Fluvoxamme (Luvox)

* Sertraline (Zoloft, Lustra!)

In certain embodiments, the TrkB inhibitor (e.g. ANA-12) and desired SSRI may be combined into one formulation for ease of patient delivery and compliance.

The pharmaceutical compositions may also contain a pharmaceutically acceptable excipient Such excipients include any phannaceutieal agent that does not itself induce the production of antibodies .harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable satis can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromid.es, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pil buffering substances, and the like., may be present hi such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991).

Kits

The present invention also provides kits comprising the components of the combinations of the invention in kit form. A kit of the present invention includes one or more components including, but not limited to, one or more TrkB inhibiting compounds (such as Ana-12) as discussed herein, in association with one or more additional components including, but not limited to a -pharmaceuticalfy acceptable earner and/or an SSRI agent, as discussed herein. Kits may also include primers, buffers, and probes along with instructions for determining elevated levels of nucleic acid, proteins., or protein fragments of a desired target.

in one embodiment, a kit includes additional compoimds/c-omposition of the invention or a pharmaceutical composition thereof in one container {e.g., in a sterile glass or plastic vial) and a second pharmaceutical composition in another container (e.g., in a sterile glass or plastic vial).

in another embodiment, the kit comprises a combination of the invention, including one or more TrkB inhibitors (e.g. ANA-12) in combination with one or more SSRI inhibitors (as described herein) along with a pharmaceutically acceptable carrier, optionally in combination with one or more additional therapeutic agent components formulated together, optionally, in a pharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above. The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

EXAMPLES

Example L Reduced levels of HDACl at Bdnf promoter 111 and increased Bdnf mRNA expression in IMS Balb/e mice exposed to cognitive tests.

The finding that activation of HDACl and HDAC3 during adolescent development abolished the WM and attention set-shifting deficits of IMS Baib/c mice (Schmauss et at, 2014) suggests that reduced acti vity of these HDACs is functionally linked to the emergence of these deficits. To test this, we first performed chromatin immunoprecipitation (ChlP) experiments to compare the levels of HDACl and H0AC3 at promotors of selected plasticity-associated genes between standard facility-reared (SFR) Balb/c controls and IMS Baib/c mice at postnatal age P60 (see Pig. i A). To this end, we focused on the promotors of the Bdnf gene because of the proposed role of Bdnf expression, in modulating synaptic plasticity in the prefrontal cortex (PFC), the anatomic regions that governs the cognitive functions affected by early life stress exposure (Lewis et at, 2005). We further targeted the promotors of two immediate-early genes that encode transcription factors known to participate in the modulation of synaptic plasticity in response to cognitive challenges. One of the targeted genes is Egr2, It was previously shown with immunocytochemicai studies on C57B1/6 mice performing an attention-set shifting task (ASST) that Egr2 immunoreactiyity was specifically increased in the ventrolateral orbital frontal cortex and the pre- and infmlimbic subregions of the medial prefrontal cortex (mPFC) during the associative lea ning phases of the ASST, and that infraiirabie Egr2 expression farther increased when mice performed the set-shifting phases of the ASST (DeSteno and Schmauss, 2008). In contrast to ASST-tesied mice, .rake that performed. a spatial WM test did not exhibit induced Egr2 gene expression (DeSte.no and Schmauss, 2008). This differs from the other gaie targeted in this study, namely the /¾$ gene. We previously showed that stimulation-induced levels ot fos gene expression in the anterior cingulate and preh^'rabic subregions of the mPFC positively cone] ate with the performance of mice in the spatial WM test. Moreover, exposure to the AS ST leads to induction of Fos gene expression in all subregions of the mPFC, and the magnitude of this induction is critical for optimal ASST performance (Glickstein et al., 2002; Clkkstein et al.,2005). in contrast to Egr2 gene induction, ASST-tesied mice also exhibit Fos gene induction in other brain regions, including orbital, motor and somatosensory cortices, hippocampus, dorsal striatum, nucleus aecmnhens, thalamus, and hypothalamus (Glickstein et al, 2005).

In the present studies, forebrain neocortical tissue that includes the entire mPFC was used to determine the levels of HDACI and H.D.AC3 on pmmoiors of the Bdnf, Egr2_f and Fm genes. As shown in Fig, 2A, in SFR Balb/e mice the highest levels of HDACI were found at Bdnf promotor III, and HDAC i association with the same promoior was significantly reduced in IMS Balb/c mice. In contrast, the levels of HDACI at Bdnf promotor IV as well as the HDACI levels at the promoters of the Egr2 and Fos genes were indistinguishable between SFR and IMS mice. Moreover, no significant differences were found for HDAC3 associations with any of the promoters examined.

The finding of reduced HDACI association with Bdnf promotor 111 in IMS mice suggests increased acefylation of hisiones marks of open chromatin and facilitated gene transcription, thus allowing increased gene transcription from this promotor either at baseline or in response to stimulation. The later option is supported by previous results showing, for example, that enhanced serotonergic stimulation led to increased enrichment of acetylated htstone H4K12 (acH4K12) and RNA Poiymerase H (Pot II) at Bdnf promotor III along with increased expression of Bdnf niRNA transcribed from this promotor in IMS Balb/c mice (Schmauss, 2015). Here we compared the expression of Bdnf transcript varian t iii mRNA. in non-tested and WM-tested SFR and IMS Balb/c mice. In this study, tested mice had completed the WM test at 20 s delay one hour prior to brain collection (see Fig. IB). Age- and sex .matched non-tested SFR and IMS mice served as controls. The .results of real-time RT-PCR experiments are shown in Fig, 2B. A 2 -way AN0VA revealed a significant main effect of rearing groups (SFR and IMS; F( 1,20) ~ 21 ,9; p < 0,0001), a significant main effect of treatment (tested versus non tested; F(1 ,20) ^::: 30.4; p ^::: 0.0001 ), and a significant interaction between these main effects (F(l,20)- 18.5, p ~ 0.0004). Post hoc Tukey BSD tests resolved these differences for WM-tesied IMS Balb/c mice thai expressed significantly more Bdnf mRNA compared with non-tested IMS mice as well as tested and nonnested SFR mice. This enhanced transcriptional activation of Bdnf promoter 11.1 is consistent with results of CMP experiments demonstrating significantly increased density of the actively elongating form of RNA polymerase II (Pol ΙΪ) at Bdnf promoter III in WM-tested compared with non- tested IMS Balb/c mice (Fig, 2C). The levels of HDACi at Bdnf promotor 111 of WM-tested mice did not differ from the baseline levels determined for IMS mice at P60 (l/2^ΔCt.:^. 2.ΊΜ1 (IMS at P60) and 2.5 ±0,9 (WM-tested IMS at P84)).

