Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification.

Creating long-lasting memories requires learning-induced changes in gene expression, which are impacted by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histones are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies implicate histone variants as novel regulators of memory, effects of PTMs on the function of histone variants are rarely considered. We previously showed that the histone variant H2A.Z suppresses memory, but it is unclear if this role is impacted by H2A.Z acetylation, a PTM that is typically associated with positive effects on transcription and memory. To answer this question, we used a mutation approach to manipulate acetylation on H2A.Z without impacting acetylation of other histone types. Specifically, we used adeno-associated virus (AAV) constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or H2A.Z.1 with impaired acetylation (acetyl-defective) by replacing the same lysines with alanine (KA). Expressing the H2A.Z.1 acetyl-mimic (H2A.Z.1KQ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1KA preferentially impacts genes involved in synaptic function, suggesting that acetyl-defective H2A.Z.1 impairs memory by altering synaptic regulation. Finally, we describe, for the first time, that H2A.Z is also involved in alternative splicing of neuronal genes, whereby H2A.Z depletion, as well as expression of H2A.Z.1 lysine mutants influence transcription and splicing of different gene targets, suggesting that H2A.Z.1 can impact behavior through effects on both splicing and gene expression. This is the first study to demonstrate that direct manipulation of H2A.Z post-translational modifications regulates memory, whereby acetylation adds another regulatory layer by which histone variants can fine tune higher brain functions through effects on gene expression and splicing.

Lost in traffic: consequences of altered palmitoylation in neurodegeneration.

One of the first molecular events in neurodegenerative diseases, regardless of etiology, is protein mislocalization. Protein mislocalization in neurons is often linked to proteostasis deficiencies leading to the build-up of misfolded proteins and/or organelles that contributes to cellular toxicity and cell death. By understanding how proteins mislocalize in neurons, we can develop novel therapeutics that target the earliest stages of neurodegeneration. A critical mechanism regulating protein localization and proteostasis in neurons is the protein-lipid modification S-acylation, the reversible addition of fatty acids to cysteine residues. S-acylation is more commonly referred to as S-palmitoylation or simply palmitoylation, which is the addition of the 16-carbon fatty acid palmitate to proteins. Like phosphorylation, palmitoylation is highly dynamic and tightly regulated by writers (i.e., palmitoyl acyltransferases) and erasers (i.e., depalmitoylating enzymes). The hydrophobic fatty acid anchors proteins to membranes; thus, the reversibility allows proteins to be re-directed to and from membranes based on local signaling factors. This is particularly important in the nervous system, where axons (output projections) can be meters long. Any disturbance in protein trafficking can have dire consequences. Indeed, many proteins involved in neurodegenerative diseases are palmitoylated, and many more have been identified in palmitoyl-proteomic studies. It follows that palmitoyl acyl transferase enzymes have also been implicated in numerous diseases. In addition, palmitoylation can work in concert with cellular mechanisms, like autophagy, to affect cell health and protein modifications, such as acetylation, nitrosylation, and ubiquitination, to affect protein function and turnover. Limited studies have further revealed a sexually dimorphic pattern of protein palmitoylation. Therefore, palmitoylation can have wide-reaching consequences in neurodegenerative diseases.

Hormone-dependent sexual responses of female mice in response to manual genital stimulation.

Although copulatory behavior is thought to have a strong innate basis in mice, there is also clear evidence that sexual experience shapes its expression. Reinforcement of behavior through rewarding genital tactile stimulation is a primary candidate mechanism for this modification. In rats, manual tactile clitoral stimulation is rewarding only when it is temporally distributed, which is hypothesized to result from an innate preference for species-typical copulatory patterning. Here we test this hypothesis using mice, which have a temporal copulatory pattern which is distinctly less temporally distributed than that of rats. Female mice received manual clitoral stimulation which was either temporally continuous every second, or stimulation which was temporally distributed, occurring every 5 s, This pattern of stimulation was paired with environmental cues in a conditioned place preference apparatus to assess reward. Neural activation in response to this stimulation was evaluated by measuring FOS immunoreactivity. Results indicated that both temporal patterns of clitoral stimulation were rewarding, but that continuous stimulation better reproduced brain activation associated with sexual reward. Furthermore, continuous, but not distributed stimulation elicited a lordosis response in some females, and this response increased within and across days. Sexual reward, neural activation and lordosis resulting from tactile genital stimulation were eliminated by ovariectomy and restored with combined 17ß-estradiol and progesterone treatment but not 17ß-estradiol treatment alone. These observations are consistent with the hypothesis that sexual reward resulting from species-typical genital tactile stimulation has a permissive effect on copulatory behavior of female mice.

