Social regulation of cortisol receptor gene expression.

In many social species, individuals influence the reproductive capacity of conspecifics. In a well-studied African cichlid fish species, Astatotilapia burtoni, males are either dominant (D) and reproductively competent or non-dominant (ND) and reproductively suppressed as evidenced by reduced gonadotropin releasing hormone (GnRH1) release, regressed gonads, lower levels of androgens and elevated levels of cortisol. Here, we asked whether androgen and cortisol levels might regulate this reproductive suppression. Astatotilapia burtoni has four glucocorticoid receptors (GR1a, GR1b, GR2 and MR), encoded by three genes, and two androgen receptors (ARα and ARß), encoded by two genes. We previously showed that ARα and ARß are expressed in GnRH1 neurons in the preoptic area (POA), which regulates reproduction, and that the mRNA levels of these receptors are regulated by social status. Here, we show that GR1, GR2 and MR mRNAs are also expressed in GnRH1 neurons in the POA, revealing potential mechanisms for both androgens and cortisol to influence reproductive capacity. We measured AR, MR and GR mRNA expression levels in a microdissected region of the POA containing GnRH1 neurons, comparing D and ND males. Using quantitative PCR (qPCR), we found D males had higher mRNA levels of ARα, MR, total GR1a and GR2 in the POA compared with ND males. In contrast, ND males had significantly higher levels of GR1b mRNA, a receptor subtype with a reduced transcriptional response to cortisol. Through this novel regulation of receptor type, neurons in the POA of an ND male will be less affected by the higher levels of cortisol typical of low status, suggesting GR receptor type change as a potential adaptive mechanism to mediate high cortisol levels during social suppression.

APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

Apolipoprotein E4 (APOE4) is an important driver of Tau pathology, gliosis, and degeneration in Alzheimer's disease (AD). Still, the mechanisms underlying these APOE4-driven pathological effects remain elusive. Here, we report in a tauopathy mouse model that APOE4 promoted the nucleocytoplasmic translocation and release of high-mobility group box 1 (HMGB1) from hippocampal neurons, which correlated with the severity of hippocampal microgliosis and degeneration. Injection of HMGB1 into the hippocampus of young APOE4-tauopathy mice induced considerable and persistent gliosis. Selective removal of neuronal APOE4 reduced HMGB1 translocation and release. Treatment of APOE4-tauopathy mice with HMGB1 inhibitors effectively blocked the intraneuronal translocation and release of HMGB1 and ameliorated the development of APOE4-driven gliosis, Tau pathology, neurodegeneration, and myelin deficits. Single-nucleus RNA sequencing revealed that treatment with HMGB1 inhibitors diminished disease-associated and enriched disease-protective subpopulations of neurons, microglia, and astrocytes in APOE4-tauopathy mice. Thus, HMGB1 inhibitors represent a promising approach for treating APOE4-related AD.

Microglia Depletion Reduces Human Neuronal APOE4-Driven Pathologies in a Chimeric Alzheimer's Disease Model.

Despite strong evidence supporting the involvement of both apolipoprotein E4 (APOE4) and microglia in Alzheimer's Disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-driven AD pathogenesis remain elusive. Here, we examined such effects utilizing microglial depletion in a chimeric model with human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) induced pluripotent stem cell (iPSC)-derived human neurons into the hippocampus of human APOE3 or APOE4 knock-in mice, and depleted microglia in half the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aß and tau pathologies in an APOE isoform-dependent manner (APOE4 > APOE3). Single-cell RNA-sequencing analysis identified two pro-inflammatory microglial subtypes with high MHC-II gene expression that are enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits.

Apolipoprotein E4 (APOE4) is the strongest known genetic risk factor for late-onset Alzheimer's disease (AD). Conditions of stress or injury induce APOE expression within neurons, but the role of neuronal APOE4 in AD pathogenesis is still unclear. Here we report the characterization of neuronal APOE4 effects on AD-related pathologies in an APOE4-expressing tauopathy mouse model. The selective genetic removal of APOE4 from neurons led to a significant reduction in tau pathology, gliosis, neurodegeneration, neuronal hyperexcitability and myelin deficits. Single-nucleus RNA-sequencing revealed that the removal of neuronal APOE4 greatly diminished neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes and microglia whose accumulation correlated to the severity of tau pathology, neurodegeneration and myelin deficits. Thus, neuronal APOE4 plays a central role in promoting the development of major AD pathologies and its removal can mitigate the progressive cellular and tissue alterations occurring in this model of APOE4-driven tauopathy.

Neuronal apoE4 induces early hyperexcitability in select populations of hippocampal neurons by altering Nell2 expression.