In contrast to the large increase in Bdnf mRNA expression in WM tested IMS Balb/c mice, Bgr2 mRNA levels did not significantly differ between WM-tested and non-tested SFR and IMS Balb/c mice (Fig. 2D), and WM test exposure did also not lead to increased recruitmenl of Pol Π to the Egr2 promotor (not shown). Finally, both SFR and IMS mice exhibited a similar test-induced induction of c-P os mRNA. expression.

Example 2, Reducing Bdnf-TrfcB signaling during adolescent development improves the cognitive functions of IMS Balb/c.

Next we asked whether overexpression of die Bdnf gene contributes to the cognitive deficits of IMS Balb/c mice. Since Bdnf protein mediates its effect through TrkB receptors (Kaplan and Miller, 2000), we took a pharmacological approach to reduce Bdni-TrkB- recepior activation using the low-molecular weight TrkB antagonist Ana- 12. Ana- 12 has been shown to penetrate the blood-brain barrier, to bind selectively to TrkB receptor, and to prevent TrkB-reeepior activation by Bdnf with high potency. Ana- 12 does not to affect TrkA and TrkC receptor function (Cazoria et al., 201 1). In the study shown in Fig, 3, we treated both SFR and IMS Balb/c mice with Ana- 1.2. Treatment was initiated at postnatal day 35 ;{P3^'5; the developmental time point at: which reduced HDAC levels were firmly established in the forebrain neocortex of IMS mice (Levine et al., 2012)), and it continued until P59 (see Fig. 1 B). At the end of this treatment, SFR and IMS mice and their non-treated IMS and SFR controls were tested in an attention set-shifting test (ASST), followed by training and testing in the spatial WM test.

Fig, 3A summarizes results obtained from the ASST. In this test, mice proceed through.4 consecutive test phases, beginning with a simple discrimination (SD) between (wo different odors or textures to find a food reward, and followed by a compound discrimination (CD) to which an additional stimulus property (one new odor or texture) is added that does not guide correct response selection. Then, a completely new set of odors and textures is presented, but the stimulus property (odor or texture) that associated with correct response selection in the CD still guides correct response selection. This is the intxadiraeasional set shifting phase (IDS) of the ASST. Finally, another set. of new odors and textures is presented, hut in this extradimensional set-shifting task (EDS), the previously irrelevant stimulus dimension (odor or texture) is now guiding correct response selection. For al! groups of mice, Repeated Measures ANOVA and post hoc Tukey-Kramer multiple comparisons showed that the number of trials to criterion was significantly higher for the EDS phase compared with a!l other test phases, including the IDS (SFR: p < 0.001; SFR-Anal2: p < 0.001 ); (IMS: p < 0.001; IMS Anal 2: p < 0.0.1). Moreover, 2- way ANOVA revealed a significant main effect of treatment (F(3,108) = 7.2, p = 0.0002), a significant main effect of test phase (F(3,108)=75.8; p < 0.0001 ), and a significant interaction between these main effects (F(9,I08)= 6, p < 0.0001 ). Post, hoc Tukey HSD teste resolved these diiierences for the EDS phase of the AS ST: Consistent with our previous findings (Mehta and Schmauss, 2011), compared to non- treated SFR mice, non-treated IMS Balb/c mice required significantly more trials to criterion to complete the EDS phase (Fig. 3.4), This deficit, however, was not detected in IMS Balb/c mice that were treated with the TrkB antagonist Ana- 12 during adolescence (Fig. 3A). Strikingly, Ana- 12 treatment of SFR mice significantly impaired their performance in the EDS phase. These findings indicate that both hypo- and hyperactivation of TrkB-receptor can cause deficits in this test.

A similar result was found in a delayed alternation spatial WM test (T maze). Here, 2- way ANOVA revealed, a significant main effect of treatment (F(3_S67) - 3.4, p ^~ 0.02), a significant main effect of delay periods (F(3,67) - 19.4, p < 0,0001), and a significant interaction between these main effects (F(6,67.) = 2,4, p - 0.03). Post hoc Tukey HSD tests resolved these differences for the groups of mice lested at 20 s inter-trial delay. At this delay, non-treated IMS mice had a significantly lower percentage of correct arms entries compared to non-treated SFR mice, but Ana- 12 treated IMS mice did not differ from non-treated SFR mice (Fig. 3B). Moreo ver, similar to the effect of Ana- .12 treatment of SFR mice on the performance in the EDS phase of the ASST, Ana- 12 treatment of SFR mice also impaired significantly their WM performance when tested at 20 s delay, again indicating that both hypo- and hyperactivation of the Bdnf-TrkB signaling pathway can cause cognitive deficits. Example 3: Reducing HI> AC I activity during adolescent development of SFR Balh/c mice also leads to cognitive deficits.

