Inventors: J. David Sweatt and Iva Zovkic

BACKGROUND

Memory formation is a multistage process that initially depends on cellular consolidation in the hippocampus, after which the memory is gradually downloaded to the cortex for maintained storage in a process termed systems consolidation (Wang, S. H. et al., Annu Rev Psycho. 61 , 49-79, C41 -44, 2010) Memory formation in the hippocampus may be referred to as recent memory while memory in the cortex may be referred to as remote memory. Epigenetic mechanisms emerged as critical regulators of both types of consolidation because of their capacity for both transient and stable transcriptional regulation (Lesbui'gueres, E. et al. Science 331,924-928, 2011; Miller, C. A. et al., Neuron 53, 857-869, 2007). In the hippocampus, epigenetic modifications are rapidly and reversibly induced by environmental stimuli, whereas delayed and stable changes in cortical DNA methylation are required to maintain remote memory (Lubin, F. D., et al., J Neurosci 28, 10576-10586, 2008; Miller, C. A. et al., Nat . NeuroscL, 2010; Gupta, S. J Neurosci 30, 3589-3599, 2010). Existing studies have focused primarily on DNA methylation and post-translational modifications of histone. Histone variant exchange is an entire branch of epigenetics that has been only peripherally studied in the brain (Pina, B. et al., Dev Bio. 123, 51-58, 1987; Schauer, T. et al., Cell Rep, Cell Rep., 5, 271-82, 2013) and has not been investigated in relation to memory and cognitive function.

Epigenetic modifications regulate DNA access, which is obscured by its packaging into nucleosomes. The nuc le o s omes consist of two each of histones H2A, H2B, H3, and H4. Nucleosome composition and gene expression can be altered through the exchange of canonical histones with their non-allelic variants. In contrast to canonical histones, histone variant synthesis is replication-independent and can thus be incoiporated in non-dividing cells, a property that is of particular relevance in post-mitotic neurons (Pina, B. et al., Dev Bio. 123, 51-58, 1987).

H2A.Z is a variant of histone H2A that has been associated with environmentally-responsive genes in a variety of non-neuronal model systems (Adam, M. et al., Mol Cell Bio., 21 , 6270-6279, 2001 ; Coleman-Derr, D. et al., PLoS Genet 8, el 002988, 2012) indicating a hypothetical potential for this variant to mediate experience-dependent neural plasticity. H2A.Z confers a number of unique · properties on the nucleosome, including altered stability and preferential association with particular modifications on neighboring histones, as well as interactions with a distinct set of binding partners compared to the canonical H2A (Coleman-Derr, D. et al, PLoS Genet 8, el002988, 2012; Draker, R. et al., PLoS Genet 8, el003047, 2012). The role of H2A.Z in gene regulation is complex and it has been implicated in gene activation, gene repression, and in maintaining a poised state for the induction of silent genes by appropriate stimuli (Bargaje, R. et al., Nucleic Acids Res 40, 8965-8978, 2012; Schauer, T. et al., Cell Rep, Cell Rep., 5, 271-82, 2013; Smith, A. P. et al., Plant Physiol, 152, 217-225, 2010).

Despite this complexity, available evidence points to acetylation of H2A.Z as a positive regulator of gene expression (Millar, C. B., et al, Genes Dev 20, 711-722, 2006; Bellucci, L., et al., PLoS One 8, e54102, 2013; Valdes-Mora, F. et al., Genome Res 22, 307-321, 2012).

In the present study, the role of H2A.Z in recent and remote memory formation using fear conditioning as a mouse model of associative learning is disclosed. H2A.Z regulation during cellular memory consolidation in the hippocampus and during systems consolidation in the cortex were investigated.

The present disclosure provides, for the first time, a role of H2A.Z in suppression of recent and remote memory formation in the hippocampus and cortex, respectively. The present disclosure provides novel compounds and compositions for use in improving memory formation and/or memory recall and/or reversing the suppression of memory formation and/or memory recall by H2A.Z. The present disclosure further provides novel compounds and compositions for use in the treatment of diseases and conditions where memory, particular deficits in memory, are involved. The present disclosure further provides methods for treating a subject to improve memory, including memory formation and/or memory recall. The present disclosure further provides methods for treating a subject wherein the subject is suffering from a disease or condition where memory formation and/or memory recall, particular deficits in the foregoing, are involved. The present disclosure further provides a therapeutic target for use in improving memory formation. The present disclosure further provides a therapeutic target for in the treatment of diseases and conditions where memory formation and/or memory recall, particular deficits in the foregoing, are involved. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A shows chromogenic IHC staining of H2A.Z in the hippocampus.

FIG. IB shows staining is absent in negative control.

FIG. 1C shows fluorescent IHC staining of H2A.Z (red) and DAPI (blue) in the CA1 and dentate gyrus (DG).

FIG. ID shows fear conditioning altered gene expression of H2 fz,a gene encoding H2A.Z. N represents naive mice (untrained controls) (n=4); C represents mice subject to context only (n=7); S represents mice subject to foot shock only without contextual learning (n=2); and CS represents mice subject to foot shock and contextual learning (n=5).

FIG. IE shows fear conditioning altered gene expression H2A.Z. N represents naive mice (untrained controls) (n=3); C represents mice subject to context only (n=3); S represents mice subject to foot shock only without contextual learning (n=4); and CS represents mice subject to foot shock and contextual learning (n=3).

FIG. IF shows fear conditioning altered promoter methylation on H2afz,& gene encoding H2A.Z. N represents naive mice (untrained controls) (n=7); C represents mice subject to context only (n=9); S represents mice subject to foot shock only without contextual learning (n=5); and CS represents mice subject to foot shock and contextual learning (n=4).

FIG.1G shows fear conditioning altered gene expression of BdnflV. *Follow up comparisons with p < 0.05.

FIG.1H shows fear conditioning altered gene expression of Egrl. * Follow up comparisons with p < 0.05.

FIG. II shows fear conditioning altered gene expression of Arc (g). *Follow up comparisons with p < 0.05.

FIG. 2A shows experimental design summary.

FIG. 2B shows the effect of fear conditioning induced changes in H2A.Z biding at the Bdnf IV, Egrl, and Arc gene promoters at the 30 min and 2 hour time points after training. The left hand graphs examine H2A.Z and the right hand graphs examine acylated H2A.Z. * indicates follow up comparisons with p<0.05.

FIG. 2C shows the effect of fear conditioning induced changes in H2A.Z biding at the Bdnf IV, Egrl, and Arc gene coding regions at the 30 min and 2 hour time points after training. The left hand graphs examine H2A.Z and the right hand graphs examine acylated H2A.Z. * indicates follow up comparisons with p<0.05.

FIG. 2D shows the effect of fear conditioning induced changes in H2A.Z biding at the -1 and +1 nucleosomes of the Npas4, Egr2 and Arc memory activator genes as well as the memory suppressor gene Ppp3ca at the 30 min (first column) and 2 hour (second column) time points after training; corresponding gene expression is shown in the third column. The left hand graphs examine H2A.Z and the right hand graphs examine acylated H2A.Z. * indicates follow up comparisons with p<0.05.

FIG. 2E shows the effect of fear conditioning induced changes in H2A.Z biding at the -1 and +1 nucleosomes of the Egrl, Fos and Bdnf IV memory activator genes as well as the memory suppressor gene Ppplcc at the 30 min (first column) and 2 hour (second column) time points after training; corresponding gene expression is shown in the third column. The left hand graphs examine H2A.Z and the right hand graphs examine acylated H2A.Z. * indicates follow up comparisons with p<0.05.

FIG. 3A shows the experimental design summary.

FIG. 3B shows fear conditioning is associated with delayed changes in H2A.Z binding to gene promoters, gene coding regions and altered acetylation patters 24h after training for the Bdnf IV, Egrl and Arc genes. * in indicates follow up comparisons with p<0.05.

FIG. 4A shows a representative image depicting AAV spread.

FIG. 4B shows H2afz-AAV decreased H2afz mRNA expression at 24 hours (n= 9 for Scr AAV; n= 10 for H2A.Z AAV). Scr AAV represents a scrambled control virus construct; H2A.Z AAV represents an H2afz specific virus construct. * indicates follow-up comparison with p<0.05.

FIG. 4C shows H2A.Z AAV enhanced memory recall in a fear conditioning test 24 hours (n=18 for Scr AAV; n= 14 for H2A.Z AAV) and 30 days (n= 5 for Scr AAV; n= 6 for H2A.Z AAV) after training Scr AAV represents a scrambled control virus construct; H2A.Z AAV represents an H2afz specific virus construct. * indicates follow-up comparison with p<0.05. FIG. 4D shows the experimental summary for gene expression experiments.

FIG. 4E shows the effect of H2A.Z AAV mediated knockdown on mRNA levels for the Fos, Arc, Egrl, Egr2, Npas4, Bdnf IV, Ppp3c and Ppplcc genes for untrained (leftmost bar in each group) and trained (right most bar in each group) 30 min after fear conditioning. For Fos, Egr2, Npas4, Ppp3ca and Ppplcc: n= 3 for Scr AAV untrained, n=3 for Scr AAV trained; n=7 for H2A.Z untrained; n-7 for H2A.Z trained. For Arc, Egrl and Bdnf IV: n= 6 for Scr AAV untrained, n=3 for Scr AAV trained; n=8 for H2A.Z untrained; n=4 for H2A.Z trained. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05. FIG. 5. shows H2A.Z exchange in the mPFC occurs shortly after training and persistent changes are found at 7 days. * in FIGS. 5A-N. indicates follow up comparisons with p<0.05. In FIGS. 5C-N, the first bar in each time series represents na ^' ive animals, the second bar in each time series represents animals exposed to context alone and the bar in each time series represents animals exposed to context and shock.

FIG. 5 A shows fluorescent IHC staining of H2A.Z (red) and DAPI (blue).

FIG. 5B shows the experimental design summary.

FIG. 5C shows at 2h, 24h and 7 days after training, fear conditioning is associated with altered acetylated H2A.Z binding at the Bdnf IV, Egrl and Arc gene promoter.

FIG. 5D shows at 2h, 24h and 7 days after training, fear conditioning is associated with altered acetylated H2A.Z binding at the Bdnf IV, Egrl and Arc gene coding region.

FIG. 5E shows the effect of fear conditioning induced changes in H2A.Z biding at the -1 and

+1 nucleosomes in cortex of the Egrl, Egr2 and Arc memory activator genes as well as the memory suppressor gene Ppp3ca at the 2 hour (first column) and 7 day (second column) and

30 day time points after training (third column). * indicates follow up comparisons with p<0.05.