The impact of apolipoprotein E4 (apoE4), the strongest genetic risk factor for Alzheimer's disease (AD), on neuronal function remains unclear. We investigated this by examining excitatory neurons in the hippocampus of young and aged human apoE4 knock-in (apoE4-KI) and apoE3-KI mice using electrophysiology and single-nucleus RNA-sequencing (snRNA-seq). In young apoE4-KI mice, we identified region-specific subpopulations of excitatory neurons with hyperexcitability underlain by reduced cell size, which were eliminated by selective removal of neuronal apoE4. Aged apoE4-KI mice showed an increased fraction of hyperexcitable granule cells, a pronounced inhibitory deficit, and E/I imbalance in the dentate gyrus, contributing to network dysfunction. snRNA-seq analysis revealed neuron type-specific and age-dependent transcriptomic changes, identifying Nell2 overexpression in apoE4-KI mice. Reducing Nell2 expression in specific neuronal types of apoE4-KI mice with CRISPRi rescued their morphological and excitability phenotypes, supporting Nell2 overexpression as a cause for apoE4-induced neuronal dysfunction. Our findings highlight the early transcriptomic and morpho-electric alterations behind the apoE4-induced neuronal dysfunction in AD. HIGHLIGHTS: ApoE4 causes hyperexcitability of select hippocampal neurons in young apoE4 mice.ApoE4 causes dentate hyperexcitability and inhibitory deficit in aged apoE4 mice.snRNA-seq reveals apoE genotype-, cell type-, and age-dependent transcriptomic changes.Nell2 overexpression identified as a cause of apoE4-induced neuronal hyperexcitability.

The APOE-R136S mutation protects against APOE4-driven Tau pathology, neurodegeneration and neuroinflammation.

Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD), leading to earlier age of clinical onset and exacerbating pathologies. There is a critical need to identify protective targets. Recently, a rare APOE variant, APOE3-R136S (Christchurch), was found to protect against early-onset AD in a PSEN1-E280A carrier. In this study, we sought to determine if the R136S mutation also protects against APOE4-driven effects in LOAD. We generated tauopathy mouse and human iPSC-derived neuron models carrying human APOE4 with the homozygous or heterozygous R136S mutation. We found that the homozygous R136S mutation rescued APOE4-driven Tau pathology, neurodegeneration and neuroinflammation. The heterozygous R136S mutation partially protected against APOE4-driven neurodegeneration and neuroinflammation but not Tau pathology. Single-nucleus RNA sequencing revealed that the APOE4-R136S mutation increased disease-protective and diminished disease-associated cell populations in a gene dose-dependent manner. Thus, the APOE-R136S mutation protects against APOE4-driven AD pathologies, providing a target for therapeutic development against AD.

Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish.

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parent's mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation.

Bioinformatics Analysis of Publicly Available Single-Nuclei Transcriptomics Alzheimer's Disease Datasets Reveals APOE Genotype-Specific Changes Across Cell Types in Two Brain Regions.

Alzheimer's Disease (AD) is a complex neurodegenerative disease that gravely affects patients and imposes an immense burden on caregivers. Apolipoprotein E4 (APOE4) has been identified as the most common genetic risk factor for AD, yet the molecular mechanisms connecting APOE4 to AD are not well understood. Past transcriptomic analyses in AD have revealed APOE genotype-specific transcriptomic differences; however, these differences have not been explored at a single-cell level. To elucidate more complex APOE genotype-specific disease-relevant changes masked by the bulk analysis, we leverage the first two single-nucleus RNA sequencing AD datasets from human brain samples, including nearly 55,000 cells from the prefrontal and entorhinal cortices. In each brain region, we performed a case versus control APOE genotype-stratified differential gene expression analysis and pathway network enrichment in astrocytes, microglia, neurons, oligodendrocytes, and oligodendrocyte progenitor cells. We observed more global transcriptomic changes in APOE4 positive AD cells and identified differences across APOE genotypes primarily in glial cell types. Our findings highlight the differential transcriptomic perturbations of APOE isoforms at a single-cell level in AD pathogenesis and have implications for precision medicine development in the diagnosis and treatment of AD.

Expression patterns and evolution of urocortin and corticotropin-releasing hormone genes in a cichlid fish.

The corticotropin-releasing hormone and urocortin family of peptides consists of five members in many vertebrates: CRH (crha/crhb in teleosts), CRH2, UCN/UTS1, UCN2, and UCN3. These genes differ in expression pattern, as well as receptor affinity, allowing them to serve a wide range of functions in a variety of species. To better understand the roles of these genes in a single species, we examined their expression patterns in the cichlid fish Astatotilapia burtoni. In situ hybridization to map mRNA expression patterns of crhb, uts1, ucn2, and ucn3 in the brain revealed conserved and distinct spatial features of expression. crhb- and uts1-expressing cells were the most broadly distributed, with several areas of co-regionalization. ucn3 was less abundant but was found in discrete regions throughout the extent of the brain, with high expression in the cerebellum, while ucn2 was restricted to only a few areas. RT-PCR showed that while crhb, uts1, and ucn3 are found in several body tissues and widespread throughout the brain, ucn2 is quite restricted in the brain, and crha is only expressed in the eye. Bayesian phylogenetic analyses identified detailed relationships and novel orthologs in the urocortin family. We found evidence for a UCN2 gene loss in some reptiles. Our detailed description of the complete family of genes in the central nervous system of a model organism will inform future studies on the function of these genes in A. burtoni and provides a foundation for comparative studies with teleosts and other vertebrates.