The results shown above indicate that, in IMS Balb/c mice, enhanced Bdnf-TrkB signaling plays a significant role in the emergence of their executive cognitive deficits. To farther test the role of reduced activity of HDACl in this process, we conducted additional studies o» SFR , Balb/c mice .hat were treated daring adolescence (P35 to PS9) with MS-275, an inhibitor of class 1 HDACs with nanomolar affinity to HDACt , a NMl-fold lower affinity to H.DAG3, and no affinity to other HDACs (Hu et al.,2003; Khan et al, 2008). As shown in Fig* 4 A, compared to non-treated SFR rake, MS-275-treated SFR mice exhibit deficits ia the EDS phase of the ASST that are similar to those found in IMS mice. Moreover, they exhibit a significant deficit in the spatial WM test at 20 s inter-trial delay (Fig. 4B). Next we compared the levels of mRNA transcribed from Bdnf promotor III and the Egr2 promotor between SFR Balb/c mice and non-tested and WM-iested SFR Balb/c mice that were treated with MS-275 using real-time RT-PCR. The tested MS-275-treated mice completed the spatial WM test (20s delay) one hour prior to brain collection (see Fig. IB). Fig. 4C illustrates that although the levels of Bdnf inRNA did not differ between SFR and non-tested MS-275-treated Baib/c mice., tested MS«275*treated Balb/c mice expressed significantly more Bdnf transcript variant III mRNA. in contrast, Egr2 mRNA levels did not significantly differ between the three groups of mice (Fig. 4D). Hence, MS-275 treatment during adolescent development of SFR mice leads to the same cognitive deficits detected in IMS Balb/c mice and the same test- induced increase of Bdnf transcript variant ill mRNA expression.

Example 4: Cognitive deficits of C57BI/6 mice fostered by Balb/c mothers during IMS exposure.

it was previously shown that C57B1/6 mice raised by Balb/e mothers and exposed to the IMS paradigm do not develop WM deficits but strikingly, they do exhibit deficits in the EDS phase of the ASST (Schmaoss et. ai.., 2014), We replicated this finding in the present study in which C57B1/6 mothers raised either their own pups with and without IMS exposure, and Balb/c mothers fostered C57B.1/6 pups during IMS exposure (see Fig. 1C).

For the ASST performance of SFR and IMS C57BI/6 mice raised by their biological mothers, Repeated Measures ANOVA and post hoc Tukey-Kramer multiple comparisons revealed that their numbers of trials to criterion were significantly higher for the EDS phase compared to ail other test phases, including the IDS phases (p < 0.01) (Fig. 5A). The EDS performance of foster IMS C57B1/6 mice raised by Balb/c mothers also differed significantly from all other test phases, including the IDS (p < 0.001) (Fig. 5A). A 2-way ANOVA further revealed a significant main effect of rearing groups (F(2.,76) =■ 10.46, p -O.OOOi), a significant main effect of test phase (F(3,76) = 34,31 , p < 0.0001), and a significant interaction between these main effects (F(6,76) - 5,0, p ~ 0.0002), Post hoc Tukey HS^'D tests resolved these differences for foster IMS C57BI/6 mice that required a significantly higher number of trials to complete the EDS phase of the ASST (Fig. 5,4). In order to test whether inter IMS C57BI/6 mice abo exhibit decreased HDAC1 anoristioii villi promoters of any of the three plasticity awocieted genes studied here at baaetine (P60; see Ftf. 1A), we performed HDACl and HDAC3 ChlPs on fbrebrain neocortkal tissues of SFR and IMS C57BI/6mice raised by their biological mothers and on foster IMS C57B1/6 mke that were raised by Balb/c mothers. No significant difFereocea were foond betwecD the three groups for the levels of HDACl and HDAC3 at flU^promotore Hi and rv and the Fov pmnolor (VI»

uvgering die Egr2 pnmnton Qmmared with SFR and IMS C57BI/6 mice, the levels of HDACl (bm not HDAC3) were significanUy lower m

We further measnred Bdnf owiscript variant 111 and Egr2 mRNA in ASST-lested and non-tested foster IMS CS7BI/6 mice by real-time RTPCR and con-pared these results to non- tested and ASST-tested IMS Balb/c mke. In these experiments, all pops raised by BaHVc inothers were foster pups, and brains of tested animals were coUecled 1 h after completion of the EDS phase of the ASST (see Fig. 1Q. For Bdnf mRNA expression, 2-way ANOVA revealed a significant main eflect of test exposure (F(l,16>»4.1, p » 0.05) and a significant interaction between strain and test exposure (F(l,16) "4.1, p-0.05). Post hoc Tukey HSD tests resolved tbsse differences for ASST-lested foster IMS Balb/c mice mat exhibited significantly increased Bdnf mRNA expression compared to all other groups of mice (fig. 6A). (The magnitude of dris increase, however, is only ~2-5-fbld compared lo the M 0-fbld increase detected in WM-tested IMS Baib/c mice (see Fig.2B)).

For £gr2 mRNA expression.2-way ANOVA also revealed significant main effects of lest exposure (F(l,18) - 6J5, p - 0.02)) and strain (F(l,18) - 4.4, p - 0.05)), and a sigm'ficant mumction between them (F - 4.36, p - 0.05). Post hoc Tukey HSD tests showed that Egr2 mRNA levels were significantly elevated in tested foster IMS C57B1/6 niice compared with all other groups of mice (Fig, <■), a rmdiag consistent with the increased levels of Pol Π at the Egr2 promotor in ASST<4ested foster IMS C57BW mke relative to non-tested foster IMS CS7B1/6 mice (Fig. *C). The HDACl levels at the Egr2 promoter of ASST-tested foster IMS C57B1/6 mice, however, did not differ from corresponding baseline levels nieasured at P60 (1/2⁴⁰: 0.42 ± 0.09 (foster IMS C57B1/6 at P60) and 0.37 ± 0.03 (ASST-tested tbsuy IMS C37BV6 at P66)X IiisumniiKy. foster IMS C57B1/6 mice that have only defidts in the ASST differ from IMS BaaVc mice by extibitmg ledaced HDACl levels at the Egr2 pcomotor rather than promotor Π1 of the Bdrtf gene, and they exhibit increased Egr2 mRNA expression laJhertfaanmcvaasedBdrf Finally, (here ere alio diffimnoes in histone modifications associated with Ikhtf promoter HI and the l¾r2 promoter between IMS BaftVc and foster IMS C57BV6 nice. Whereas IMS Balb/c mice have increased acH4KI2 levels associated with Bctyytottxwt ΙΠ compared with SFR BaftVc mke (Schmanss.2015), the kveb of scH4K12 at this promoter did not differ between SFR C57B1/6 controls and foster IMS C57B1/6 mice (1/2^. 3.01 ± 1.03 (SFR C57B1/6) and 3.18 ± 0.9 (foster IMS C57B1/6) (p-0.9)). For the -¾r2 promotor, however, we observed a significant reduction of die levels of a repressive mark of gene transcription, namely H3K9me3, in foster IMS C57BI/6 mice. Since reduced levels of acH3K9tne3 were not found in IMS BaBVc mice, we ccedocted additional ChlPs targeting die Egr2 promoter using an antibody directed against acH3K9, a marker of active gene inscription, and we expressed these results as the ratio of acH3K9/H3K9me3 levels associated with the Egr2 promotor. As shown in Fig. e"D, foster IMS CS7B1/6 mice (raised by BaBVc mothers) had a ngm^csntiy higher acH3K9/H3K9me3 ratio compared to SFR and IMS C57B1/6 and BaBVc mke (raised by their biological mothers). Hence, although reduced promoter-associated levels of HDAC1 am a common denominator for BaJb/c and C57B1/6 mice raised by BaBVc mothers during IMS exposure, the genes affected and the histone modification profiles at die promotonoftheafiecfirf genes are also influenced by the genetic backgnnmds of die pups.

