Functionally Distinct Neuronal Ensembles within the Memory Engram.

Memories are believed to be encoded by sparse ensembles of neurons in the brain. However, it remains unclear whether there is functional heterogeneity within individual memory engrams, i.e., if separate neuronal subpopulations encode distinct aspects of the memory and drive memory expression differently. Here, we show that contextual fear memory engrams in the mouse dentate gyrus contain functionally distinct neuronal ensembles, genetically defined by the Fos- or Npas4-dependent transcriptional pathways. The Fos-dependent ensemble promotes memory generalization and receives enhanced excitatory synaptic inputs from the medial entorhinal cortex, which we find itself also mediates generalization. The Npas4-dependent ensemble promotes memory discrimination and receives enhanced inhibitory drive from local cholecystokinin-expressing interneurons, the activity of which is required for discrimination. Our study provides causal evidence for functional heterogeneity within the memory engram and reveals synaptic and circuit mechanisms used by each ensemble to regulate the memory discrimination-generalization balance.

Genomic Decoding of Neuronal Depolarization by Stimulus-Specific NPAS4 Heterodimers.

Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both a neuron's action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if distinct types of electrical activity can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in mouse hippocampal neurons trigger two spatially segregated and molecularly distinct induction mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate in the formation of stimulus-specific NPAS4 heterodimers that exhibit distinct DNA binding patterns. Thus, NPAS4 differentially communicates increases in a neuron's spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity along the somato-dendritic axis of a neuron.

Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.

Endocardial identity is established during early somitogenesis by Bmp signalling acting upstream of npas4l and etv2.

The endocardium plays important roles in the development and function of the vertebrate heart; however, few molecular markers of this tissue have been identified and little is known about what regulates its differentiation. Here, we describe the Gt(SAGFF27C); Tg(4xUAS:egfp) line as a marker of endocardial development in zebrafish. Transcriptomic comparison between endocardium and pan-endothelium confirms molecular distinction between these populations and time-course analysis suggests differentiation as early as eight somites. To investigate what regulates endocardial identity, we employed npas4l, etv2 and scl loss-of-function models. Endocardial expression is lost in npas4l mutants, significantly reduced in etv2 mutants and only modestly affected upon scl loss-of-function. Bmp signalling was also examined: overactivation of Bmp signalling increased endocardial expression, whereas Bmp inhibition decreased expression. Finally, epistasis experiments showed that overactivation of Bmp signalling was incapable of restoring endocardial expression in etv2 mutants. By contrast, overexpression of either npas4l or etv2 was sufficient to rescue endocardial expression upon Bmp inhibition. Together, these results describe the differentiation of the endocardium, distinct from vasculature, and place npas4l and etv2 downstream of Bmp signalling in regulating its differentiation.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family.

Neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4) is a protective transcriptional regulator whose dysfunction has been linked to a variety of neuropsychiatric and metabolic diseases. As a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor family, NPAS4 is distinguished by an ability to form functional heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, both of which are also bHLH-PAS family members. Here, we describe the quaternary architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers in complexes involving DNA response elements. Our crystallographic studies reveal a uniquely interconnected domain conformation for the NPAS4 protein itself, as well as its differentially configured heterodimeric arrangements with both ARNT and ARNT2. Notably, the PAS-A domains of ARNT and ARNT2 exhibit variable conformations within these two heterodimers. The ARNT PAS-A domain also forms a set of interfaces with the PAS-A and PAS-B domains of NPAS4, different from those previously noted in ARNT heterodimers formed with other class I bHLH-PAS family proteins. Our structural observations together with biochemical and cell-based interrogations of these NPAS4 heterodimers provide molecular glimpses of the NPAS4 protein architecture and extend the known repertoire of heterodimerization patterns within the bHLH-PAS family. The PAS-B domains of NPAS4, ARNT, and ARNT2 all contain ligand-accessible pockets with appropriate volumes required for small-molecule binding. Given NPAS4's linkage to human diseases, the direct visualization of these PAS domains and the further understanding of their relative positioning and interconnections within the NPAS4-ARNT and NPAS4-ARNT2 heterodimers may provide a road map for therapeutic discovery targeting these complexes.

Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

BACKGROUND: Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. METHODS: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. RESULTS: Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. CONCLUSIONS: These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke.

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion.

We show here that the transcription factor Npas4 is an important regulator of medium spiny neuron spine density and electrophysiological parameters and that it determines the magnitude of cocaine-induced hyperlocomotion in mice. Npas4 is induced by synaptic stimuli that cause calcium influx, but not dopaminergic or PKA-stimulating input, in mouse medium spiny neurons and human iPSC-derived forebrain organoids. This induction is independent of ubiquitous kinase pathways such as PKA and MAPK cascades, and instead depends on calcineurin and nuclear calcium signalling. Npas4 controls a large regulon containing transcripts for synaptic molecules, such as NMDA receptors and VDCC subunits, and determines in vivo MSN spine density, firing rate, I/O gain function and paired-pulse facilitation. These functions at the molecular and cellular levels control the locomotor response to drugs of abuse, as Npas4 knockdown in the nucleus accumbens decreases hyperlocomotion in response to cocaine in male mice while leaving basal locomotor behaviour unchanged.

NPAS4 Exacerbates Pyroptosis via Transcriptionally Regulating NLRP6 in the Acute Phase of Intracerebral Hemorrhage in Mice.

Intracerebral hemorrhage (ICH) is a severe cerebrovascular disease with a high disability rate and high mortality, and pyroptosis is a type of programmed cell death in the acute phase of ICH. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is a specific transcription factor highly expressed in the nervous system, yet the role of NPAS4 in ICH-induced pyroptosis is not fully understood. NLR family Pyrin-domain-containing 6 (NLRP6), a new member of the Nod-like receptor family, aggravates pyroptosis via activating cysteine protease-1 (Caspase-1) and Caspase-11. In this study, we found that NPAS4 was upregulated in human and mouse peri-hematoma brain tissues and peaked at approximately 24 h after ICH modeling. Additionally, NPAS4 knockdown improved neurologic dysfunction and brain damage induced by ICH in mice after 24 h. Meanwhile, inhibiting NPAS4 expression reduced the levels of myeloperoxidase (MPO)-positive cells and Caspase-1/TUNEL-double-positive cells and decreased cleaved Caspase-1, cleaved Caspase-11, and N-terminal GSDMD levels. Consistently, NPAS4 overexpression reversed the above alternations after ICH in the mice. Moreover, NPAS4 could interact with the Nlrp6 promoter region (-400--391 bp and -33--24 bp) and activate the transcription of Nlrp6. Altogether, our study demonstrated that NPAS4, as a transcription factor, can exacerbate pyroptosis and transcriptionally activate NLRP6 in the acute phase of intracerebral hemorrhage in mice.

Theanine, a Tea-Leaf-Specific Amino Acid, Alleviates Stress through Modulation of Npas4 Expression in Group-Housed Older Mice.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine's stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish.

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

NPAS4 suppresses propofol-induced neurotoxicity by inhibiting autophagy in hippocampal neuronal cells.

Propofol, a general intravenous anesthetic, has been demonstrated to cause a profound neuroapoptosis in the developing brain followed by long-term neurocognitive impairment. Our study aimed to examine the neuroprotective effect of neuronal PAS domain protein 4 (NPAS4), an activity-dependent neuron-specific transcription factor, on propofol-induced neurotoxicity in hippocampal neuronal HT22 cells. The differentially expressed genes in HT22 cells after treatment with propofol were screened from Gene Expression Omnibus dataset GSE106799. NPAS4 expression in HT22 cells treated with different doses of propofol was investigated by qRT-PCR and Western blot analysis. Cell viability, lactate dehydrogenase (LDH) release, caspase-3 activity, and apoptosis were evaluated by MTT, a LDH-Cytotoxicity Assay Kit, a Caspase-3 Colorimetric Assay Kit, and TUNEL assay, respectively. The protein levels of LC3-I, LC3-II, Beclin 1, p62 and NPAS4 were detected using Western blot analysis. Propofol treatment concentration-dependently decreased NPAS4 expression in HT22 cells. Propofol treatment inhibited cell viability, increased LDH release and caspase-3 activity, and induced apoptosis and autophagy in HT22 cells. NPAS4 overexpression suppressed propofol-induced cell injury and autophagy in HT22 cells. Mechanistically, autophagy agonist rapamycin attenuated the neuroprotective effect of NPAS4 in propofol-treated HT22 cells. In conclusion, NAPS4 overexpression protected hippocampal neuronal HT22 cells against propofol-induced neurotoxicity by reducing autophagy.