FIG. 5F shows the effect of fear conditioning induced changes in H2A.Z biding at the -1 and +1 nucleosomes in cortex of the Npas4, Fos and Bdnf IV memory activator genes as well as the memory suppressor gene Ppplcc at the 2 hour (first column) and 7 day (second column) and 30 day time points after training (third column). * indicates follow up comparisons with p<0.05.

FIG. 5G shows the effect of fear conditioning induced changes in acetylated H2A.Z biding at the -1 and +1 nucleosomes in cortex of the Egrl, Egrl, Arc, Npas4, Fos and Bdnf IV memory activator genes as well as the memory suppressor genes Ppp3ca and Ppplcc at the 2 hours after training. * indicates follow up comparisons with p<0.05.

FIG. 5H shows the effect of fear conditioning induced changes in acetylated H2A.Z biding at the -1 and +1 nucleosomes in cortex of the Egrl, Egr2, Arc, Npas4, Fos and Bdnf IV memory activator genes as well as the memory suppressor genes PppSca and Ppplcc at the 2 hours after training. * indicates follow up comparisons with p<0.05.

FIG. 6A shows the experimental design summary.

FIG. 6B shows a representative image depicting AAV spread.

FIG. 6C shows H2afz AAV reduced H2A.Z protein and mRNA expression (t3 = 6.91, p = 0.006; ScrA AV n=2; H2A.Z AAV n=3). Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05.

FIG. 6D shows the effect of H2A.Z AAV mediated knockdown on cognitive function in mice tested 24 hours (n=5), 7 days (n=5) or 30 days (n=8) after training. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05. FIG. 6E shows the experimental design summary.

FIG. 6F shows the effect of H2A.Z AAV mediated knockdown on mRNA levels for the Fos, Arc, Egrl, Egr2, Npas4, Bdnf IV, Ppp3ca and Ppplcc genes for untrained (leftmost bar in each group) and trained (right most bar in each group) 30 min after fear conditioning. For all genes: n= 5 for Scr AAV untrained, n=3 for Scr AAV trained; n=4 for H2A.Z untrained; n=4 for H2A.Z trained. For Arc, Egrl and Bdnf IV: n= 6 for Scr AAV untrained, n=3 for Scr AAV trained; n=8 for H2A.Z untrained; n=4 for H2A.Z trained. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05.

FIG. 7A shows the summary of experimental design.

FIG. 7B shows the effect of AAV exposure to open field tests, including locomotor activity, movement velocity, vertical activity and the time spent in the center between H2A.Z mice and scramble controls. n= 8 mice per group. Data are expressed as mean + s.e.m.

FIG. 8A shows that associative memory decreases with age, as evident by reduced freezing in aged compared to young mice. N= 4 mice per group.

FIG. 8B shows the summary of experimental design.

FIG. 8C shows the effect of H2A.Z AAV mediated knockdown on cognitive function in aged mice tested 24 hours (n=4) after training. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05.

FIG. 9A shows exemplary siRNA nucleic acid constructs.

FIG. 9B shows the effect of siRNA on H2afz mRNA in primary rat cortical neurons. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05.

FIG. 9C shows exemplary ASO nucleic acid constructs. The X* indicates phosphorothioated DNA (the middle 10 nucleotides) and mX* indicates phosphorothioated 2'O-Methyl RNA (the first and last 5 nucleotides).

FIG. 9D shows the effect of ASO on H2afz mRNA in primary rat cortical neurons. Data are expressed as mean + s.e.m. * indicates follow-up comparison with p<0.05.

FIG. 10A shows the sequence of the mRNA encoding H2A.Z from rat, mouse and human. FIG. 10B shows exemplary consensus sequences for regions to which ASOs of the present disclosure bind

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound that decreases the expression and/or activity of H2A.Z. In a second aspect, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject, wherein the improved cognitive function is a result, at least in part, of improved memory formation, improved memory recall or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a third aspect, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject, wherein the improved cognitive function is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2 A.Z.

In a fourth aspect, the present disclosure provides a method or a use of a medicament for improving memory formation in a subject. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a fifth aspect, the present disclosure provides a method or a use of a medicament for improving memory formation in a subject, wherein the improved memory formation is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a sixth aspect, the present disclosure provides a method or a use of a medicament for improving memory recall in a subject. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a seventh aspect, the present disclosure provides a method or a use of a medicament for improving memory recall in a subject, wherein the improved memory recall is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In an eight aspect, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where cognitive function, particular deficits in cognitive function, is involved. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a ninth aspect, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where cognitive function is impaired. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. In such an aspect, the impaired cognitive function may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In a tenth aspect, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where memory formation is impaired. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. In one embodiment of such an aspect, the impaired memory formation may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In an eleventh aspect, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where memory recall is impaired. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. In one embodiment of such an aspect, the impaired memory recall may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In a twelfth aspect, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a thirteenth aspect, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z, wherein the suppression of cognitive function is a result, at least in part, of suppressed memory formation, suppressed memory recall or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a fourteenth aspect, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z, wherein the suppression of cognitive function is a result, at least in part, of suppressed cellular consolidation in the hippocampus, suppressed systems consolidation in the cortex or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a fifteenth aspect, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. In another embodiment of this aspect, the H2A.Z may play a role in the suppression of cognitive function.

In a sixteenth aspect, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved, wherein H2A.Z plays a role in the suppression of memory formation, memory recall or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a seventeenth aspect, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved, wherein H2A.Z plays a role in impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing. In one embodiment of this aspect, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z.

In an eighteenth aspect, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z.

In a nineteenth aspect, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is a nucleic acid compound. In certain embodiments, the nucleic acid compound is RNA based or DNA based; the nucleic acid compound may also contain both RNA and DNA elements. In the foregoing, the RNA or DNA based elements may be naturally occurring elements or may be modified.

In a twentieth aspect, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is a small interfering RNA (siRNA).

In a twenty-first aspect, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is an antisense oligonucleotide (ASO).

In a twenty-second aspect, the foregoing compounds or medicaments is used in the methods or uses described above.

DETAILED DESCRIPTION

Definitions

The terms "treatment", "treat" and "treating" as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics or administered prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to delay the delay or reduce the onset of such symptom, aspect or characteristic. Such treating need not be absolute to be useful. In one embodiment, the symptom, aspect or characteristic is cognitive function, memory formation or memory recall.

The term "in need of treatment" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

The term "individual", "subject" or "patient" as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

The term "therapeutically effective amount" as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial. Memory formation is a multi-faceted consolidation process in which molecular events transform transient experiences into long-lasting memories, in part by increasing the expression of memory-associated genes in response to experience. Genes involved in memory formation are packaged into nucleosomes, which are composed of an octamer of histone proteins that restrict access to DNA. The histone octamer consists of two each of histones H2A, H2B, H3, and H4, which can be replaced by distinct histone variants to alter gene expression. The pre s ent dis cl o sure inve sti gate s the ro l e o f hi s tone v ari ants , p articul arl y hi stone H2A . Z , in cognitive function.

The present disclosure demonstrates that histone H2A.Z acts as a suppressor of cognitive function, including memory formation and/or recall, such that a reduction of its levels, expression or activity serves to improve cognitive function, including memory formation and/or recall . The present disclosure shows that reduction of H2A.Z polypeptide levels in two memory-associated brain regions improves cognitive function, at least in part, by improving memory formation, memory recall and increasing the expression of memory- associated genes. Specifically, mice with reduced levels of H2A.Z exhibit increased expression of memory-associated genes compared to mice with normal levels of H2A.Z. This effect is specific to learning, since differences in gene expression were not found in untrained mice with reduced levels of H2A.Z compared to controls.

As a result, H2A.Z is identified as a novel target for use in improving cognitive function, including memory formation and memory recall and for use in the treatment of diseases and conditions involving cognitive function, particularly deficits in cognitive function.

Prior to the present disclosure, the role of H2A.Z in cognitive function was unknown. The present disclosure is the first to demonstrate that manipulation of any histone variant is involved in memory and specifically, that reducing H2A.Z expression results in memory enhancement.

Methods of Treatment

As disclosed herein, the present application discloses for the first time that histone variant exchange in involved in cognitive function. Further, the present disclosure shows that histone variant H2A.Z is involved in cognitive function and that reduction in H2A.Z expression and/or activity is associated with increased memory formation and/or memory recall both in the hippocampus and the cortex.

The present disclosure thereby provides for methods, uses of medicaments as well as compounds and compositions for use in improving cognitive function and treating diseases and conditions in which cognitive function, particularly deficits in cognitive function, is involved.

In one embodiment, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject, wherein the improved cognitive function is a result, at least in part, of improved memory formation, improved memory recall or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving cognitive function in a subject, wherein the improved cognitive function is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving memory formation in a subject.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving memory formation in a subject, wherein the improved memory formation is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving memory recall in a subject.

In another embodiment, the present disclosure provides a method or a use of a medicament for improving memory recall in a subject, wherein the improved memory recall is a result, at least in part, of increased or more efficient cellular consolidation in the hippocampus, increased or more efficient systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where cognitive function, particular deficits in cognitive function, is involved.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition where cognitive function is impaired. In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein cognitive function is impaired, wherein the impaired cognitive function may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein cognitive function is impaired, wherein the impaired cognitive function may be the result of impaired memory formation, impaired memory recall or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein memory formation is impaired.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein memory formation is impaired, wherein the impaired memory formation may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein memory recall is impaired.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a subject having a disease or condition wherein memory recall is impaired, wherein the impaired memory recall may be the result of impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z.

In another embodiment, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z, wherein the suppression of cognitive function is a result, at least in part, of suppressed memory formation or suppressed memory recall.

In another embodiment, the present disclosure provides a method or a use of a medicament for reversing the suppression of cognitive function by H2A.Z, wherein the suppression of cognitive function is a result, at least in part, of suppressed cellular consolidation in the hippocampus, suppressed systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved, wherein H2A.Z plays a role in the suppression of memory formation, memory recall or a combination of the foregoing.

In another embodiment, the present disclosure provides a method or a use of a medicament for treating a disease or condition in which H2A.Z is involved, wherein H2A.Z plays a role in impaired cellular consolidation in the hippocampus, impaired systems consolidation in the cortex or a combination of the foregoing.

In another embodiment, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z. In a particular embodiment, the expression of H2A.Z is decreased.

In another embodiment, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is a nucleic acid compound. In certain embodiments, the nucleic acid compound is RNA based or DNA based; the nucleic acid compound may also contain both RNA and DNA elements. In the foregoing, the RNA or DNA based elements may be naturally occurring elements or may be modified. In a particular embodiment, the expression of H2A.Z is decreased.