Fiaaiplf St Twalauatsfor Adafcsces* and YaejafAdnJmwttl. flfraar I.ads*«jd/Retnted CefadrJvela^ainBeat

The below outfine is flhistiativeofa nxtlMd for improving cogmnve flatrtwumg m a patient in need thereof conyrismg adrmnistering a therapeutically effective ainount of at toast one oiiin-derived neurotrofiric factor (BdnfHropomyosine kinase B (TrkB) inhibitor to the A population of patients that wiflben^

the IbJhiwing: an adolescent (typically 10-18) or young adult patiem (18-24) with a mood/emotional disorder which can be an anxiety disorder or stress- induced disorder, inchiding early life stress, or a cornbmation thereof. Additionally, the mood disorder can also be a panic disorder, an obsessive ccmpfilsrve disorder, a post-tFenmtic stress disorder, or any ty^ft^ftw ."flwoA In. certain einbcdnnontt} it is. wrpgcteii mat adnhs could also benefit ftpin twtsjhT-cnts described herein.

The patiax win be treated whn an erre^

of crossing the blood brain barrier. H V QQI06SBQL vBSQQ BPvfl IBC XDQQH 1BDQSO flUI 9BQWII OB-PBUL ΙΕΗΚΊΟΒ mOBTB ΙΗΜΠΗΙ patient will exfaint at least a 10% iutprovwnaai of cognitive functioning, compered with a fratfKnf cognitive functioning reference or fflfftrof In ffilsiii instances it is expected that the cogmtive fhretionin^

an effective amount of a TrfcB inhibitor such at ΛΝΛ-12. It is also expected thai tbe treated human patient will exhibit an impravement in one or more of die typical indicators of cognitive fiucaVxiing inchiding wotting timmmy mtfuftimi set*sbifttng. The treatnieat will be continued for » period of time nntiJ these inyOvements are observed and mamtained and this time can vary fiom several week^ to several monms. h u expected that these trealed patients will be less likely to experience a relapse.

m edm¾onal instances, me patient will be evahiated (either simuhancously or prior to TrkB mhibitor treatment, or sobsequent to TrkB mnflHtor treatment) fix symptoms mat would indicate treatment with an SSRI would be indicated (See Millan et aL 2012 and Mclntyre el ■L 2013). m these mstances where treatment with one or mete SSRI are indicated during treatment with at least; one TrkB inhibitor, the patient win be treated with at least one selective serotonin renptake inhibitor (SSRI) selected fiom the group consisting of Citalopram (Ceiexa); Escitalopram (Lcxapro, Cipralex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoountine (Luvox); Sertrsline (Zoloft, Lustra!), or any cctiftination thereof. U is expected that Jbr patients treated with both a TrkB inhibitor and al least one SSIR, that both their cognitive and emotional phenotypes will exhibit improvement and will be less likely to experience a relapse. The treatment will be continued for a period of tune until these inaxwemenls are observed and maintained and Pus time can vary Scorn several weeks, to several months. Qptinial cognitive task perfanjance' depends upon fineljMuned babjace between neuronal activation and uthibmon, a that is by tightly regulated gene induction and repression (Rao et al, 2000; Millan et aL, 2012; Pezze et aL, 2014). Since HDACs are in a strong position to modulate the expression of plasticity-related genes nnpKcatad in the control of cognitive Junctions, the present study asked whether die cognitive deficits found in nuce exposed to early Efe stress can., be expstined by reduced o¾ pi ration of class I HDACs 1 and/or 3 that we previously detected in IMS BaUWc mice (Uvine et aL, 2012). We found that IMS Baftvc mice exhibit reduced association of HDAC1 (but not HDAC3) specifically with Bdnf promoter IU, and mat cognitive task exposure leads to increased density of the tmnacrinuonafly active form of Pol Π at this premolar, a raiding consistent with the large increase in Bdnf transcript variant III expression found after cognitive testing. Moreover, when non-stressed Balb/c mice were treated during adolescence with the HD AC] -preferring inhibitor MS-275, they exhibit the same cognitive deficits found in IMS mice as well as the same enhancement of test-induced Bdnf gene expression. The results of these pharmacological studies indicate clearly that the cognitive deficits and the associated change in Bdnf gene expression are due to reduced HDAC1 activity.

Our finding that blunting the Bdnf-TrkB receptor signaling during adolescence abolishes both the WM. and attention set-shifting deficits in IMS Balb/c mice indicates that overexpression of the Bdnf gene contributes substantially to the emergence of these deficits. However, we found that. SFR mice treated with the TrkB receptor antagonist. Ana- 12 also exhibit deficits in spatial WM and the EDS phase of the ASST. While this result was unexpected, it is consistent with the prevalent view that both hypo- and hyperactivation can cause cognitive deficits (Rao et al._< 2000; Pezze et al, 2014), i.e., both reduced and enhanced Bdnf-TrkB receptor signaling can have detrimental effects on cognitive functions.