Phenotypic analysis of catastrophic childhood epilepsy genes.

Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen ∼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

Experimental and real-world evidence supporting the computational repurposing of bumetanide for APOE4-related Alzheimer's disease.

The evident genetic, pathological, and clinical heterogeneity of Alzheimer's disease (AD) poses challenges for traditional drug development. We conducted a computational drug repurposing screen for drugs to treat apolipoprotein (apo) E4-related AD. We first established apoE-genotype-dependent transcriptomic signatures of AD by analyzing publicly-available human brain database. We then queried these signatures against the Connectivity Map database containing transcriptomic perturbations of >1300 drugs to identify those that best reverse apoE-genotype-specific AD signatures. Bumetanide was identified as a top drug for apoE4 AD. Bumetanide treatment of apoE4 mice without or with Aß accumulation rescued electrophysiological, pathological, or cognitive deficits. Single-nucleus RNA-sequencing revealed transcriptomic reversal of AD signatures in specific cell types in these mice, a finding confirmed in apoE4-iPSC-derived neurons. In humans, bumetanide exposure was associated with a significantly lower AD prevalence in individuals over the age of 65 in two electronic health record databases, suggesting effectiveness of bumetanide in preventing AD.

Social status regulates kisspeptin receptor mRNA in the brain of Astatotilapia burtoni.

The brain controls reproduction in response to relevant external and internal cues. Central to this process in vertebrates is gonadotropin-releasing hormone (GnRH1) produced in neurons of the hypothalamic-preoptic area (POA). GnRH1 released from the POA stimulates pituitary release of gonadotropins, which in males causes sperm production and concomitant steroid hormone release from the testes. Kisspeptin, a neuropeptide acting via the kisspeptin receptor (Kiss1r), increases GnRH1 release and is linked to development of the reproductive system in mammals and other vertebrates. In both fish and mammals, kiss1r mRNA levels increase in the brain around the time of puberty but the environmental and other stimuli regulating kisspeptin signaling to GnRH1 neurons remain unknown. To understand where kiss1r is expressed and how it is regulated in the brain of a cichlid fish, Astatotilapia burtoni, we measured expression of a kiss1r homolog mRNA by in situ hybridization and quantitative reverse transcription-PCR (qRT-PCR). We found kiss1r mRNA localized in the olfactory bulb, specific nuclei in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, as well as in GnRH1 and GnRH3 neurons. Since males' sexual physiology and behavior depend on social status in A. burtoni, we also tested how status influenced kiss1r mRNA levels. We found higher kiss1r mRNA levels in whole brains of high status territorial males and lower levels in low status non-territorial males. Our results are consistent with the hypothesis that Kiss1r regulates many functions in the brain, making it a strong candidate for mediating differences in reproductive physiology between territorial and non-territorial phenotypes.

Behavioral Comorbidities and Drug Treatments in a Zebrafish scn1lab Model of Dravet Syndrome.

Loss-of-function mutations in SCN1A cause Dravet syndrome (DS), a catastrophic childhood epilepsy in which patients experience comorbid behavioral conditions, including movement disorders, sleep abnormalities, anxiety, and intellectual disability. To study the functional consequences of voltage-gated sodium channel mutations, we use zebrafish with a loss-of-function mutation in scn1lab, a zebrafish homolog of human SCN1A. Homozygous scn1labs552/s552 mutants exhibit early-life seizures, metabolic deficits, and early death. Here, we developed in vivo assays using scn1labs552 mutants between 3 and 6 d postfertilization (dpf). To evaluate sleep disturbances, we monitored larvae for 24 h with locomotion tracking software. Locomotor activity during dark (night phase) was significantly higher in mutants than in controls. Among anticonvulsant drugs, clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved at night for scn1labs552 mutant larvae. To monitor exploratory behavior in an open field, we tracked larvae in a novel arena. Mutant larvae exhibited impaired exploratory behavior, with increased time spent near the edge of the arena and decreased mobility, suggesting greater anxiety. Both clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved and increased time spent in the center of the arena. Counting inhibitory neurons in vivo revealed no differences between scn1labs552 mutants and siblings. Taken together, our results demonstrate conserved features of sleep, anxiety, and movement disorders in scn1lab mutant zebrafish, and provide evidence that a zebrafish model allows effective tests of treatments for behavioral comorbidities associated with DS.