Sex-specific effects of the histone variant H2A.Z on fear memory, stress-enhanced fear learning and hypersensitivity to pain.

Emerging evidence suggests that histone variants are novel epigenetic regulators of memory, whereby histone H2A.Z suppresses fear memory. However, it is not clear if altered fear memory can also modify risk for PTSD, and whether these effects differ in males and females. Using conditional-inducible H2A.Z knockout (cKO) mice, we showed that H2A.Z binding is higher in females and that H2A.Z cKO enhanced fear memory only in males. However, H2A.Z cKO improved memory on the non-aversive object-in-place task in both sexes, suggesting that H2A.Z suppresses non-stressful memory irrespective of sex. Given that risk for fear-related disorders, such as PTSD, is biased toward females, we examined whether H2A.Z cKO also has sex-specific effects on fear sensitization in the stress-enhanced fear learning (SEFL) model of PTSD, as well as associated changes in pain sensitivity. We found that H2A.Z cKO reduced stress-induced sensitization of fear learning and pain responses preferentially in female mice, indicating that the effects of H2A.Z depend on sex and the type of task, and are influenced by history of stress. These data suggest that H2A.Z may be a sex-specific epigenetic risk factor for PTSD susceptibility, with implications for developing sex-specific therapeutic interventions.

Histone H2A.Z is required for androgen receptor-mediated effects on fear memory.

Epigenetic factors translate environmental signals into stable outcomes, but how they are influenced by regulators of plasticity remain unclear. We previously showed that androgen receptor overexpression inhibited fear memory in male mice and increased expression of the histone variant H2A.Z, a novel epigenetic regulator of memory. Here, we used conditional-inducible H2A.Z knockout mice to investigate how H2A.Z deletion influences androgenic regulation of fear memory. We showed that conditional inducible H2A.Z deletion blocked memory-enhancing effects of androgen depletion (induced by gonadectomy), and of pharmacological inhibition of the androgen receptor with flutamide. Similarly, H2A.Z deletion blocked the memory-reducing effects of DHT, and DHT treatment in cultured hippocampal neurons altered H2A.Z binding, suggesting that AR is an H2A.Z regulator in neurons. Overall, these data show that fear memory formation is regulated by interactions between sex hormones and epigenetic factors, which has implications for sex differences in fear-related disorders.

Both neural and global androgen receptor overexpression affect sexual dimorphism in the mouse brain.

Testosterone is the main endocrine mechanism mediating sexual differentiation of the mammalian brain, although testosterone signalling is complex and important mechanistic questions remain. Notably, the extent to which testosterone acts via androgen receptors (AR) in this process remains unknown and it is also not clear where testosterone acts in the body to produce sexual dimorphisms in neuroanatomy. To address these questions, we used a transgenic mouse model of Cre/loxP-driven AR overexpression in which AR was induced selectively in neural tissue (Nestin-cre) or in all tissues (CMV-cre). We then studied sexually dimorphic features of several well-characterised sexual dimorphisms: calbindin-immunoreactive neurones in the medial preoptic area (CALB-SDN), tyrosine hydroxylase neurones in the anteroventral periventricular nucleus, and vasopressin-immunoreactive neurones originating in the bed nucleus of the stria terminalis and their projections in the lateral septum. We additionally evaluated oestrogen receptor α immunoreactivity in these nuclei. Briefly, we found that global but not neural overexpression of AR resulted in masculinisation of CALB-SDN nucleus volume, cell number and cell size in transgenic females. Furthermore, neural AR overexpression resulted in increased oestrogen receptor α staining in females compared to males in the medial preoptic area. AR overexpression did not affect other measures. Overall, the results of the present study provide support for the hypothesis that androgenic mechanisms external to the nervous system can affect sexual differentiation of the brain.

Blocking H2A.Z Incorporation via Tip60 Inhibition Promotes Systems Consolidation of Fear Memory in Mice.

Memory formation is a protracted process that initially involves the hippocampus and becomes increasingly dependent on the cortex over time, but the mechanisms of this transfer are unclear. We recently showed that hippocampal depletion of the histone variant H2A.Z enhances both recent and remote memories, but the use of virally mediated depletion reduced H2A.Z levels throughout testing, making its temporally specific function unclear. Given the lack of drugs that target histone variants, we tested existing drugs for efficacy against H2A.Z based on their targeting of known H2A.Z regulators. The Tip60 (part of H2A.Z deposition complex) inhibitor Nu9056 reduced H2A.Z binding, whereas the histone deacetylase (HDAC) inhibitor Trichostatin-A increased H2A.Z acetylation without influencing total H2A.Z in cultured hippocampal neurons. Tip60 (but not HDAC) inhibition 23 h after learning enhanced remote (tested at 7 d) and not recent (tested at 24 h) contextual fear memory in mice. In contrast, Tip60 inhibition 30 d after learning impaired recall of remote memory after 1 h, but protected the memory from further decline 24 h later. These data provide the first evidence of a delayed postlearning role for histone variants in supporting memory transfer during systems consolidation.