Strikingly, in IMS Balb/c mice, the same HDAC1 -dependent increase in Bdnf-TrkB signaling has been shown to enhance the antidepressant efficacy of adolescent treatment, with fluoxetine (Schmauss, 201.5), However, the present findings can explain why adolescent treatment of IMS Balb/c mice with fluoxetine (an exemplary SSR1), although effectively improving their emotional phenoiype, does not improve their cognitive deficits (Schmauss et at, 2014). This raises the intriguing question whether antidepressant drug treatments that enhance the Bdnf/TrkB-signaling effects benefit those patients that also have cognitive deficits, a question particularly germane to findings that patients in remission from depression that continue to have cognitive deficits are more likely to relapse (Mil km et al., 2012; Melntyre et a!., 2013; Trivedi and Greer, 2014).

Although studies illustrated that the effect of IMS exposure on pups is due to the

IMS-triggered alteration in maternal care (Huot et al., 2004; Schmauss et al, 2014), we found that IMS-triggered cognitive deficits as well as the reduced HDAC1 association with promoters of distinct plasticity-related genes are influenced by the genetic backgrounds of the pups. IMS C57B1/6 mice raised by Balb/c mothers during IMS exposure exhibit deficits in attention set-shifting, but no WM deficits. Moreover, while their levels of HDACl at Bdnf promoter ill are unaltered, they exhibit decreased HDACl levels at the promotor of the Egr2 gene, a gene selectively induced in the ventrolateral orbital frontal cortex and the pre- and infralimhic suhregions of the rnPFC during ASST (but not WM) exposure (DeSteno and Schmauss, 2008), Indeed, only foster IMS C57B1/6 mice exhibit a robust (-5 fold) increase of tnmscription of the Egr2 gene upon ASST exposure. Since we previously showed Act mice with enhanced ASST paribraamce expressed lower levels of Egr2 than those with diffwiHitwtf ASSnr pCedfocisttDOC (DcStcoo vtd. SduMRiss» 2O08)« tfac pveseot fiwdifij^ Iwidy farther support for the conchision thai overaxpression of the Egr2 gene associates specifically with ASST selective cognitive deficits.

GMnpered with IMS Balb/c mke, foster IMS C57BV6 mice also exhibit a different hwtone ipodificinon phenotype at file E&2 proniotor that is characterized by an incraased ratio of acetylated H3K9 (a histone mark of open chromatin and active gene transcription) over H3K9me3 (a madrar of gene represtjon).While IMS Balb/c mice exhibit mcreased acetylation of bJstone H4 protein, especially histone H4K12 acetylatm atJ-^promotor HI (Schmauss, 2015), increased aoetyiation of histone H3 protein was not detected in these mice (Levine et aL, 2012). Hence, IMS Balb/c and foster IMS C57BI/6 mke do not only exhibit reduced HDACl levels at promoters of different genes, (he pnunotors of the affected genes also difler in their histone modification profiles. Nevertheless, despite vie cognitive task- induced enhanced ttnnsuiption of diflerenl plasticiry-ielated genes in bom strains of mice, reduced HDACl association with the respective promoters is a common epigenetic phenotype. myonantty. just as ongoing cognitive experience has been shown to regulate the molecular consequences of HDAC inhibition (Sewai et aL, 2015), the present study shows mat the effect of reduced promotofHtssociated HDACl levels on gene expression is potently unmasked by cognitive challenge. Moreover, in mice exposed to eady life stress, reduced HDACl levels are not found on promotors of all plasticnyntisociated genes fsatmmwi here, and cognitive challenge exerts different, gene specific effects in different genetic backgrounds. This finding points to a sensitive mleraction between genomic sequence variations (acting in cis) and the stressHDodulated epigcnome, an Ttrtwitrtyjn unlikely to be mrniidfod with studies on genetically modified mice harboring a conm¾onal HDACl deficiency.

Ccmrary to the present study mat uncovered a role of reduced HDACl activity in the emergence of early-life stress triggered cognitive deficits mat are largely governed by the PFC, several other studies have d½ronsh¾ed that HDAC inhibitors can amdiorate age- related cognitive fafltot CPelea et aJL 20101. or memosv ^wt^'wlt m Beuvodeeenerative disorders, UL, junctions that aw piimaiily governed by the hipp>x:sii¾ws (Fischer et al„ 2007; Kilgore et al., 2010; GitfF et aL, 2012). For example, in an animal model of inducible severe neurocleganeration and synapse loss, die non selective HDAC inhibitor sodium butyrate mcreased histone H3 and H4 acetyl anon in (he hippocainpns, promoted associative and spatial learning, ami increased expression of synaptic proteins in remaking neurons (Fischer et al, 2007): Similarly, in an animal model of Alzheimer's Disease with early onset contextual memory impairment, three non-selective HDAC inhibitors (sodium butyrate, sodium valproate, and suberoy^'ianilide hydroxarak acid (SAHA)) rescued memory deficits and led to stably maintained consolidated memory for two weeks (Kilgore ei al, 2010). Yet, the study of Kilgore et al (2010) also showed with biochemical assays that all. three HDAC inhibitors share in common a potent inhibition of class I HDACs 1-3, and other studies pointed to a specific ro!e of BDAC2 in the modulation of distinct cognitive functions. For example, studies on HDACl and HDAC2 overexpressing mice revealed that, although both lines of mice exhibit decreased acetylation of histone H.4K12 in the hippocampus, only HDAC2 overexpressing mice have deficits in associative spatial learning (a hippocampal function) and WM (a PFC function) (Guan et al, 2009), Conversely, HDAC2 knockout mice with increased histone H4 and H2B acetylation in the hippocampus exhibit increased associative learning and improved WM, and they have increased hippocampal spine density and synaptic terminals (Guan et al., 2009). HDAC2 deficient mice also exhibit increased acetylation of histone H3 and H4 at Bdnf promoter MI, Egr2, and Fox, and they are refractory to the effect of SAHA on synaptic plasticity and learning (Guan et at, 2009),

A specific role of HDAC2 in memory impairment is former supported by findings of increased HDAC2 expression in the hippocampal CA1 subfiekl and the PFC in an animal model of severe neurodegeneration. In this model, shRNA-mediaied. HDAC2 knockdown lead to increased acetylation of histone H4K12 at promotors of plasticity-associated genes along with increased expression of these genes, re-instated morphological and synaptic plasticity of surviving neurons, and improved associative and spatial memory (Graff ei al., 2012).