Circular RNA circ_0003420 mediates inflammation in sepsis-induced liver damage by downregulating neuronal PAS domain protein 4.

OBJECTIVE: Our aim was to investigate whether circular RNA (circRNA) circ_0003420 mediates inflammation in sepsis-induced liver damage and to determine the mechanism involved. MATERIALS AND METHODS: Liver tissue samples from patients with sepsis and healthy subjects were used to identify differentially expressed circRNAs. Additionally, Kupffer cells were treated with lipopolysaccharide (LPS) to establish an in vitro model of sepsis-induced liver damage. Cell viability and proliferation were measured with a cell counting kit-8 and 5-ethynyl-2'-deoxyuridine (EdU) labeling, respectively. Relative mRNA and protein levels of IL-6, IL-1ß, tumor necrosis factor (TNF)-α, and neuronal PAS domain protein 4 (NPAS4) were determined via reverse-transcription quantitative PCR and western blotting, respectively. RESULTS: We observed circ_0003420 upregulation accompanied by NPAS4 downregulation in liver samples from patients with sepsis-associated damage and in Kupffer cells treated with LPS. Results of in vitro experiments indicated that LPS treatment reduced cell viability and induced well-pronounced apoptosis and inflammatory signs. Circ_0003420 silencing counteracted LPS's influence on cell proliferation, apoptosis, and inflammation signs. Bioinformatics and a dual-luciferase reporter assay revealed that circ_0003420 targets NPAS4 mRNA and negatively correlates with NPAS4 expression. Moreover, NPAS4 knockdown recovered the apoptosis rate and expression levels of inflammatory cytokines in the LPS-treated circ_0003420 knockdown cells, whereas NPAS4 overexpression had similar effects on Kupffer cell properties as circ_0003420 silencing. CONCLUSION: We demonstrate that circ_0003420 targets NPAS4 mRNA thereby mediating the cell damage and inflammation caused by LPS. This study provides a possible target for treatment of liver damage induced by sepsis.

Single Exposure to the Cathinones MDPV and α-PVP Alters Molecular Markers of Neuroplasticity in the Adult Mouse Brain.

Synthetic cathinones have gained popularity among young drug users and are widely used in the clandestine market. While the cathinone-induced behavioral profile has been extensively investigated, information on their neuroplastic effects is still rather fragmentary. Accordingly, we have exposed male mice to a single injection of MDPV and α-PVP and sacrificed the animals at different time points (i.e., 30 min, 2 h, and 24 h) to have a rapid readout of the effect of these psychostimulants on neuroplasticity in the frontal lobe and hippocampus, two reward-related brain regions. We found that a single, low dose of MDPV or α-PVP is sufficient to alter the expression of neuroplastic markers in the adult mouse brain. In particular, we found increased expression of the transcription factor Npas4, increased ratio between the vesicular GABA transporter and the vesicular glutamate transporter together with changes in the expression of the neurotrophin Bdnf, confirming the widespread impact of these cathinones on brain plasticity. To sum up, exposure to low dose of cathinones can impair cortical and hippocampal homeostasis, suggesting that abuse of these cathinones at much higher doses, as it occurs in humans, could have an even more profound impact on neuroplasticity.

The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development.

The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.

Central and peripheral immune responses to low-dose lipopolysaccharide in a mouse model of the 15q13.3 microdeletion.