In another embodiment, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is a small interfering RNA (siRNA). In a particular embodiment, the expression of H2A.Z is decreased. In another particular embodiment, the siRNA has the sequence shown in SEQ ID NOS: 1 or 2.

In another embodiment, the present disclosure provides a compound or medicament that decreases the expression and/or activity of H2A.Z, wherein the compound or medicament is an antisense oligonucleotide (ASO). In a particular embodiment, the expression of H2A.Z is decreased. In another particular embodiment, the siRNA has the sequence shown in SEQ ID NOS: 3 or 4.

In another embodiment, the foregoing compounds or medicaments are used in the methods or uses described above. In the foregoing methods and uses, the method or use comprises, consists of or consists essentially of administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. In certain embodiments, the method or use comprises administering to the subject a compound or medicament that decreases the expression and/or activity of H2A.Z. Such methods or use may further comprise, consist of or consist essentially of indentifying a subject in need of treatment. Such methods or use may further comprise, consist of or consist essentially of administering an additional agent(s) useful in the treatment of a disease or condition which the subject may be suffering. In certain embodiment, the expression of H2A.Z is decreased.

The methods of the present disclosure as described above treatment comprises the step initiating in said subject a treatment regimen that decreases H2A.Z function. In one embodiment, the method comprises inhibiting the expression of a histone variant, including but not limited to, H2A.Z. In another embodiment, the methods comprise inhibiting the binding of a histone variant, including but not limited to H2A.Z, to the promoter region of a gene involved in cognitive function. In another embodiment, the methods comprise inhibiting the binding of a histone variant, including but not limited to H2A.Z, to the coding region of a gene involved in cognitive function. In one embodiment, the present disclosure provides for increasing cognitive function by a combination of the foregoing.

In the foregoing methods and uses, the compound or medicament may be administered alone or as a part of a pharmaceutically acceptable composition. In certain embodiments, the compound or medicament is administered as a part of a pharmaceutically acceptable composition. In certain embodiment, when a pharmaceutically acceptable composition is used, the compound or medicament will be present in an amount of about 0.1 to 95%, 0.5 to 50%, 1 to 25% or 1 to 10% (the foregoing weight to weight based on the total weight of the composition).

In the foregoing methods and uses, the compound or medicament may be administered using a variety of dosing regimens. In one embodiment, the compound or medicament, whether alone or as a part of a pharmaceutically acceptable composition, is administered in a therapeutically effective amount. The therapeutically effective amount and the dosage administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health and weight of the recipient; the severity and stage of the disease state or condition; the kind of concurrent treatment; the frequency of treatment; and the effect desired. The total amount of the compound administered will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one skilled in . the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

In the foregoing methods and uses, the compound or medicament may be administered by a variety of routes. The compound or medicament can be administered enterally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as milk, elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms. The compound or medicament of the present disclosure can also be administered intranasally (nose drops) or by inhalation via the pulmonary system, such as by propellant based metered dose inhalers or dry powders inhalation devices. Other dosage forms include topical administration, such as administration transdermally, via patch mechanism or ointment. Representative routes of enteral administration include, but are not limited to, oral, sublingual and rectal. Representative routes of parenteral administration include, but are not limited to, subcutaneous, intrathecal, intracerebral, intraventricular, intravenous, and intramuscular administration. In another embodiment, the compound or medicament may be delivered by nasal or pulmonary inhalation. In a particular embodiment, the route of administration is intrathecal, intraventricular or intracerebral.

In the foregoing methods and uses, the disease or condition recited may be any disease or condition in which cognitive function is impaired or in which it is desired to increase cognitive function. Further, the disease or condition may be any disease or condition in which memory recall or memory formation is impaired or in which it is desired to increase memory recall or memory formation. In certain aspects, the disease or condition, H2A.Z is responsible, at least in part, for the impaired cognitive function, memory formation and/or memory recall. In certain diseases or conditions, H2A.Z, may be overexpressed, however, overexpression is not required. In one embodiment of the foregoing methods or uses, the disease or condition is Alzheimer's disease. In another embodiment of the foregoing methods or uses, the disease or condition is mental retardation. In another embodiment of the foregoing methods or uses, the disease or condition is autism or a condition in the autism spectrum (as defined in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5)). In another embodiment of the foregoing methods or uses the disease or condition is aging or senescence.

The present disclosure describes methods to treat and prevent disorders that are related, at least in part, to decreased or impaired cognitive function. In one embodiment, the effects on cognitive function are expressed through effects on memory formation, memory recall or a combination of the foregoing. In one embodiment, the effects on cognitive function, including but not limited to, memory formation and memory recall, occur in the hippocampus, including but not limited to the CAl region, the cortex or a combination of the foregoing.

In one embodiment, the present disclosure provides methods for increasing cognitive function by inhibiting the expression of a histone variant, including but not limited to, H2A.Z. As a result, the association of H2A.Z with various genes involved in cognitive function is modulated. In a particular embodiment, cognitive function is improved through improvements in memory formation, memory recall, alteration in the expression of genes related to cognitive function or a combination of the foregoing. In another embodiment, the present disclosure provides for increasing cognitive function by inhibiting the binding of a histone variant, including but not limited to H2A.Z, to the promoter region of a gene involved in cognitive function. In another embodiment, the present disclosure provides for increasing cognitive function by increasing the binding of a histone variant, including but not limited to H2A.Z, to the promoter region of a gene involved in cognitive function. In still another embodiment, the present disclosure provides for increasing cognitive function by inhibiting the binding of a histone variant, including but not limited to H2A.Z, to the coding region of a gene involved in cognitive function. In still another embodiment, the present disclosure provides for increasing cognitive function by increasing the binding of a histone variant, including but not limited to H2A.Z, to the coding region of a gene involved in cognitive function. In one embodiment, the present disclosure provides for increasing cognitive function by a combination of the foregoing.

In the foregoing methods and uses, the gene involved in cognitive function is an activator of cognitive function. In certain aspects, the gene is the BdnflV gene, the Egrl gene, the Egr2 gene, the Npas4 gene, the Fos gene and/or the Arc gene. In certain aspects, the gene is the Egrl gene, the Arc gene or the Bdnf IV gene. When the gene is an activator of cognitive function, in certain embodiments the expression of the gene is increased, in one embodiment by the modulation of H2A.Z association with the gene.

In the foregoing methods and uses, the gene involved cognitive function is a suppressor of cognitive function. In certain aspects the gene is the Ppp3ca gene or the Ppplcc gene. When the gene is a suppressor of cognitive function, in certain embodiments the expression of the gene is decreased, in one embodiment by the modulation of H2A.Z association with the gene.

Antagonist of H2A.Z

In one embodiment, an antagonist of H2A.Z function is used. The antagonist for use in the methods of treatment and prevention may be a small molecule, a pharmaceutical, a specific antagonist of H2A.Z expression, a specific antagonist of H2A.Z binding to a promoter or coding region of a gene involved in cognitive function, an antagonist of a signaling pathways that results in increased H2A.Z expression, a nucleic acid construct, an oligopeptide, or an antibody or portions thereof. Such antagonist may be provided as a pharmaceutical composition in a pharmaceutically acceptable carrier as described herein. In a specific embodiment, the antagonist is administered in a therapeutically effective amount. Such administration would thereby increase cognitive function and treat and/or prevent those diseases and/or conditions associated with cognitive function, particularly loss of cognitive function. As discussed above, the treatment/prevention need not be absolute to provide benefit in the methods disclosed.

In one embodiment, the antagonist of H2A.Z function decreases the expression of H2A.Z. In such an embodiment the antagonist may be a nucleic acid construct as discussed herein. The nucleic acid compound may be a RNA or DNA based nucleic acid compound; the nucleic acid compound may also contain both RNA and DNA elements. In particular embodiment, the nucleic acid construct is a siRNA, a non-coding RNA, an antisense nucleotide or combinations of the foregoing.

In one embodiment, the antagonist of H2A.Z function is an antibody directed to H2A.Z. In one embodiment, the antibody is a blocking antibody that fully or partially blocks the function or activity of H2A.Z. In one embodiment, the antibody blocks the interaction of H2A.Z with its target, including but not limited to, a nucleic acid sequence on a gene involved in cognitive function, a factor that binds to such a nucleic acid sequence and to which H2A.Z binds and/or a factor that is required for H2A.Z binding to a nucleic acid sequence on a gene involved in cognitive function. In an alternative embodiment, the antibody is directed to a co-factor that is required for H2A.Z function or a binding target of H2A.Z.

As used herein, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including antagonist, e.g. neutralizing antibodies and agonist antibodies as discussed below), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), as well as antibody fragments. Specifically included are "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see for example, U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984, each of the foregoing hereby incorporated by reference for such teachings). The monoclonal antibodies further include "humanized" antibodies or antibody fragments thereof (such as, but not limited to, Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or FR sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062, 1995, which is hereby incorporated by reference herein for such teaching), single-chain antibody molecules and multi-specific antibodies formed from antibody fragments. Suitable antibodies to H2A.Z are available and include antibodies from Active Motif (Cat. No. 39943), Cell signaling technology (Cat. No. 2718), Millipore (Cat. Mo. 07-594), Abbexa (Cat. No. abxl3376), Proteintech Group (Cat. No. 16441-AP), Thermo Fischer Scientific, Inc. (Cat. No.PA5- 21928, -21923 and 17336), Amsbio (Cat. No.TA308894 and 308893),Biorbyt (Cat. No.orbl 18270, 41428, 29203m and 41433), Aviva Systems Biology (Cat. No.ARP61353 P050, 61354 P050, OAGA00665 and OAGO00660), Gene Tex(Cat. No.GTX108298 and 108273), Novus Biologicals (Cat. No.NBPl-95981 and NBP1-70893) and Santa Cruz Biotechnology (Cat. No.sc-67218, sc-54387 and sc-54388).

In an alternate embodiment, the antagonist of H2A.Z function is an oligopeptide that binds to H2A.Z or to a nucleic acid sequence that H2A.Z binds. Oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. Such oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 10, 25, 50, 75 or 100 amino acids in length. Such oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, for example, U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al, J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol, 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668; each of the foregoing hereby incorporated by reference for such teachings).

In yet another alternate embodiment, the antagonist of H2A.Z function is an organic molecule that binds to H2A.Z or to a nucleic acid sequence that H2A.Z binds. In certain case, the organic molecule may block the function of H2A.Z or those agents that stimulate the expression of activity of H2A.Z. In one embodiment, the antagonists blocks the acetylation of H2A.Z. Such an organic molecule may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585, which is hereby incorporated by reference for such teaching). Given the teachings of the prior art, such organic molecules may be identified without undue experimentation using well known techniques.