Finally, studies also identified specific roles for HDACl and HDAC3 in distinct hippocampal-dependent cognitive processes. One ^'study showed thai vims-mediated overexpression of HDAC l in the hippocampus did not affect WM and short- and long-term memory. However, it facilitated the extinction of fear memories and, conversely, hippocampal injection of MS-275 or knockdown of HDACl with siRNA significantly impaired fear extinction. The authors linked these effects to binding of HDAC l to Fas and. Egr2^' promotors where it affects histone H3K9 acetylation (Bahari-Javan et al., 2012). Another study on mice with a focal deletion of dorsal hippocampal HDAC3 and increased histone H4K8 acetylation (thai correlated with mcreased Fos and Nr4a2 gene expression) revealed a cognitive phenotype of facilitated long-term memory (McQuown et al, 2011.). It is evident from the studies sunanarked. above that class I HDACs 1-3 play distinct roles in. several cognitive functions. However, while reducing the activity of hippocarnpal HDACs 1-3 (and possibly also prefrontal cortical HDAC2) can exert a plethora of pro- cognitive effects, the present study revealed that early-life stress-triggered cognitive deficits arise primarily from, reduced HDAC1 association with promotors of distinct plasticity- associated genes. Whether reduced IIDACI activity in mice exposed to early life stress only affects executive cognitive function governed by the PFC, or whether Mppoearapal-relaied functions are also affected by reduced HDAC^'I (or HDAC3 activity) remains to be determined. However, since the HDAC-dependent epigenetk response of IMS Balb/e mice is only firmly established by mid-adolescence (P35) (Levine et a!., 2012) it is conceivable that it predominantly affects functions governed by late-maturing brain regions such as the PFC (Pechiel and Pkzagaili 2011).

Regardless of this physiological determination, as the present data using validated mouse models for these early stress conditions and corresponding cognitive deficits illustrate, thai pharmacological treatment of a patient with a TrkB inhibitor such as ANA- 12 will lead to improvement of the cognitive deficits and improvement in cognitive functioning (including improvement to working memory and attention set-shifting), which may also serve to benefit certain emotional phenotypes or conditions such as MOD (major depressive disorder) without any additional pharmacological intervention. In certain alternative instances, it is expected that in addition to treating a patient with a TrkB inhibitor such as ANA- S 2, that treating with one or more SSRi (including any combination of: Citaloprara (Celexa); Bscitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxatnine (Luvox); Sertraline (Zoloft, Lustra!}} would result in improvement to both the cognitive deficits and emotional p^'henotype- e.g., depressive condition/s.

Conclusions

The present results demonstrate for the first time that an epigenetk response to early life stress exposure contributes the emergence of working memory and attention set-shifting deficits, i.e. deficits that often accompany mental disorders and that are largely resistant to treatment. The results show that reduced histone deacetylase 1 levels at promotors of distinct plasticity-related genes allow- for abnormally increased, expression of these genes upon, cognitive testing, an effect that influences cognitive task performance negatively. The genes affected by reduced HDACI activity differ in genetically different strains of mice, indicating that genomic sequence variations and their differential interaction with the stress-modulated epigenome contribute to and determine the outcome of early-life stress-triggered cognitive impairment.

GENERAL METHODS

Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2^nd Edition, 2001 3* Edition) Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001 ) Current Protocols in Molecular Biology, Vols. I- 4, John Wiley and Sons. Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol.4).

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. /, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubei, et al. (2001 ) Current Protocols in Molecular Biology. Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigraa-Aldrich, Co. (2001) Products for Life Science Research, St Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in immunology, Vol. J, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001 ) Current Protocols in Immunology, Vol 4, John Wiley, Inc., New York).

Materials and Methods

Animals

Balb/cJ and C57BV6J mice were housed in a temperature- and light controlled barrier facility with free access to food and water. All experiments involving the animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committees at Columbia University and the New York State .Psychiatric Institute,

infant maternal separation (IMS)

In these experiments, Balb/c mothers either raised their own pups or fostered Balb/c or C57B1/6 pups from other litters from the clay of birth. Al! pups exposed to the IMS paradigm were separated from their mothers daily for three hours (from 1 :00 to 4:00 PM), starting at postnatal age P2 and ending at P15, They were weaned at P28 and group housed by sex (see Fig. 1A). Other pups of Balb/c or C57B1/6 mothers were left undisturbed with their mothers and were also weaned at P28. These mice are referred to as standard-facility reared (SFR). Housing and. husbandry conditions were identical for SFR and IMS mice. Drug treatments

Drugs were administered to mice (males and females) starting at P35 and ending at P59 (see Fig. IB). All drugs were administered via the drinking water. Drug intake was monitored daily and drugs were replenished every 48 h (MS-275; Sigma Aidrich, St. Louise, MO) or 24 h (Ana- 12; Calbiochem; BiMeriea, MA), MS-275 is also known as Entinostat, or SNDX-275 and is a benzaraide histone deacelylase inhibitor undergoing clinical trials for treatment of various cancers. Entinostat inhibits class 1 HDACl and HDAC3 with ICso of 0.51 μΜ and 1.7 uM, respectively, and is capable of penetrating the blood brain barrier weakly (See, Hooker J.M., et af, Histone deacetylase inhibitor, MS-275, exhibits poor brain penetration: PK studies of [C]MS-275 ttsing Positron Emission Tomography. ACS Chem Neurosci. 2010;I( l):65-73). It has the formula: C21H2ON4Q3.