Carriers of the human 15q13.3 microdeletion (MD) present with a variable spectrum of neuropathological phenotypes that range from asymptomatic to severe clinical outcomes, suggesting an interplay of genetic and non-genetic factors. The most common 2 MB 15q13.3 MD encompasses six genes (MTMR10, FAN1, TRPM1, KLF13, OTUD7A, and CHRNA7), which are expressed in neuronal and non-neuronal tissues. The nicotinic acetylcholine receptor (nAChR) α7, encoded by CHRNA7, is a key player in the cholinergic anti-inflammatory pathway, and the transcription factor KLF13 is also involved in immune responses. Using a mouse model with a heterozygous deletion of the orthologous region of the human 15q13.3 (Df[h15q13]/+), the present study examined peripheral and central innate immune responses to an acute intraperitoneal (i.p.) injection of the bacteriomimetic, lipopolysaccharide (LPS) (100 µg/kg) in adult heterozygous (Het) and wildtype (WT) mice. Serum levels of inflammatory markers were measured 2 h post injection using a Multiplex assay. In control saline injected animals, all measured cytokines were at or below detection limits, whereas LPS significantly increased serum levels of interleukin 1beta (IL-1ß), tumor necrosis factor alpha (TNF-α), IL-6 and IL-10, but not interferon-γ. There was no effect of genotype but a sexual dimorphic response for TNF-α, with females exhibiting greater LPS-induced TNF-α serum levels than males. In situ hybridization revealed similar increases in LPS-induced c-fos mRNA expression in the dorsal vagal complex in all groups. The hippocampal expression of the pro-inflammatory cytokines was evaluated by real-time quantitative PCR. LPS-treatment resulted in significantly increased mRNA expression for IL-1ß, IL-6, and TNF-α compared to saline controls, with no effect of genotype, but a significant sex-effect was detected for IL-1ß. The present study provided no evidence for interactive effects between the heterozygous 15q13.3 MD and a low-dose LPS immune challenge in innate peripheral or central immune responses, although, sex-differential effects in males and females were detected.

CD8+ T-cells infiltrate Alzheimer's disease brains and regulate neuronal- and synapse-related gene expression in APP-PS1 transgenic mice.

Neuroinflammation is a major contributor to disease progression in Alzheimer's disease (AD) and is characterized by the activity of brain resident glial cells, in particular microglia cells. However, there is increasing evidence that peripheral immune cells infiltrate the brain at certain stages of AD progression and shape disease pathology. We recently identified CD8+ T-cells in the brain parenchyma of APP-PS1 transgenic mice being tightly associated with microglia as well as with neuronal structures. The functional role of CD8+ T-cells in the AD brain is however completely unexplored. Here, we demonstrate increased numbers of intra-parenchymal CD8+ T-cells in human AD post-mortem hippocampus, which was replicated in APP-PS1 mice. Also, aged WT mice show a remarkable infiltration of CD8+ T-cells, which was more pronounced and had an earlier onset in APP-PS1 mice. To address their functional relevance in AD, we successfully ablated the pool of CD8+ T-cells in the blood, spleen and brain from 12 months-old APP-PS1 and WT mice for a total of 4 weeks using an anti-CD8 antibody treatment. While the treatment at this time of disease stage did neither affect the cognitive outcome nor plaque pathology, RNAseq analysis of the hippocampal transcriptome from APP-PS1 mice lacking CD8+ T-cells revealed highly altered neuronal- and synapse-related gene expression including an up-regulation for neuronal immediate early genes (IEGs) such as the Activity Regulated Cytoskeleton Associated Protein (Arc) and the Neuronal PAS Domain Protein 4 (Npas4). Gene ontology enrichment analysis illustrated that the biological processes "regulation of neuronal synaptic plasticity" and the cellular components "postsynapses" were over-represented upon CD8+ T-cell ablation. Additionally, Kegg pathway analysis showed up-regulated pathways for "calcium signaling", "long-term potentiation", "glutamatergic synapse" and "axon guidance". Therefore, we conclude that CD8+ T-cells infiltrate the aged and AD brain and that brain CD8+ T-cells might directly contribute to neuronal dysfunction in modulating synaptic plasticity. Further analysis will be essential to uncover the exact mechanism of how CD8+ T-cells modulate the neuronal landscape and thereby contribute to AD pathology.

Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain-containing protein 4 (NPAS4).

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic ß-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.

The Bdnf and Npas4 genes are targets of HDAC3-mediated transcriptional repression.

BACKGROUND: Histone deacetylase-3 (HDAC3) promotes neurodegeneration in various cell culture and in vivo models of neurodegeneration but the mechanism by which HDAC3 exerts neurotoxicity is not known. HDAC3 is known to be a transcriptional co-repressor. The goal of this study was to identify transcriptional targets of HDAC3 in an attempt to understand how it promotes neurodegeneration. RESULTS: We used chromatin immunoprecipitation analysis coupled with deep sequencing (ChIP-Seq) to identify potential targets of HDAC3 in cerebellar granule neurons. One of the genes identified was the activity-dependent and neuroprotective transcription factor, Neuronal PAS Domain Protein 4 (Npas4). We confirmed using ChIP that in healthy neurons HDAC3 associates weakly with the Npas4 promoter, however, this association is robustly increased in neurons primed to die. We find that HDAC3 also associates differentially with the brain-derived neurotrophic factor (Bdnf) gene promoter, with higher association in dying neurons. In contrast, association of HDAC3 with the promoters of other neuroprotective genes, including those encoding c-Fos, FoxP1 and Stat3, was barely detectable in both healthy and dying neurons. Overexpression of HDAC3 leads to a suppression of Npas4 and Bdnf expression in cortical neurons and treatment with RGFP966, a chemical inhibitor of HDAC3, resulted in upregulation of their expression. Expression of HDAC3 also repressed Npas4 and Bdnf promoter activity. CONCLUSION: Our results suggest that Bdnf and Npas4 are transcriptional targets of Hdac3-mediated repression. HDAC3 inhibitors have been shown to protect against behavioral deficits and neuronal loss in mouse models of neurodegeneration and it is possible that these inhibitors work by upregulating neuroprotective genes like Bdnf and Npas4.

Neuronal PAS domain protein 4 (Npas4) controls neuronal homeostasis in pentylenetetrazole-induced epilepsy through the induction of Homer1a.

Neuronal intrinsic homeostatic scaling-down of excitatory synapse has been implicated in epilepsy pathogenesis to prevent the neuronal circuits from hyperexcitability. Recent findings suggest a role for neuronal PAS domain protein 4 (Npas4), an activity-dependent neuron-specific transcription factor in epileptogenesis, however, the underlying mechanism by which Npas4 regulates epilepsy remains unclear. We herein propose that limbic seizure activity up-regulates Npas4-homer1a signaling in the hippocampus, thereby contributing to epileptogenesis in mice. The expression level of Npas4mRNA was significantly increased after the pentylenetetrazol (PTZ) treatment. Npas4KO mice developed kindling more rapidly than their wild-type littermates. The expression of Homer1a in the hippocampus increased after seizure activity. Npas4 increased Homer1a promoter activity in COS7 cells. The PTZ-stimulated induction of Homer1a was attenuated in the hippocampus of Npas4KO mice. The combination of fluorescence in situ hybridization and immunohistochemical analyses revealed that Homer1amRNA co-localized with the Npas4 protein after the convulsive seizure response. PTZ reduced excitatory synaptic transmission at the associational/commissural fibers-CA3 synapses through the Npas4-mediated down-regulation of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in hippocampal CA3 neurons. The adeno-associated virus (AAV)-mediated expression of Homer1a resulted in lower α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor GluA1 subunit levels in the hippocampal plasma membrane fraction than in that from AAV-EGFP-transfected Npas4KO mice. The development of kindling was more strongly suppressed in AAV-Homer1a-microinjected Npas4KO mice than in AAV-EGFP-microinjected Npas4KO mice. These results indicate that Npas4 functions as a molecular switch to initiate homeostatic scaling and the targeting of Npas4-Homer1a signaling may provide new approaches for the treatment of epilepsy.