In still a further embodiment, the antagonist of H2A.Z function is a nucleic acid construct (which may be referred to herein as a polynucleotide). Representative nucleic acid constructs include, but are not limited to, ASOs, siRNA and other forms of non-coding RNA. Antisense oligomers are generally designed to bind in a sequence specific manner to DNA or RNA regions and alter the expression of disease-causing proteins. Requirements for successful implementation of antisense therapeutics include (a) stability in vivo, (b) sufficient membrane permeability and cellular uptake, and (c) a good balance of binding affinity and sequence specificity. Any nucleic acid construct that specifically hybridizes to a target nucleic acid construct may be used in the methods and uses described herein. Specific regions of nucleic acid encoding for the H2A.Z polypeptide are disclosed herein. In one embodiment, the target sequence is a sequence of SEQ ID NOS; 9 and/or 10. A polynucleotide "specifically hybridizes" to a target polynucleotide if it hybridizes to the target under physiological conditions, with a T _m greater than 37 degrees Celsius, greater than 45 degrees Celsius, preferably at least 50 degrees Celsius, and typically 70 degrees Celsius, 80 degrees Celsius, 90 degrees Celsius or higher. The T _m of an oligomer is the temperature at which 50% hybridizes to a complementary target polynucleotide. T _m is determined under standard conditions in physiological saline, as described, for example, in Miyada et al., Methods Enzymol. 154:94-107 (1987). Such hybridization may occur with near or substantial complementary of the antisense oligomer to the target sequence, as well as with exact complementarity. Polynucleotides are described as "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base- pairing rules. In one embodiment, the complementarity between a nucleic acid construct and the target sequence is 100%. In oanother embodiment, the complementarity between a nucleic acid construct and the target sequence at least 85%, 90%, 95% or higher. In one embodiment, the nucleic acid construct may have no more than 5 mismatches with the target sequence. In one embodiment, the nucleic acid construct may have no more than 4, 3 or 2 mismatches with the target sequence. As used herein, the term mismatch means that a nucleotide in the nucleic acid construct cannot bind in a complementary manner to its corresponding nucleotide partner in the target sequence.

In one embodiment, such nucleic acid constructs decreases expression of H2A.Z. In another embodiment, such nucleic acid constructs binds to a nucleic acid sequence that H2A.Z binds, thereby inhibiting H2A.Z binding. In another embodiment, the nucleic acid construct may provide an alternate target for H2A.Z bind that does not result in the expression of H2A.Z. A decrease in expression may include a decrease in transcription of the gene and/or translation of the mRNA or may involve destruction of the mRNA or other coding sequence that directs the expression of H2A.Z. Such nucleic acid constructs may bind to a nucleic acid (DNA or RNA) encoding H2A.Z and trigger the destruction of such nucleic acid. Nucleic acid construct may be of about 10-50 nucleotides in length, 10 to 30 nucleotides in length or 15 to 31 nucleotides in length. In the case of reduced protein expression, the nucleic acid may directly block expression of a given gene, or contribute to the accelerated breakdown of the RNA transcribed from that gene. Morpholino oligomers as described herein are believed to act via the former (steric blocking) mechanism. Preferred antisense targets for steric blocking oligomers include the ATG start codon region, splice sites, regions closely adjacent to splice sites, and 5'-untranslated region of mRNA, although other regions have been successfully targeted using morpholino oligomers. When such a nucleic acid constructs decreases expression or activity of H2A.Z, such decrease is not required to be absolute. Decreases of any amount may be useful in the methods and uses described herein. In one embodiment, the decrease is 5% or greater, 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 75% or greater or more.

Many polynucleotide analogues have been developed in which the phosphodiester linkages of native nucleic acid are replaced by other linkages that are resistant to nuclease degradation (Barawkar, D. A. et al, Proc. Na't'l Acad. Sci. USA 95, 19, 11047-521, 1998; Linkletter, B. A. et al., Nucleic Acids Res. 29, 11, 2370-6 2001; Micklefield, J., Curr. Med. Chem. 8, 10, 1157-79, 2001). Antisense oligonucleotides having other various backbone modifications have also been prepared (Crooke, S. T., Antisense Drug Technology: Principles, Strategies, and Applications, New York, Marcel Dekker 2001; Micklefield, J., Curr. Med. Chem. 8, 10, 1157-79 2001; Crooke, S. T., Antisense Drug Technology, Boca Raton, CRC Press 2008). In addition, oligonucleotides have been modified by peptide conjugation in order to enhance cellular uptake (Moulton, H. M. et al., Bioconjug Chem 15, 2, 290-9 2004; Nelson, M. H. et al., Bioconjug. Chem. 16, 4, 959-66, 2005; Moulton, H. M. et al., Biochim Biophys Acta 2010). Additional nucleic acid constructs are described in US Patent Publication 2014/0330006. Such nucleic acid construct may comprise modified sugar- phosphodiester backbones or other sugar linkages, such as but not limited to phosphorothioate linkages and linkages as described in WO 91/06629 (which is hereby incorporated by reference for such teaching), methylphosponate linkages, N3'->P5' phosphoramidate linkages, morpholino linkages or peptide nucleic acid linkages (as described in Dias, et al., Mol Cancer Ther, 1, 347-355, 2002, which is hereby incorporated by reference for such teachings). In addition, the ribose base may be modified at the 2' position by an O-alkyl group, such as, but not limited to, methyl or methoxyethyl.

In a particular embodiment, the nucleic acid construct is an ASO, siRNA, a miRNA, short hairpin RNA or other RNA species that mediates RNA interference. The use of such techniques to regulate gene expression has been described (see US Publication Nos: 2005/0261219, 2005/0182007 and 2005/0143333, which are hereby incorporated by reference for such teaching).

The sequence of the mRNA encoding H2A.Z from rat, mouse and human is shown in FIG 10A (SEQ ID NOS: 6-8, respectively). In a particular embodiment, the nucleic acid construct is a siRNA having the sequence of SEQ ID NOS: 1 and/or 2 as shown in FIG. 9A. In another particular embodiment, the nucleic acid construct is a ASO having the sequence of SEQ ID NOS: 3 and/or 4 as shown in FIG. 9C. Such nucleic acid constructs, either alone or in combination, are shown to be effective in decreasing expression of H2A.Z. As shown in FIG. 9C, the ASOs shown incorporate phosphrothioate linkages throughout the length of the sequence and further incorporate 2'O-mehtoxyethyl modification. While such modifications may be beneficial, they are not mandatory in every embodiment and the sequence without modifications or with other modifications known in the art may be used.

In one embodiment, the nucleic acid construct is a decoy oligonucleotide. In this approach, synthetic double stranded oligonucleotides that mimic the consensus binding site within the cis-acting elements of its target genes and attenuate the binding of the transcription factor to promoter regions of its target genes to block their expression, have been tested successfully in a clinical trial. In another embodiment, the nucleic acid construct is an aptamer. An aptamer is a short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into specific three-dimensional (3D) structures with dissociation constants usually in the pico- to nano-molar range. In contrast to other nucleic acid molecular probes, aptamers interact with and bind to their targets through structural recognition a process similar to that of an antigen-antibody reaction.

The nucleic acid construct of the present disclosure may target any desired region of the nucleic acid encoding H2A.Z. In one embodiment, the ASO target the degradation of mRNA coding for H2A.Z by binding to specific sequence in the mRNA. In certain embodiment, the specific sequence is 10 to 50 nucleotides in length, 10 to 30 nucleotides in length or 15 to 31 nucleotides in length. In certain embodiments, the nucleic acid constructs binds to a coding region of the mRNA; in certain embodiment, the nucleic acid construct binds to a non-coding region of the mRNA. In certain embodiments, the specific sequence is nucleotides 357 to 377 of SEQ ID NO: 6 or the corresponding positions in SEQ ID NOS: 7 and 8 and the nucleic acid construct is a sequence complementary to such sequence. The consensus sequence for these region for these areas is shown in FIG. 10B.

Compositions

Compositions suitable for enteral administration may be liquid solutions, such as a therapeutically effective amount of the compound or medicament dissolved in diluents, such as milk, water, saline, buffered solutions, infant formula, other suitable carriers, or combinations thereof. The compound or medicament can be mixed to the diluent just prior to administration or may be prepared prior to administration. In an alternate embodiment, formulations suitable for enteral administration may be capsules, sachets, tablets, lozenges, and troches. In each embodiment, the composition may contain a predetermined amount of the compound or medicament of the present disclosure, as solids or granules, powders, suspensions and suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.

Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

Formulations suitable for topical administration include creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art. Compositions suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the patient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound or medicament can be administered in a physiologically acceptable diluent in a pharmaceutically acceptable carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl .beta.-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The compound or medicament of the present disclosure can be formulated into aerosol formulations to be administered via nasal or pulmonary inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen. Such aerosol formulations may be administered by metered dose inhalers. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

The compound or medicament of the present disclosure, alone or in combination with other suitable components, may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Systems for dispensing liquids as a nasal spray are disclosed in U.S. Pat. No. 4,511,069. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,511,069. Additional aerosol delivery forms may include, for example, compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the active agent dissolved or suspended in a pharmaceutical solvent, for example, water, ethanol, or a mixture thereof.

Nasal and pulmonary solutions typically comprise the compound or medicament to be delivered, optionally formulated with a surface-active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments, the solution further comprises a propellant. The pH of the nasal spray solution is optionally between about pH 3.0 and 6.0, preferably 4.5.+-.0.5. Suitable buffers for use within these compositions are as described above or as otherwise known in the art. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol, benzylalkonimum chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.

Within alternate embodiments, nasal and pulmonary formulations are administered as dry powder formulations comprising the active agent in a dry, usually lyophilized, form of an appropriate particle size, or within an appropriate particle size range, for intranasal delivery. Minimum particle size appropriate for deposition within the nasal or pulmonary passages is often about 0.5 μιη. mass median equivalent aerodynamic diameter (MMEAD), commonly about 1 μηι MMEAD, and more typically about 2 μη MMEAD. Maximum particle size appropriate for deposition within the nasal passages is often about 10 μηι MMEAD, commonly about 8 μιη MMEAD, and more typically about 4 μηι MMEAD. Intranasally and pulmonaryly respirable powders within these size ranges can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like. These dry powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhaler (DPI), which relies on the patient's breath, upon pulmonary or nasal inhalation, to disperse the power into an aerosolized amount. Alternatively, the dry powder may be administered via air-assisted devices that use an external power source to disperse the powder into an aerosolized amount, e.g., a piston pump.

The compound or medicament of the present disclosure may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, magnesium carbonate, and the like.

Compositions of the present disclosure can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants.