ANA- 12 is a selective, small-molecule non-competitive antagonist of TrkB (Kd ^::: 10 sM and 12 uM for the high- and low-affinity sites, respectively), the mam receptor of brain- derived neurotrophic factor (BDNF/Bdnf) (Cazorla 2011). The compound crosses the blood- brain-barrier and exerts central TrkB blockade, producing effects as early as 30 minutes (-400 nM) and as long as 6 hours (- 10 .oM) following iiUraperitoneal injection in mice (Cazorla 201 .1). It blocks the neurotrophic actions of BDNF without compromising neuron survival (Cazorla 201 1). ANA- 12 produces rapid antidepressant- and anxiolytic- like effects in animal models, the former of which have been elucidated to be mediated by blockade of B^'DNF signaling in the nucleus accumbens. (Zhang JC 2015 and Shirayarna Y 2015). It has also been found to alleviate methamphetamine-induced depression-like behavior (including anhedonia), behavioral sensitization, and nucleus accumbens aeuropiasticity changes with subchronic (1.4-day) administration, in mice, whereas the TrkB agonist 7,8-dihydroxyflavone was ineffective in doing so (Ren Q 2015). IMS Batb/dmce fhst received Ana-12 consiimeri 1 iiu/kg/cley, and SFR mice that received MS-275 nomwiwl 15 uM/day. We previously shewed thai iieimer trettraent attend the emotional behavior of these mice when tested at the end of this treatment (Schmeass, 2015). However, at the dose administered, Ana-12 effectively blocked the antidepressant efiixts of fluoxetine in IMS Balb/c mice (Schmanss, 2015) and, in SFR BaJb/c mice, MS-275 increased the levels of acH4K12 and Pol Π at Bdnf promoter III only when coHKhnmktefed with fluoxetine (Schmauss, 2015).

CegaMve teste

To avoid fitter effects, one male and one finale moose of a total of at least 4 titters per treatment group were assigned to cognitive testing. Similar to a previous study (Mehta and Sdunanst, 2011), no significant sex diflerences in cognitive task performance of SFR and IMS mice were found Henoe, males end females are equally leprcsenied in die groups of mice studied here. Starting at postnatal age P60, mice were mod restricted such that they gmdually (over the period of 4 to 5 days) lost 10-15% of their free-feeding body weights. Prior to ASST testing, mice tailed to dig for food buried dee

filled wiA familiar bedd

the ASST. beginning with a simple discrimination (SD) between odor (seemed terra cotta pots) or texture (different digging media), and followed by a oonawund discrimination (CD) in which another stimulus property (a second odor or texture) was introdoced that was not a refaable predictor of rood reward. The third phase required an intredimensional shift of attention (IDS), ie., bom relevant and irrelevant stinmhis properties were changed, but the relevant stimulus m^'mension used in the SD and CD (odor or texture) remained the same. Finally, the Iboneriy irrelevant stimulus dimension became relevant and required an extredmMnstonal shift of attention (EDS). In all test irhasfit. -wHwt« had to reach a criterion of 6 consecutive correct trials, and the mimber of trials to criterion was referred to as respofise accuracy.

Some of the BaftVc mice that completed the ASST were further trained for alternate arm entries in the T-maze until they reached more than 70% correct arm entries (in 10 trials per day) on 2 consecutive days with 5 s inter-trial delay periods. Then, mice performed the testwnlL2.fai.Bjv

then working memory. (All groups of mice did not roaimam spatial WM at 30 s imeHriei delay). Only correct arm entries were rewarded with food, and the percentage of correct arm entries in the total number of 10 trials per deary period was taken as a measure of response accuracy. One hour ifler completion of cither the AiSST or the WM test, fcrabaan ofocortk-jrt tissue was dissected from male and femak mice using the mesodkncerjbalk junction as the anatomic landmark for me caudal border of the forebram. ASST-tested mke wan 66 days old (see Fig. 1Q, and their ηοα-tesied controls ware matched for age and sex. The age of WM-tested mke and their nonnested controls (also derived from at least 4 different litters pogrom?) ranged from P81 to P84 (see Fig. 1Q. Total RNA was extracted using guanidme/ccsram chloride uhiacentrifiigaoon. Fizslretiand cDNA was synmesized using Murine Moloney Leukemia Virus reverse bwncriptase (USB, Cleveland, QH) in conjunction with oHgo dTlS primers. Real time PGR was performed using die iQ Real Time PCR detection System (Bio-Rad, Hercules, CA) and SYBR Green (Bio-RadX Bdnf mRNA was amplified using me primers targeting Bdnf transcript variant m reported by Tsankova et al. (2006). Egr2 mRNAwas ampliiSed using the primer

pair 5'-ATGAACGGAOTOGCXKX3A-3' (SEQ ID NO:l); and S'- AOTAGACKiTOGTCCAOTT-3' (SEQ ID NO:2) and c-Fos mRNA was amplified using the primer pair 5'-ATCATGTTCTCGGOTTTGAA-3' (SEQ ID NO:3) and 5X^ACXXrrOOGGATAAAGTTOCK3' (SEQ ID NO:4). Cyck tlireshoUs (a) of anplifkation (normalized to those obtain for β actio) were expressed as 1/2?* values so mat higher numbers reflect higher expression.

Ckrwumi lmninnepit«lpltarteni (CU?)

CbJP was performed on forebnun neocorucal tissue of groups of male and female mke (selected from different titters as described above) either at P60 (baseline) or alter cognitive testing wim and without drug treatment (see Fig. 1). Dissected tissue was fixed with disrupter (Misomx, Fanningdak, NY) to an average DNA length of 200 to 400 base pairs. Samples were centrifuged al 15,000 xg, and 50 ul atiquots of fhe supernatant were iiiMU-i^eciuUated overnight with 20 ul protein A magnetic beads (Millipore, Temeeula, CA) and ChlP-grade nstibodies directed either against HDACl (MiluporeX HDAC3 (Abeam mc, Cambridge, MA), RNA Porymerase Π CTD repeat YSPTSPS (AbcamX trirnethyl- Mstone H3fc¾ (MffliporeX or acetyMibtone H3K9 (MiO-pore). Beads were treated according to me mstruction of flw mamiftctnrer. manunourei: iphtted DNA and « serial dilation of IX input were analyzed by SYBR-Greea real-tone PCR nsing primers targeting Bdnf promoter ΠΙ (original nomenclature) adopted from Tsankova et al (2006) and. the promoters of the Eg.r2 md Pos genes adopted from Bahari-Javao et al, (2012).