Androgen receptor is a negative regulator of contextual fear memory in male mice.

Although sex-hormones have a well-documented role in memory formation, most literature has focused on estrogens, whereas the role of androgens and their receptor (the androgen receptor; AR) in fear memory is relatively unexplored. To address this gap, we used a transgenic mouse model of AR overexpression (CMV-AR) to determine if AR regulates fear memory, and if this effect can be reversed either by the removal of circulating androgens via gonadectomy, or by antagonising AR activity with flutamide. We found that AR overexpression results in reduced freezing in response to foot shock, and that this difference is reversed with both gonadectomy and flutamide treatment. Differences between genotypes were reinstated by testosterone replacement in gonadectomized mice, suggesting that reduced fear memory in mutants results from AR activation by testosterone and is not secondary to group differences in circulating testosterone. Potential transcriptional mechanisms by which CMV-AR exerts its effects on fear memory were assessed by quantitating the expression of memory-related genes in area CA1 of the hippocampus. Several genes that are altered with AR inhibition and activation, including genes that encode for the histone variant H2A.Z, cholinergic receptors, glutamate receptors, and brain-derived neurotrophic factor. Overall, our findings suggest that AR is a negative regulator of fear memory and identify potential gene targets through which AR may mediate this effect.

Nonneural Androgen Receptors Affect Sexual Differentiation of Brain and Behavior.

Testosterone, acting via estrogenic and androgenic pathways, is the major endocrine mechanism promoting sexual differentiation of the mammalian nervous system and behavior, but we have an incomplete knowledge of which cells and tissues mediate these effects. To distinguish between neural and nonneural actions of androgens in sexual differentiation of brain and behavior, we generated a loxP-based transgenic mouse, which overexpresses androgen receptors (ARs) when activated by Cre. We used this transgene to overexpress AR globally in all tissues using a cytomegalovirus (CMV)-Cre driver (CMV-AR), and we used a Nestin-Cre driver to overexpress AR only in neural tissue (Nes-AR). We then examined whether neural or global AR overexpression can affect socio-sexual behaviors using a resident-intruder paradigm. We found that both neural and global AR overexpression resulted in decreased aggressive behaviors and increased thrusting during mounting of intruders, consistent with a neural site of action. Global, but not neural, AR overexpression in males led to an increase in same-sex anogenital investigation. Together, these results suggest novel roles for nonneural AR in sexual differentiation of mice, and indicate that excess AR can lead to a paradoxical reduction of male-typical behavior.

Distinct Etiological Roles for Myocytes and Motor Neurons in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy.

Polyglutamine (polyQ) expansion of the androgen receptor (AR) causes Kennedy's disease/spinobulbar muscular atrophy (KD/SBMA) through poorly defined cellular mechanisms. Although KD/SBMA has been thought of as a motor neuron disease, recent evidence indicates a key role for skeletal muscle. To resolve which early aspects of the disease can be caused by neurogenic or myogenic mechanisms, we made use of the tet-On and Cre-loxP genetic systems to selectively and acutely express polyQ AR in either motor neurons (NeuroAR) or myocytes (MyoAR) of transgenic mice. After 4 weeks of transgene induction in adulthood, deficits in gross motor function were seen in NeuroAR mice, but not MyoAR mice. Conversely, reduced size of fast glycolytic fibers and alterations in expression of candidate genes were observed only in MyoAR mice. Both NeuroAR and MyoAR mice exhibited reduced oxidative capacity in skeletal muscles, as well as a shift in fast fibers from oxidative to glycolytic. Markers of oxidative stress were increased in the muscle of NeuroAR mice and were reduced in motor neurons of both NeuroAR and MyoAR mice. Despite secondary pathology in skeletal muscle and behavioral deficits, no pathological signs were observed in motor neurons of NeuroAR mice, possibly due to relatively low levels of polyQ AR expression. These results indicate that polyQ AR in motor neurons can produce secondary pathology in muscle. Results also support both neurogenic and myogenic contributions of polyQ AR to several acute aspects of pathology and provide further evidence for disordered cellular respiration in KD/SBMA skeletal muscle.