The compounds and compositions of the present disclosure can be presented in unit- dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Suitable unit doses, i.e., therapeutically effective amounts, may be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will, of course, vary depending on the desired clinical endpoint. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutically acceptable carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, Eds., 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., 622-630 (1986).

When nucleic acid constructs are used, the compositions of the present disclosure may be formulated specifically for use with such constructs. Suitable compositions include lipid delivery vectors, such as, but not limited to, liposomes. Liposomes can be neutral or cationic, depending on the nature of the phospholipids and other components. The nucleic acid construct may be delivered to the target cell by being contained in the interior compartment or through electrostatic interactions with components of the liposome membrane. Such lipid delivery vehicles may, by virtue of their high affinity for cellular membranes. In addition, as liposomes use the endosomal pathway for nucleic acid delivery, he liposomes may contain additional agents (for example, chloroquine and l,2-dioleoyl-sn-glycero-3- phosphatidylethanolamine) to allow escape from the endosomal pathway and transit to the nucleus.

A range of polymers may also be used as delivery vectors. Suitable polymer delivery vectors include cationic polymers, such as but not limited to, poly-L-lysine, PAMAM dendrimers, polyalkylcyanoacrylate nanoparticles, and polyethyleneimine.

The use of vectors in antisense drug delivery in vivo does not require the use of specific delivery vectors as the antisense naked oligonucleotides are in many cases sufficient. A delivery vehicle does not appear to be needed as endosomal/lysosomal sequestration, and lack of nuclear localization do not appear to be a problem. Such nucleic acid constructs may be delivered via enteral or parenteral administration using the formulations described herein. In certain cases permeability enhancers when co-administered with the nucleic acid constructs may increase the efficiency of delivery. When delivery vectors are used, they may include liposomes and polymers as described above as well as nanostructures (Mol. Pharmaceutics, 2013, 10, 9, 3514—3518). Other delivery strategies are described in Pakunlu et al (J. Controlled Release, 114, 2, 2006, 153- 162).

One skilled in the art will appreciate that suitable methods of administering a compound or medicament of the present invention to an patient are available, and, although more than one route can be used to administer a particular compound or medicament, a particular route can provide a more immediate and more effective reaction than another route. EXAMPLES

Example 1- H2A.Z is inhibited during consolidation

As a first step in investigating the role of H2A.Z in cognitive function, immunohistochemistry (IHC) was used to confirm that H2A.Z is expressed throughout the hippocampus, including the area CA1 and the dentate gyrus (FIG. 1 A-C). It has been previously shown that epigenetic modifications in the CA1 are critical for consolidating contextual fear conditioning, a rodent model of associative learning and memory (Lubin, F. D., et al, JNeurosci 28, 10576-10586, 2008; Miller, C. A. et al., Neuron 53, 857-869, 2007). To determine if H2A.Z is also involved in CA1 -mediated consolidation, H2A.Z expression wa s ex ami n e d 30 min or 2h after contextual fear conditioning in mice. In these experiments, mice were trained as described herein and the CA 1 region of the hippocampus was collected 30 min and 2 hours later. IHC showed that expression of H2afz, a gene encoding H2A.Z, was inhibited at 30 min (F _2,6 = 6.30, p = 0.03) after training (Figure ID) and returned to baseline 2 hours after training (data not shown) consistent with previous observations of transient epigenetic changes in the CA1. FIGS. IE and IF show, respectively, that H2A.Z protein expression was inhibited 30 min after training and that promoter methylation at the Η2 gene was increased. The decreased expression of H2A.Z at 30 min was unexpected and is typical of learning-induced down-regulation of memory suppressor genes, including protein phosphatase 1 and calcineurin, generating the hypothesis that H2A.Z is a negative regulator of memory in the CNS.

Over the same time course, the expression of memory-promoting genes was increased, with higher levels of Egrl and Arc observed at 30 min (Egrl: F _2>6 = 19.87, p = 0.002; ^r : F 2,6 = 24.66, p = 0.001 ) and 2h (Egrl; F ₃ , ₁₄ = 8.72, p = 0.003 ; Arc: F _2i i ₄ = 6.03 , p = 0.01) and increased BdnflV expression observed at 2h (¾ΐ4 = 15.16, p < 0.001).

Example 2- Training induces H2A.Z exchange in CA1

H2A.Z. positioning around the transcriptional start site TSS) is strongly associated with transcription. In order to examine the exchange of H2A.Z in nucleic acid, chromatin immunoprecipitation (ChiP) was us ed to determine whether fear conditioning induces an active exchange of H2A.Z at memory-associated genes during consolidation in the hippocampus (FIG. 2A). Based on evidence for distinct functional outcomes of H2A.Z incorporation on gene promoters and coding regions (Coleman- Derr, D. et al., PLoS Genet 8, e l 002988, 2012), H2A.Z binding was investigated on both genic regions of the memory-associated genes Bdnf TV, Arc, and Egrl as a whole. The results are shown in FIG. 2B. In FIG. 2B at each time point the bars in each graph represents (from left to right) naive mice (N, untrained controls), mice subject to context only (C) and mice subject to foot shock and contextual learning (CS).

Training produced gene-, time-, and region-specific changes in binding. With the exception of increased H2A.Z binding at the Arc promoter (F ₂ , ₂ 3 = 5.3, p = 0.01) and decreased binding at the Egrl coding region (T¾.23 ⁼ 4.19, p = 0.03) at 30 min, H2A.Z exchange was not evident until 2h after training (FIG. 2). At this time point, H2A.Z binding decreased at the promoters of all three genes (Bdnf IV: F ₂ ,i6 =5.52, p = 0.02; Egrl: F _¾ io = 21.32, p < 0.001 ; Arc: , _½ = 27.10, p < 0.001) and the coding regions of Egrl (F _2>10 = 6.15, p = 0.02) and Arc (F ₂ ,i6 = 13.37, p < 0.001). In contrast, H2A.Z binding increased at the Bdnf IV coding region at 2h (F _{2> 1} 6 = 9.77, p = 0.002) (FIGS. 2B and C).

In additional experiments, the H2A.Z exchange rate specifically at the -1 nucleosome (the first nucleosome upstream of the TSS) and the +1 nucleosome (first nucleosome downstream of the TSS) of memory associated genes during consolidation in the hippocampus 30 min and 2 hours after training (FIGS. 2D and 2E, showing Npas4, Egr2, Arx and Ppp3ca genes and showing Egrl, Fos, Bdnf IV and Ppplcc genes, respectively). In FIGS. 2D and 2E, N represents naive mice, C represents mice subject to context only, S indicates mice subject to foot shock only with contextual learning and CS represents mice subject to foot shock and contextual learning. For FIG. 2D, at the 30 minute time point n: =7 for the N group; =5 for the C group; =4 for the S group; and =6 for the CS group for Npas4, Egr2 and Arc and n: =4 for the N group; =3 for the C group; =3 for the S group; and =3 for the CS group for Ppp3ca. For FIG. 2D, at the 2 hour time point n: =10 for the N group; =2 for the C group =6 for the S group; and =4 for the CS group for Npas4, Egrl and Arc and n; = 6 for the N group; =2 for the C group; =2 for the S group; and =4 for the CS group for Ppp3ca. For the gene expression studies, n" = 5 for the N group; =6 for the C group; =2 for the S group; and =6 for the CS group for all genes. For FIG. 2E, at the 30 min time point, n: =7 for the N group; =5 for the C group; =4 for the S group; and =6 for the CS group for Egrl and Bdnf IV and n: =4 for the N group; =3 for the C group; =3 for the S group; and =3 for the CS group for Fos and Ppplcc. For FIG. 2E at the 2 hour time point, n: =10 for the N group; =2 for the C group; =4 for the S group; and =6 for the CS group for all genes. For the gene expression studies, n: -5 for the N group; =6 for the C group; =2 for the S group; and =6 for the CS group for all genes.

At 30 min after training, H2A.Z binding was reduced at the +1 nucleosome of memory-promoting genes (Npas4: Welch's F _{3j4 98} = 67.10, PO.001 ; Arc: Welch's F _{3 ; 6} .8 = 153.95, PO.001 ; Egrl : Welch's F _3;7 . ₈₆ = 282.71, PO.001 ; Egr2: F ₃ ,i 8 = 3.50, P=0.04; Fos: F _3>9 = 39.61 , PO.0001), and the expression of corresponding genes was increased during this time (Npas4: F _3>15 = 22.38, PO.001; Arc: F ₃ ,i 5 = 16.34, PO.001 ; Egrl: F _3>15 = 12.55, PO.001 ; Egr2: F _3>15 = 9.72, PO.001; Fos: F _3>6 = 60.71, PO.001). In contrast, H2A.Z incorporation for the memory suppressor PppSca increased at the +1 nucleosome (F _{3 >} 9 = 5.83, PO.02) when gene expression was reduced (F _3jl7 = 4.07, PO.03) (FIGS. 2D and E). This data suggests that H2A.Z at the +1 nucleosome restricts transcription. These findings are consistent with reports of stimulus-induced H2A.Z eviction and evidence for the +1 nucleosome acting as a transcriptional barrier. Given that the data are normalized to histone H3 to correct for potential changes in nucleosome occupancy, the data show that H2A.Z eviction in particular is associated with activity- induced gene expression.

At the -1 nucleosome, H2A.Z binding increased for both memory promoting and memory-suppressing genes (Npas4: F _3>8 = 12.89, PO.002; Eg :Welch's F _{3 ;3} . ₃ 9 = 9.18, PO.04; Egrl: F ₃ , ₈ = 7.47, PO.01 ; Fos: F _3>8 = 8.23, PO.008; PppSca: F _3;8 = 9.20, PO.004) at 30 min, irrespective of changes in gene expression (FIGS. 2D and 2E). Various studies have associated H2A.Z binding in the -1 nucleosome with steady-state gene activity, however the present data suggest that stimulus-induced changes in H2A.Z binding do not correlate with transcription at this time point.

H2A.Z binding returned to baseline levels within 2 h, except for a delayed increase in Bdnf IV expression (F _3>17 = 15.09, P O.001) and a concomitant reduction in H2A.Z binding at the +1 nucleosome (F _3>18 = 3.72, PO.03) (FIG. 2E). Of note, H2A.Z was evicted in context-only mice, even though gene expression increased only with context and shock pairing. This may reflect the 2 h time point, since Bdnf IV expression is typically elevated 1 h after training.