Statistical analyses

Repeated Measures ANOVA and one-way ANOVA were performed using GraphPad

Software (InStat; La .folk, CA), Two-way ANOVA was performed using the VassarStats Statistical Computation website (American Library Association, February 27, 2012).

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TNCmPORATfON BY REFERENCE

AU references cited herein are

15 inmvidual publication, database entry (e.g Genbank seqoences or GenelD entries), patent application, or patent, was apeciikally and indivkhially mdiratrd to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.FJL §1^7(bXlX to relate to each and every mdtrvidnal publication, database entry (e.g. Genbank sequences or GeoelD entries), patent application, or patent, each of which is

20 clearly identified in compliance with 37 CJF.R. f 1.57(b)(2), even if such citation is not inariediatery adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of uicorpofjttioo by reference, if any, within die specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not ml ended as an admission that the reference is pertinent prior art, nor

25 doftt if fflnsHftrtt— y nft^foifrwi at to ito wwtPto ο»* date nfihmm wiWkariont or dpfef*¹?

ΤΙιβ present invention is not to be faiittd in scope by the specific enibodiments described herein. Indeed, various modifications of die invention in addition to those described berem will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the

The foregoing written spcttiicjtjon is

in the art to practice die invention. Various modifications of the invention in addition to those shown and described herein will become appelant to those skilled in die art from die foregoing description and fall within the scope of the appended claims. The entire disclosure or eacu ot toe patent oocanwntt, nicioaing certuicaies at oonecuoa, patent application doetnneuts, scientific articles, govniunental reports, websites, ml other references referred to herein is mcoipontted by reference herein in its entirety for til purposes. In case of a conflict m terminology, tbe present specificatioo controls.

5 The invention can be embodied in other specific forms without douarhiig front die spirit or essential characteristics thereof. The fuiegoing efnbodiroents are to be considered in all respects illustrative rather than titrating on the invention described herein. Further, it should be ojideratood that the order of steps or order for perforating certain actions is immaterial so long as die invention remains operable. Moreover, two or more steps or actions

10 can be conducted shnuhnneoush/.

Unless defined otherwise, all technical and scientific terms used herein have me same meaning as commonly understood by one of primary skill in the art to which this mvention belongs, ta the case of conflict, u¼ present spe

Claims

WHAT IS CXAIMEB IS:

1 . A .method of treating cognitive defects in. a. mood disorder in a patient in need thereof comprising administering a therapeutically effective amount of at least, one brain-derived, neurotrophic factor (Bdnft-tropomyosine kinase B (TrkB) inhibitor to the patient.

2. The method of claim 1 , wherein the treating comprises at least a 10% improvement of cognitive functioning, compared with a baseline cogni tive functioning reference or control.

3, The method of claim 2, wherein the cognitive Sanctioning .improvement is greater than about 50%.

4. The method of claim 1, wherein the TrkB inhibitor is a TkrB antagoni st.

5. The method of claim I, wherein the TrkB inhibitor is administered by mouth, topically, reetally, or intravenously.

6. The method of claim I , wherein the mood disorder is an anxiety disorder or stress- induced disorder including early life stress, or a. combination thereof.

7. The method of claim 1 , wherein the mood disorder is a panic disorder, an obsessive compulsive disorder, a post-traumatic stress disorder, or any combination thereof.

8. The method of claim 1, wherein the patient is an adolescent or young adult.

9. The method of claim 1, wherein the TkrB inhibitor comprises ANA- 12.

10. The method of claim 1, further comprising administering at least one selective serotonin reuptake inhibitor (SSRI) selected from the group consisting of Citajopram

(Celexa); Escitalopram (Lexapto, Cipraiex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxamine (LUVOX); Sertraline (Zoloft, Lustra!), and combinations thereof.

Π. The method of claim 1, wherein the patient is a mammal

12. The method of claim 11, wherein the patient, is a human.

13. The method of claim I. , wherein the patient, exhibi is a cognitive deficit in one _.or more of the following features; working memory or attention set-shi fting.

14. A method of improving cognitive functioning in a patient in need thereof comprising administering a therapeutically effective amount of at least one brain-derived neurotrophic factor (Bdnf)-rropomyosme kinase B (TrkB) inhibitor to the patient,

1.5. The method of claim 14, wherein the improvement comprises at least a 10% improvement, of cognitive functioning,- compared with a baseline -cognitive functioning reference or control,

16. The method of claim 15, wherein the improvement is greater than about 50%,

17. The method of claim 14, wherein the TrkB inhibitor is a TkrB antagonist.

18. The method of claim 14, wherein the TrkB inhibitor is adn«imiere$ by month, topically, rectalty, or mtravenously.

19. The method of claim 14, wherein the patient ^'exhibits ^'a. mood disorder comprising m anxiety disorder or stress-induced disorder including early life stress, or a combination thereof.

20. The method of claim 14„ wherein the patient exhibits a mood disorder comprisi ng a panic disorder, a» obsessive corapuiss ve disorder, a posMraumatic stress disorder, or any combination thereof.

21. The method of claim 14, wherein the patient is an adolescent or young adult, 22. The method of claim 14, wherein the TkrB inhibitor comprises ANA-12.

23. The method of claim 14, further comprising administering at ieast one selective serotonin reuptake inhibitor <SSRi) selected from the group consisting of CitaJopram

(Celexa); Escitalopram (Lexapro, Cipralex); Paroxetine (Paxil, Seroxat); Fluoxetine (Prozac); Fluvoxamine (Luvox); Sertraline (Zoloft, Lustrai), and combinations thereof.

24. The method of claim 14, wherein the patient exhibits a cognitive deficit in one or more of the following features: working memory or attention set-shifting.