Three Distinct Glutamate Decarboxylase Genes in Vertebrates.

Gamma-aminobutyric acid (GABA) is a widely conserved signaling molecule that in animals has been adapted as a neurotransmitter. GABA is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). Two vertebrate genes, GAD1 and GAD2, encode distinct GAD proteins: GAD67 and GAD65, respectively. We have identified a third vertebrate GAD gene, GAD3. This gene is conserved in fishes as well as tetrapods. We analyzed protein sequence, gene structure, synteny, and phylogenetics to identify GAD3 as a homolog of GAD1 and GAD2. Interestingly, we found that GAD3 was lost in the hominid lineage. Because of the importance of GABA as a neurotransmitter, GAD3 may play important roles in vertebrate nervous systems.

Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

Divergent evolution of two corticotropin-releasing hormone (CRH) genes in teleost fishes.

Genome duplication, thought to have happened twice early in vertebrate evolution and a third time in teleost fishes, gives rise to gene paralogs that can evolve subfunctions or neofunctions via sequence and regulatory changes. To explore the evolution and functions of corticotropin-releasing hormone (CRH), we searched sequenced teleost genomes for CRH paralogs. Our phylogenetic and synteny analyses indicate that two CRH genes, crha and crhb, evolved via duplication of crh1 early in the teleost lineage. We examined the expression of crha and crhb in two teleost species from different orders: an African cichlid, Burton's mouthbrooder, (Astatotilapia burtoni; Order Perciformes) and zebrafish (Danio rerio; Order Cypriniformes). Furthermore, we compared expression of the teleost crha and crhb genes with the crh1 gene of an outgroup to the teleost clade: the spotted gar (Lepisosteus oculatus). In situ hybridization for crha and crhb mRNA in brains and eyes revealed distinct expression patterns for crha in different teleost species. In the cichlid, crha mRNA was found in the retina but not in the brain. In zebrafish, however, crha mRNA was not found in the retina, but was detected in the brain, restricted to the ventral hypothalamus. Spotted gar crh1 was found in the retina as well as the brain, suggesting that the ancestor of teleost fishes likely had a crh1 gene expressed in both retina and brain. Thus, genome duplication may have freed crha from constraints, allowing it to evolve distinct sequences, expression patterns, and likely unique functions in different lineages.

A second corticotropin-releasing hormone gene (CRH2) is conserved across vertebrate classes and expressed in the hindbrain of a basal neopterygian fish, the spotted gar (Lepisosteus oculatus).

To investigate the origins of the vertebrate stress-response system, we searched sequenced vertebrate genomes for genes resembling corticotropin-releasing hormone (CRH). We found that vertebrate genomes possess, in addition to CRH, another gene that resembles CRH in sequence and syntenic environment. This paralogous gene was previously identified only in the elephant shark (a holocephalan), but we find it also in marsupials, monotremes, lizards, turtles, birds, and fishes. We examined the relationship of this second vertebrate CRH gene, which we name CRH2, to CRH1 (previously known as CRH) and urocortin1/urotensin1 (UCN1/UTS1) in primitive fishes, teleosts, and tetrapods. The paralogs CRH1 and CRH2 likely evolved via duplication of CRH during a whole-genome duplication early in the vertebrate lineage. CRH2 was subsequently lost in both teleost fishes and eutherian mammals but retained in other lineages. To determine where CRH2 is expressed relative to CRH1 and UTS1, we used in situ hybridization on brain tissue from spotted gar (Lepisosteus oculatus), a neopterygian fish closely related to teleosts. In situ hybridization revealed widespread distribution of both crh1 and uts1 in the brain. Expression of crh2 was restricted to the putative secondary gustatory/secondary visceral nucleus, which also expressed calcitonin-related polypeptide alpha (calca), a marker of parabrachial nucleus in mammals. Thus, the evolutionary history of CRH2 includes restricted expression in the brain, sequence changes, and gene loss, likely reflecting release of selective constraints following whole-genome duplication. The discovery of CRH2 opens many new possibilities for understanding the diverse functions of the CRH family of peptides across vertebrates.

Animal models in epilepsy research: legacies and new directions.

Human epilepsies encompass a wide variety of clinical, behavioral and electrical manifestations. Correspondingly, studies of this disease in nonhuman animals have brought forward an equally wide array of animal models; that is, species and acute or chronic seizure induction protocols. Epilepsy research has a long history of comparative anatomical and physiological studies on a range of mostly mammalian species. Nonetheless, a relatively limited number of rodent models have emerged as the primary choices for most investigations. In many cases, these animal models are selected on the basis of convenience or tradition, although technical or experimental rationale does, and should, factor into these decisions. More complex mammalian brains and genetic model organisms including zebrafish have been studied less, but offer substantial advantages that are becoming widely recognized.