Indeed, the association between gene expression and H2A.Z binding was no longer evident 2 h after training. Whereas H2A.Z binding returned to baseline, gene expression remained elevated (Arc: Welch's F _{3 j} 9. ₂₇ = 12.16, P=0.001 ; Egrl: Welch's

F ₃ ,8. ₂ = 56.68, P=0.01 ; Egr2: Welch's F _3>4 . ₇₇ = 11.13, P=0.01 ; Fos: F _3;17 = 5.54,

P=0.008). For Ppp3ca, H2A.Z binding increased 2 h after training at -1 (F _3jl o ⁼

28.35, P<0.001) and +1 (F _3;10 = 4.10, P=0.04) nucleosomes, even though gene expression returned to baseline. Thus, H2A.Z exchange is uncoupled from gene expression during the late stages of transcription, consistent with evidence that

H2A.Z exchange is primarily involved in transcription initiation.

These data demonstrate that H2A.Z binding at memory-associated genes is

2

dynamically regulated during hippocampus-dependent memory consolidation in a manner consistent with active exchange of H2A. Although the binding pattern varied in a gene and region-specific manner, there was an overall reduction of H2A.Z binding across genes and regions 2h after training. Given that the genes of interest were up-regulated 30 min and 2h after training, the observation of reduced H2A.Z binding at 2h is consistent with reports of H2A.Z eviction from active genes and H2A.Z accumulation in inactive genes.

H2A.Z has a complex role in gene regulation and has been associated with both negative and positive effects on gene expression, with evidence suggesting that H2A.Z has positive effects on transcription when acetylated. No effect of training on H2A.Z acetylation in the promoter regions of memory-associated genes was observed 30 min after training, although decreased levels of AcH2A.Z were found at the Egrl coding region (F¾23 = 5.71, p =0.01). Consistent with reduced H2A.Z binding 2h after training, reduced AcH2A.Z binding was also observed at all promoters (Bdnf IV: F2,8 = 13.45, p = 0.003 ; Egrl: F2,8 = 8.54, p = 0.0;1 Arc: F2 _> 8 = 10.14, p = 0.006) and coding regions (Bdnf IV: F2,8 = 5.22, p = 0.04; Egrl: F2,8 = 7.73, p = 0.02; Arc: F2,8 = 5.50, p = 0.03) at this time point. Of note, increased H2A.Z binding was not associated with increased acetylation, and in the case of Bdnf IV coding region, there was actually a decrease in acetylation when H2A.Z binding was increased. At 30 min after training, when H2A.Z exchange is most pronounced, acetylated H2A.Z (H2A.Zac) binding increased at the - 1 nucleosome of Egrl (F _{3 ; 8} = 1 1 .07, P=0.03) and Fos (F _{3 > 8} = 3 .92, P=0.05) . Consistent with H2A.Z eviction from the + 1 nucleosome at 30 min, a subset of genes also exhibited reduced acetylation at the + 1 nucleosome at this time point (Egr2 : F _{3 ; 8} = 4.03 , P=0.05 ; Egrl : F _{3 j 8} = 14. 1 3 , P=0.001 ) (data not shown). Based on previous evidence that the increased ratio of acetylated to total H2A.Z has a positive effect on transcription, the results are consistent with a repressive effect of H2A.Z incorporation on transcriptional regulation in area CA1 of the hippocampus.

Example 3- Delayed H2A.Z binding in the CA1 at 24h

In contrast to the transient nature of most epigenetic modifications previously reported in area CA1 changes in H2A.Z and AcH2A.Z binding 24h after fear conditioning were also observed (FIG. 3A). Surprisingly, the sites with increased H2A.Z binding did not have altered acetylation and the sites with increased acetylation did not have altered H2A.Z binding (FIG. 3B). Specifically, increased H2A.Z binding at the BdnflVpromoter (F2,9 = 9.49, p = 0.006) and the Arc coding region (F2.9 = 50.98, p < 0.0001) were observed and increased AcH2A.Z binding at Egrl

(F2,s = 5.26, p = 0.04) and Arc (F2,s = 5.02, p = 0.04) promoters, as well as Bdnf!V(F2,8 = 12.06,

p = 0.004) and Egrl (F2,8 = 6.62, p = 0.02) coding regions (FIG. 3 B-D). These results are consistent with delayed re-loading of H2A.Z on promoter and coding regions of genes after the initial eviction observed 2h after training. The implications of increased H2A.Z loading at 24h are suggest that re-loading ofH2A.Z on genomic loci is associated with re-repression of recently activated genes, with some evidence suggesting that this increased loading causes the genes to be poised for later activation (22, 23).

Example 4- H2A.Z reduction enhances memory

Overall, the results above directly demonstrate that fear conditioning training triggers H2A.Z subunit exchange in the hippocampus, implicating a role for this process in cognitive function. To directly investigate H2A.Z's involvement in memory formation, an AAV mediated H2A.Z knockdown construct was used . It was confirmed that this construct was selectively expressed in the pyramidal cell layer in the dorsal Cal (FIG. 4 A). The construct reduced H2A.Z expression as measured by mRNA levels (t ₁₇ = -4.76, p < 0.0001) (FIG. 4B) and produced a 55.8% reduction in H2A.Z protein levels (data not shown). Two weeks after the AAV mediated H2A.Z knockdown, mice were trained with a mild, single-shock contextual fear conditioning protocol (0.5 mA, 2 sec) and freezing behavior was quantified as an index of long- term memory at 24 hours and 30 days after training. Mice with reduced levels of H2A.Z froze significantly more than the mice injected with a scramble control virus at 24 hours (t ₃₀ = 2.28, p = 0.04) and 30 days (t9 = -2.31, p=0.05) after training, indicating that lowering levels of H2A.Z enhances hippocampus-dependent recall of recent memory (FIG. 4C).

Next, it was determined whether AAV-mediated reduction of H2A.Z altered the expression of plasticity-associated genes 30 min after fear conditioning (experimental set up FIG. 4D; results in FIG. 4E). No differences in baseline gene expression were observed in untrained scramble and H2A.Z mice. H2A.Z depletion increased Bdnf IV [Virus (Scramble, H2afz) X Training (Naive, Fear conditioned) interaction (F _ljl7 =8.31, p=0.01)] and Arc [Virus X Training interaction (F _1>17 =6.03, p=0.025)] expression 30 min after training, whereas the expression of other memory promoting genes increased only as a function of fear conditioning, irrespective of H2A.Z manipulation [Main effect of Training (Npas4 F _1>12 =7.12, p=0.02; Egrl F _ljl7 =16.23, p=0.001; Egr2: F _U2 =38.53, pO.OOl; Fos; F _1;12 =93.69, pO.001)]. The memory suppressor genes Ppp3ca and Ppplcc were not altered by training or by H2A.Z manipulation, suggesting that the effects of H2A.Z depletion are gene-specific. These data strongly indicate that H2A.Z is a novel regulator of gene transcription in neurons in the CNS. The absence of baseline differences in gene expression indicates that H2A.Z is not sufficient for transcriptional regulation on its own and it instead promotes increased gene expression in response to environmental stimuli, in a manner consistent with reports of an inverse correlation between H2A.Z binding and gene expression under induced conditions. Overall, the data suggest that H2A.Z is a memory suppressor that reduces the magnitude of gene expression and memory under normal circumstances and that reducing levels of H2A.Z enhances memory through a mechanism involving an increase in training-induced gene expression.

Example 5- H2A.Z exchange occurs in the cortex

Whereas memory consolidation is dependent on transient epigenetic modifications in the

hippocampus (3-5, 7) memory maintenance is supported by persistent changes in cortical DNA methylation. In addition, post-translational modifications of histones may serve as epigenetic tags that support memory transfer from the hippocampus to the cortex shortly after training (2).

Given the finding of persistent H2A.Z subunit exchange in the area CA1, it was hypothesized that H2A.Z in the cortex supports systems consolidation of remote memory.

The presence of H2A.Z in the mPFC was confirmed using IHC (FIG. 5A). Next, ChiP was us ed to measure H2A.Z binding to Egrl , Bdnf IV and Arc gene promoter and coding regions 2h, 24h and 7 days after training to encompass the early period of remote memory consolidation and the subsequent transfer of memory to the cortex. The experimental set-up i s shown in FIG. 5 B and the results in FIGS . 5 C (gene promoter) and 5 D (gene coding regions) . In FIGS . 5 C and 5 D , at each time point the bars in each graph represents (from left to right) naive mice (N, untrained controls), mice subject to context only (C) and mice subject to foot shock and contextual learning (CS).

At 2h, H2A.Z binding increased at Egrl (F _2>6 = 6.18, p = 0.04) and Arc (F ₂ , ₆ = 12.42, p = 0.007) promoters and AcH2A.Z binding decreased at Bdnf IV and Egrl promoters (Bdnf IV: F _¾6 = 6.32, p = 0.03; Egrl: F _2,6 = 10.60, p = 0.01) and coding regions (BdnflV: F _2,6 = 13.12, p = 0.006; Egrl: F _2>6 = 11.03, p = 0.01). At the intermediate 24h time point, H2A.Z binding was increased only at the Arc promoter (F _2j 8 = 10.29, p = 0.006), although increased AcH2A.Z binding was found at both Egrl (F _2> i2 = 5.70, p = 0.02) and Arc (F _2>12 = 4.72, p = 0.03) promoters (FIG. 5C). These data suggest that H2A.Z subunit exchange occurs in the cortex in response to behavioral training and that H2A.Z may be involved in mediating the initial stages of systems consolidation of remote memory.

At 7 days, when the memory becomes increasingly dependent on the cortex, a number of differences in H2A.Z binding emerged that were not seen at the earlier time points. Specifically, H2A.Z binding increased at the Bdnf/V (F _2il9 = 8.76, p = 0.002) promoter without a change in

AcH2A.Z, whereas H2A.Z (F _2j i ₉ = 5.10, p = 0.02) and AcH2A.Z (F _2>10 = 4.34, p = 0.04) binding decreased at the Bdnf IV coding region. Increased H2A.Z F _2> i9 = 6.06, p = 0.009) binding also emerged for the first time on the Egrl coding region without a change at the promoter. A stable change at all sampled time points, including 7 days, was only found at the Arc promoter, wherein H2A.Z (F ₂ ,i9 = 5.50, p = 0.03) and AcH2A.Z (F ₂ , ₁₀ = 4.54, p = 0.04) binding was increased throughout the sampled time period.

In additional experiments, the H2A.Z exchange rate specifically at the -1 nucleosome (the first nucleosome upstream of the TSS) and the +1 nucleosome (first nucleosome downstream of the TSS) of memory associated genes during consolidation in the cortex 2 hours, 7 days and 30 days after training (FIGS. 5E and 5F showing Egrl, Egr2, Arc and Ppp3ca genes and showing Npas4, Fos, Bdnf IV and Ppplcc genes, respectively). In FIGS. 5E and 5F, the dashed line represents naive (N) mice, C represents mice subject to context only, S indicates mice subject to foot shock only with contextual learning and CS represents mice subject to foot shock and contextual learning. For FIG. 5E, at the 2 hour time point n= to: 4 for the N group, 2 for the C group, 4 for the S group and 6 for the CS group; at the 7 day time point n= to: 4 for the N group, 4 for the C group, 4 for the S group and 4 for the CS group; at the 30 day time point n= to: 2 for the N group, 3 for the C group, 3 for the S group and 3 for the CS group. For FIG. 5F, at the 2 hour time, 7 day and 30 day time points n= to: 2 for the N group, 4 for the C group, 3 for the S group and 5for the CS group.

H2A.Z binding at the +1 nucleosome was reduced 2h after training (Arc: F _{3j l2} =4.05, p=0.03; Egrl : F _{3; 12} =3.53, p=0.049; Egr2: F ₃ , ₁₂ =5.36, p^O.01), whereas H2A.Z binding increased at the +1 nucleosome of the memory suppressor Ppp3ca (F _3; i ₂ =4.06, p=0.03). Changes in H2A.Z binding at the -1 nucleosome were found only for Ppp3ca (F ₃₎ i ₂ =13.84, p<0.001), where less H2A.Z was present at 2h (Extended Data Figures 5 and 6). Training- induced H2A.Z eviction at 2h implicates H2A.Z in the early stages of systems consolidation.

At 7 days, when memory becomes increasingly dependent on the cortex, H2A.Z binding increased at the -1 nucleosome of memory-promoting genes (Arc: F _3,2 i=3.03, p=0.05; Egrl : F _{3> 12} =5.46, p=0.01 ; Egr2: F ₃ ,i ₂ =3.66, p=0.04; Bdnf IV: F _3j i ₂ =4.21 , p=0.03) and the -1 nucleosome of the memory suppressor Ppp3ca (F _3j2 i=5.98, p=0.004). These changes were no longer evident at 30 days, indicating that TSS-flanking H2A.Z is associated with systems consolidation, but perhaps not with memory maintenance, consistent with a recent study of cortical histone acetylation.

The effect of H2A.Z acetylation was also examined in FIGS. 5G and 5H at the 2 hour and 7 day time points for the Egrl, Egrl, Arc, Npas4, Fos, Bdnf IV, Ppp3ca and Ppplcc genes. In FIGS. 5G and 5H, the dashed line represents naive (N) mice, C represents mice subject to context only, S indicates mice subject to foot shock only with contextual learning and CS represents mice subject to foot shock and contextual learning. For FIG. 5G, n= to: 2 for the N group, 4 for the C group, 3 for the S group and 5 for the CS group for all genes. For FIG. 5H, n= to: 4 for the N group, 3 for the C group, 4 for the S group and 4 for the CS group for all genes.

In contrast to observations of cortical H2A.Z exchange at 2h, no differences in AcH2A.Z binding at this time were found (FIG. 5G). In contrast, at 7 days, AcH2A.Z binding was reduced at a subset of -1 (Egrl : F ₃ ,n=4.96, p=0.02; Egr2: F _3;11 =3.58, p=0.05; Bdnf IV: F _{3; 1 1} =6.83, p=0.007; Ppp3ca: F _{3> u} =4.05, p=0.03; Ppplcc: F ₃ ,n=3.57, p=0.05) and +1 (Egrl : F _{3) 1} i=3.59, p=0.046; Bdnf IV: F _{3> 1 1} =4.17, p=0.03; Ppplcc: F _{3> 1 1} =3.56, p=0.05) nucleosomes (FIG. 5H). Reduced AcH2A.Z binding suggests that an activity- associated modification is removed during systems consolidation in the cortex.

The data presented show that H2A.Z in the mPFC is transiently altered within hours of training, when the recall of memory is still dependent on the hippocampus. A second wave of H2A.Z exchange occurs at later time points, as the memory becomes increasingly dependent on the cortex. These data show that changes in H2A.Z binding reflect a general role of H2A.Z in cortex-mediated process of systems consolidation and memory maintenance and specifically that H2A.Z may be an epigenetic regulator supporting cortical memory transfer.

Example 6- H2A.Z reduction enhances remote memory

H2A.Z AAV was infused into the mPFC to investigate its role in cortex- dependent remote-memory. Mice were fear conditioned 2 weeks after the AAV infusion and tested 24 h later, when recall of recent memory is still dependent on the hippocampus, or 7 days and 30 days later, when the recall of remote memory is dependent on the cortex. The experimental set-up is shown in FIG. 6 A. FIGS. 6B and C confirmed a reduction in H2afz mRNA, as well as a 68.34% reduction in protein. H2A.Z depletion did not affect fear memory during the hippocampus-dependent 24h time point, whereas significantly higher freezing was observed in H2A.Z depleted mice at the two remote time points (30 days: t ₁₄ =-5.28, pO.0001 ; and 7 days: t ₈ =-3.07, p=0.02) (FIG. 6D). A previous study has reported that the administration of HDAC inhibitors for 4 days after training mice on a social transmission of food preference paradigm enhanced the recall of remote memory 30 days after training, indicating that the initial few days after training are essential for systems consolidation.

Next, it was determined whether AAV-mediated reduction of H2A.Z altered the expression of plasticity-associated genes 30 min after fear conditioning. The experimental set-up is shown in FIG. 6E. Γη separate mice, H2A.Z knockdown enhanced Fos expression irrespective of training 30 min after fear conditioning (Main effect of Virus: Fi _, i ₂ =4.77, p=0.049) and reduced the expression of Ppp3ca (Training X Virus Interaction; p<0.002) in untrained H2A.Z compared to untrained scrambled mice. The expression of remaining genes increased only as a function of fear conditioning (Npas4: Fi,i2=108.65, pO.001; Arc: F ₂ =156.00, pO.001; Egrl: F _U2 =10.73, p=0.007; Egr2 F _1>12 =115.03, pO.001, Fos: F _U2 =63.77, pO.001) (FIG. 6F). Although the virus was present throughout consolidation and maintenance stages, the emergence of memory enhancement at 7 days, and altered Fos and PppSca expression at 30 min, are consistent with an early role in systems consolidation.

The data disclosed show a role for cortical H2A.Z in mediating remote memory. The observations of improved memory with AAV treatment in the hippocampus and the cortex show that H2A.Z has a restrictive effect on both recent and remote memory, consistent with its potential broad role as a memory suppressor.

Given the prolonged exposure to AAV in these mice, additional tests of their activity in the open field were conducted to ensure that the H2A.Z AAV did not produce non-specific effects on freezing behavior. The experimental set-up is given in FIG. 7A. No differences between H2A.Z and scramble mice in open field test, including locomotor activity, movement velocity, vertical activity, or time spent in the center, a commonly used index of anxiety, were noted (FIG. 7B). Overall, these data indicate that inhibiting H2A.Z expression enhances mPFC-dependent recall of remote memory by strengthening the memory during systems consolidation.

Example 7- H2A.Z reduction enhances cognitive function in aged mice

The previous examples have demonstrated the role of H2A.Z in cognitive function. The experiments in Examples 1 -6 were performed using young adult mice. In this example, an experiment was conducted to determine the effect of H2A.Z depletion in aged mice. FIG. 8A shows that associative memory decreases with age, as evident by reduced freezing in aged compared to young mice. In this experiment, young mice (approximately 5 months of age) and old mice (approximately 13 months of age) were subject to fear conditioning and the freezing behavior was determined.

To determine whether the age-related deficit observed in FIG. 8A was regulated by H2A.Z, H2A.Z was depleted from the hippocampus of aged mice (approximately 13 months of age) and memory was assessed in response to fear conditioning as described with the exception that a 0.7 mA rather than a 0.5 mA shock, with the memory test occurring 24h after training (experimental set-up in FIG. 8B). H2A.Z depletion improved memory in response to the training protocol, indicating that H2A.Z can enhance memory in aged mice in response to salient stimuli (FIG. 8C).

Example 8- Reduction of H2afz mRNA by nucleic acid constructs

This example demonstrates the ability of nucleic acid constructs to decrease expression of H2A.Z.

FIG. 9A illustrates the siRNA nucleic acid constructs used. FIG. 9B shows the mRNA encoding H2A.Z was decreased by transfection of siRNA into primary rat cortical neurons using the Lonza nucleofactor following the manufacturer's directions. The required number of cells (4 - 5 x 10 ⁶ cells per sample) were centrifuged at 80xg for 5 minutes at room temperature. The cell pellet is carefully resuspended in 100 μΐ room temperature Nucleofector Solution per sample. 100 μΐ of cell suspension was combined with 300 nM siRNA and the mixture transferred into a certified cuvette. The appropriate Nucleofector program (0-005) is selected) and the selected program applied. 500 μΐ of the pre-equilibrated neuronal culture medium is added to the cuvette and the mixture is gently transferred into a prepared culture dish. 24 hours after nucleofection, H2afz mRNA were determined. FIG. 9B shows the results when both of the siRNA molecules shown in FIG. 9A are used. FIG. 9B shows that the combined siRNA significantly decreased the mRNA encoding H2A.Z as compare to a scrambled control. Furthermore, the effect of the siRNA was specific to H2A.Z as the mRNA coding for H2A.V was not affected. Each siRNA shown in FIG. 9A when used separately also resulted in decreased mRNA encoding H2A.Z.

FIG. 9C illustrates the ASO nucleic acid constructs used. FIG. 9D shows the mRNA encoding H2A.Z was decreased by addition of ASO into primary rat cortical neurons. ASOs were added to primary rat cortical neuronal cultures as described in Carroll et.al., Mol Therapy, Dec; 19(12):2178-85, 2011 (which reference is hereby incorporated by reference for such teaching). Briefly, for neuronal treatments, ASOs were resuspended in sterilized phosphate-buffered saline (PBS) to a concentration of 150uM and the aliquots of the stock solution was added to supplementary media on DIV2 to a final concentration of 1.5uM. 24 hours after ASO addition, H2afz mRNA levels were determined. The ASO used in this experiment is that shown in FIG. 9A. FIG. 9D shows the results when both of the ASO molecules shown in FIG. 9C are used. FIG. 9D shows that the combined ASOs significantly decreased mRNA encoding H2A.Z as compare to a scrambled control. Furthermore, the effect of the ASO was specific to H2A.Z as the mRNA coding for H2A.V was not affected. Each ASO shown in FIG. 9C when used separately also resulted in decreased mRNA encoding H2A.Z.

DISCUSSION

The present disclosure provides the first evidence for H2A.Z involvement in cognitive function and introduces histone variant exchange as a novel mechanism contributing to the molecular basis of cognitive function. Converging data from experiments on gene expression, H2A.Z incorporation, and viral interference ar e p re s ente d that demonstrate H2A.Z's activity as a memory suppressor in the hippocampus and the cortex. Specifically, training-induced reduction of hippocampal H2A.Z expression and H2A.Z binding to memory-associated genes shows that H2A.Z is transiently suppressed during the normal process of memory consolidation. This finding is further supported by observations of enhanced memory recall and increased expression of memory- associated genes in response to direct manipulation of H2A.Z levels through AAV -mediated reduction of H2A.Z expression in both the hippocampus and the cortex.

Of particular note is the finding of delayed alterations in H2A.Z binding at 24h in the CA1 and stable alterations in cortical H2A.Z binding up to 7 days after training. Typically, epigenetic changes in the CA1 are transient and thus far, DNA methylation is the only epigenetic mark found to be stably altered in the cortex during memory maintenance. Based on reports for an antagonistic relationship between DNA methylation and H2A.Z incorporation in various DNA methylation to regulate memory maintenance. Indeed, some evidence suggests that pharmacological inhibition of DNA methylation is partly mediated by alterations in H2A.Z binding. This is particularly relevant for memory, as pharmacological inhibition of DNA methylation in the CA1 and the mPFC impairs memory recall. The extent to which this deficit is mediated by increased H2A.Z incorporation remains to be determined.

Overall, H2A.Z has been identified as a novel target for regulation of cognitive function and the present disclosure introduces histone variant exchange as an additional level of epigenetic regulation that contributes to the complex coordination of gene-specific and temporally distinct gene expression required for memory formation. In addition, H2A.Z antagonists are a novel therapeutic target for memory disorders.

METHODS

Animals

Male C57BL/6J mice (Jackson Laboratories) of approximately 9-12 weeks of age were used for the experiments. Mice were pair housed upon arrival and food and water were available ad libitum. The mice were given at least one week to habituate to the colony before inclusion in experiments before cage mates were randomly assigned to the behavioral treatment group, such that mice in the same cage always belonged to the same test group. All protocols complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Alabama Animal Care Committee. All animals were handled for 4 days prior to fear conditioning.

Fear conditioning

For ChiP and gene expression experiments, mice were placed into the training chamber and given 2 min to explore the novel context. After 2 min, mice in the context + shock group received 3 electric footshocks (0.7 mA, 2 sec) administered 1 min apart, with an additional minute allowed for exploration before removal from the chamber. Mice in the context only group were exposed to the same procedure without footshock delivery. Brains were collected at the indicated times after training and processed for ChiP.

For AAV injected mice, a milder fear conditioning paradigm was used. For mice with AAV injections in the area CA1, the procedure consisted of 3 min of exploration, and a single footshock (0.5 mA, 2 sec), with an additional 30 sec for exploration. This protocol was increased to include 3 shocks for gene expression experiments. To ensure the development of a persistent memory trace over 30 days, mice with AAV injections in the mPFC were trained using a moderate protocol consisting of two footshocks (0.5 mA, 2 sec). For behavioral testing, mice were returned to the testing chamber for 4 min and freezing behavior was measured as an index of fear memory at the indicated time after training. Mice that received intra-cortical AAV injections and were tested 24h after training were re-tested 7 days after training for the assessment of memory at the transition between recent and remote memory. Stereotaxic surgery for viral delivery

Viral knockdown of H2A.Z was achieved using a commercially available and validated H2afz shRNA packaged in AAV2/9 (3.7xl0 ¹³ gc/ml) fused to the U6 promoter and labeled with eGFP (driven by a CMV promoter; Vector Biolabs). H2afz is the primary of two genes coding for H2A.Z and both produce protein products indistinguishable with antibodies in the applicant's laboratory. The following shRN A sequence was employed: CCGG- C G AC AC C TG A A ATCTAGGA-C ACTCG- AGTGTCCTAGATTTCAGGTGTCG-

TTTTTG (SEQ ID NO: 5). A commercially available scramble shRNA expressing eGFP was used a control (Vector Biolabs).

Mice were anesthetized with isoflurane and secured in a Kopf stereotaxic apparatus.

Viral particles were bilaterally delivered into the CA1 (AP -1; ML ±1.5; DV - 1.6; 1.5 ul/hemisphere) or the anterior cingulate cortex (AP +1.9; ML ±0.4; DV -1.8; 1 ul/hemisphere) at a rate of 225 nl/min, with 2 weeks allowed for recovery. These parameters were selected to avoid an altered behavioral profile observed with delivery of larger volumes. Only mice with correctly targeted injections were included in the analysis. Stereotaxic coordinates were based on Paxinos and Franklin.

Immunohisto chemistry ( ^" IHO

Mice were perfused with 10% formalin and embedded in paraffin. Paraffin embedded slides were dried at 55-60°C for one hour in the oven and put through sequential 5 minute washes with xylene, 100%, 95%, 70%, 50% and 30% ethanol, water, and PBS. For antigen retrieval, slides were immersed in 10 mM sodium citrate (pH 6.0) in a plastic Copland jar for 1 1 min in the microwave at 95° (550 watts). After cooling to room temperature in a running cold water bath, sections were put through a sequential rinse in PBS with 1% H202, and again in PBS before a lh blocking step (10% normal goat serum, 0.3% TX-PBS, 1% Bovine Serum Albumin). Sections were incubated in 1 :200 concentration of H2A.Z antibody (Cell Signaling Cat# 2718S) overnight at room temperature in humidified chamber with 5% normal goat serum with 0.3% TX- PBS and 1% Bovine Serum Albumin. The next day, sections were washed with PBS before incubating with secondary anti-rabbit antibody (1 :500). For DAB staining, sections were rinsed with PBS, then incubated in ABC for lh at RT, rinsed with PBS, immersed in DAB and rinsed with PBS. The stained sections were dehydrated with ethanol, then placed in xylene and coverslipped with DPX. For fluorescent staining, sections were incubated in AlexaFluor 594 for 2h at RT, rinsed in PBS and coverslipped with mounting media.

Chromatin immunoprecipitation (ChiP)

The brains were collected at the indicated time after training and kept at - 80 until processing. Samples were cross-linked with 1% formaldehyde, washed 6 times with ice-cold PBS and a cocktail of protease inhibitors (Roche). Samples were then homogenized in 500 μΐ of SDS lysis buffer [(50mMTris, pH 8.1, 10 mM EDTA, 1% SDS, Protease inhibitor tablets (Roche)] and chromatin was sheared by sonication at 40% power (6 times for 10 sec, 50 sec rest between sonications). Lysate was centrifuged at 5500 rcf for 5 min at 4°C, aliquoted, and diluted 1 :10 with ChiP dilution buffer (EMD Millipore). The aliquots were treated with 25 ul of Magna ChiP Protein G magnetic beads (EMD Millipore) and 4 ul of H2A.Z (Cell Signaling Cat# 2718S), AcH2A.Z (Abeam Cat# ab l 8262), or H3 (Cell Signaling Cat# 2650S) antibody and incubated overnight at 4°C. The next day, beads were pelleted with a magnetic separator and washed sequentially with low-salt, high-salt, LiCL immune complex, and TE buffers (EMD Millipore). Immune complexes were extracted using IX TE buffer and proteinase K (EMD Millipore ChiP kit) and heating at 65°C for 2h, followed by 95°C for 10 min. Samples were purified with the Qiagen PCR cleanup kit and quantified using PCR. With the exception of Bdnf IV coding region, all primers were obtained from Graff et al.

mRNA expression and real-time PCR

RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) and cDNA was synthesized using iScript (BioRad). H2afz, Ppp3ca, Ppplcc and Egr2 mRNA was quantified using pre-designed probes available from Applied Biosystems, using ActinB and HPRT as a control. Arc, Egrl, Bdnf IV, Fos, Npas4, as well as HPRT and Rpll3a (control genes) were designed in lab and ordered from IDT.

Methylated DNA immunoprecipitation (MeDIP) The brains were collected 30 min after training and kept at -80 until processing. DNA was extracted using the QIAamp DNA Micro Kit (Qiagen). 2ug of DNA was diluted in 800 ul of buffer EB (Qiagen PCR cleanup kit) and sonicated at 40% power (8 times for 10 sec, 30 sec rest between sonications). Sonicated DNA was incubated at 95 °C for 15 min and used as input control. 300 ul of DNA was diluted in 300 ul of IP dilution buffer (EMD Millipore), incubated with 4ul of 5mC antibody (Epigentek Cat# A1014) and 25ul of Protein A beads (Invitrogen) at 4°C for lh. The beads were pelleted with a magnetic separator and washed with IX bind/wash buffer 2 times for 3 min each. After the final wash, beads were resuspended in lOOul lx TE buffer (lOmM Tris-HCl, ImM EDTA, pH 8.0 with 1% SDS and Proteinase K) and heated at 65°C for 2 hours, then at 95°C for 10 min. The beads were again pelleted with a magnetic separator and the DNA was purified using the Qiagen PCR cleanup kit and quantified using PCR.

Western blots

Histones were extracted from snap frozen tissue using the EpiQuick Total Histone extraction kit (Epigentek) and the 8 g of the extracts were run resolved on a 15%) gel at 75V and transferred to a PVDF membrane at 75V for 2h. The membrane was blocked in Li-Cor Odyssey blocking buffer for lh at room temperature and incubated in H2A.Z primary antibody (1.T000, Cell Signaling) at 4°C overnight. The next day, membranes were washed 3 times in 0.1% TBS-T and incubated for lh in goat anti-rabbit AlexaFluor 800 (1 :15,000, Li-Cor), washed 3 times in 0.1 % TBS-T and imaged on the Li-Cor Odyssey fluorescent imaging system. The membranes were then incubated in actin primary antibody (1 :2000, Abeam) at room temperature and in goat anti-mouse AlexaFluor 800 for lh before washing in 0.1% TBS-T and imaging on the Li-Cor Odyssey.

Statistics

Sample sizes were determined using freely available online power analysis software (http://www.stat.ubc.ca/~rollin/stats/ssize/n2.html), assuming a moderate effect size of 0.5.

Analyses were conducted using one-way and two-way Analysis of Variance (ANOVA). Follow-up analyses were conducted using Fisher's Least Significant Difference and independent samples t-tests, where appropriate. The p value for all cases was set to 0.05 and follow-up analyses for specific comparisons were only conducted when the omnibus ANOVA was significant, given that ANOVA is robust to potential violations of normality. Homogeneity of variance was confirmed using Levene's test for equality of variances. When the assumption was violated, comparisons were conducted using Welch ANOVA and significant results were followed up using Games-Howell post hoc tests, which is appropriate for unequal variances, unequal group sizes and small sample sizes. The use of Welch ANOVA is specified next to the F value for the appropriate comparison in the text. All of the comparisons were conducted using 2-tailed tests of significance.