Acute Hypobaric Hypoxia Causes Alterations in Acetylcholine-Mediated Signaling Through Varying Expression of Muscarinic Receptors in the Prefrontal Cortex and Cerebellum of Rats' Brain.

Sharma, Narendra Kumar, Mansi Srivastava, Tikam Chand Dakal, Vipin Ranga, and Pawan Kumar Maurya. Acute hypobaric hypoxia (HH) causes alterations in acetylcholine-mediated signaling through varying expression of muscarinic receptors in the PFC and cerebellum of rats' brain. High Alt Med Biol. 00:00-00, 2024. Background: Muscarinic receptor (CHRM) proteins are G-protein-associated acetylcholine receptors found in neuronal membranes. Five major subtypes, CHRM1-CHRM5, modulate acetylcholine in central nervous system signaling cascades. CHRM1, CHRM3, and CHRM5 are linked to Gαq/Gα11 proteins, whereas CHRM2 and CHRM4 are linked to Gαi/Gαo proteins. Objective: Limited research has been conducted to explore the impact of HH on CHRM gene expressions. It is caused by low oxygen availability at high altitudes, which impairs neurotransmission, cognitive performance, and physiological functions. Previous studies have shown that exposure to hypoxia leads to a reduction in CHRM receptors, which in turn causes alteration in signal transduction, physiological responses, cognitive deficits, and mood alterations. Method: In the present study, we have used semiquantitative PCR to measure muscarinic receptor gene expression after 6, 12, and 24 hours of HH exposure at 25,000 feet using a decompression chamber in rat brain's PFC and cerebellum. Result: We have found that CHRM1-CHRM5 downregulated after acute exposure to hypoxia until 12 hours, and then, the expression level of these receptors increased to 24 hours when compared with 12 hours in PFC. All subtypes have shown a similar pattern in PFC regions under hypoxia exposure. On the other hand, these receptors have shown altered expression at different time points in the cerebellum. CHRM1 and CHRM4 acutely downregulated, CHRM2 and CHRM5 downregulated, while CHRM3 upregulated after hypoxia exposure. Conclusion: Our study, for the first time, has shown the altered expressions of muscarinic receptors under temporal hypoxia exposure. The altered expression pattern has shown an association with acclimatization and protection against necrosis due to hypoxia. This study may pave further investigations for understanding and addressing the cognitive, behavioral, and physiological impacts of hypoxia and therapeutic development.

A global transcriptional atlas of the effect of acute sleep deprivation in the mouse frontal cortex.

Sleep deprivation (SD) has negative effects on brain and body function. Sleep problems are prevalent in a variety of disorders, including neurodevelopmental and psychiatric conditions. Thus, understanding the molecular consequences of SD is of fundamental importance in biology. In this study, we present the first simultaneous bulk and single-nuclear RNA sequencing characterization of the effects of SD in the male mouse frontal cortex. We show that SD predominantly affects glutamatergic neurons, specifically in layers 4 and 5, and produces isoform switching of over 1500 genes, particularly those involved in splicing and RNA binding. At both the global and cell-type specific level, SD has a large repressive effect on transcription, downregulating thousands of genes and transcripts. As a resource we provide extensive characterizations of cell-types, genes, transcripts, and pathways affected by SD. We also provide publicly available tutorials aimed at allowing readers adapt analyses performed in this study to their own datasets.

Overexpression of the schizophrenia risk gene C4 in PV cells drives sex-dependent behavioral deficits and circuit dysfunction.

Fast-spiking parvalbumin (PV)-positive cells are key players in orchestrating pyramidal neuron activity, and their dysfunction is consistently observed in myriad brain diseases. To understand how immune complement pathway dysregulation in PV cells drives disease pathogenesis, we have developed a transgenic line that permits cell-type specific overexpression of the schizophrenia-associated C4 gene. We found that overexpression of mouse C4 (mC4) in PV cells causes sex-specific alterations in anxiety-like behavior and deficits in synaptic connectivity and excitability of PFC PV cells. Using a computational model, we demonstrated that these microcircuit deficits led to hyperactivity and disrupted neural communication. Finally, pan-neuronal overexpression of mC4 failed to evoke the same deficits in behavior as PV-specific mC4 overexpression, suggesting that perturbations of this neuroimmune gene in fast-spiking neurons are especially detrimental to circuits associated with anxiety-like behavior. Together, these results provide a causative link between C4 and the vulnerability of PV cells in brain disease.

Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.

There are no cures for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Emerging evidence suggests the gut microbiota plays a role in their pathogenesis, though the influences of specific bacteria on disease-associated proteins remain elusive. Here, we reveal the effects of 229 human bacterial isolates on the aggregation and toxicity of Aß1-42, α-synuclein, and polyglutamine tracts in Caenorhabditis elegans expressing these culprit proteins. Our findings demonstrate that bacterial effects on host protein aggregation are consistent across different culprit proteins, suggesting that microbes affect protein stability by modulating host proteostasis rather than selectively targeting disease-associated proteins. Furthermore, we found that feeding C. elegans proteoprotective Prevotella corporis activates the heat shock response, revealing an unexpected discovery of a microbial influence on host proteostasis. Insight into how individual bacteria affect PCD proteins could open new strategies for prevention and treatment by altering the abundance of microbes.

The direct and indirect pathways of the basal ganglia antagonistically influence cortical activity and perceptual decisions.

The striatum, the main input nucleus of the basal ganglia, receives topographically organized input from the cortex and gives rise to the direct and indirect output pathways, which have antagonistic effects on basal ganglia output directed to the cortex. We optogenetically stimulated the direct and indirect pathways in a visual and a working memory task in mice that responded by licking. Unilateral direct pathway stimulation increased the probability of lick responses toward the contralateral, non-stimulated side and increased cortical activity globally. In contrast, indirect pathway stimulation increased the probability of responses toward the stimulated side and decreased activity in the stimulated hemisphere. Moreover, direct pathway stimulation enhanced the neural representation of a contralateral visual stimulus during the delay of the working memory task, whereas indirect pathway stimulation had the opposite effect. Our results demonstrate how these two pathways influence perceptual decisions and working memory and modify activity in the dorsal cortex.

HDAC1 and HDAC2 orchestrate Wnt signaling to regulate neural progenitor transition during brain development.

Tightly controlled neurogenesis is crucial for generating the precise number of neurons and establishing the intricate architecture of the cortex, with deficiencies often leading to neurodevelopmental disorders. Neuroepithelial progenitors (NPs) transit into radial glial progenitors (RGPs) to initiate neural differentiation, yet the governing mechanisms remain elusive. Here, we found that histone deacetylases 1 and 2 (HDAC1/2) mediated suppression of Wnt signaling is essential for the NP-to-RGP transition. Conditional depletion of HDAC1/2 from NPs upregulated Wnt signaling genes, impairing the transition to RGPs and resulting in rosette structures within the neocortex. Multi-omics analysis revealed that HDAC1/2 are critical for downregulating Wnt signaling, identifying Wnt9a as a key target. Overexpression of Wnt9a led to an increased population of NPs and the disruption of cortical organization. Notably, Wnt inhibitor administration partially rescued the disrupted cortical architecture. Our findings reveal the significance of tightly controlled Wnt signaling through epigenetic mechanisms in neocortical development.

Reiterated male-to-female violence disrupts hippocampal estrogen receptor ß expression, prompting anxiety-like behavior.

Intimate partner violence (IPV) is a significant public health concern whose neurological/behavioral sequelae remain to be mechanistically explained. Using a mouse model recapitulating an IPV scenario, we evaluated the female brain neuroendocrine alterations produced by a reiterated male-to-female violent interaction (RMFVI). RMFVI prompted anxiety-like behavior in female mice whose hippocampus displayed a marked neuronal loss and hampered neurogenesis, namely reduced BrdU-DCX-positive nuclei and diminished dendritic arborization in the dentate gyrus (DG): effects paralleled by a substantial downregulation of the estrogen receptor ß (ERß). After RMFVI, the DG harbored reduced brain-derived neurotrophic factor (BDNF) pools and tyrosine kinase receptor B (TrkB) phosphorylation. Accordingly, ERß knockout (KO) mice had heightened anxiety and curtailed BDNF levels at baseline while dying prematurely during the RMFVI procedure. Strikingly, injecting an ERß antagonist or agonist into the wild-type (WT) female hippocampus enhanced or reduced anxiety, respectively. Thus, reiterated male-to-female violence jeopardizes hippocampal homeostasis, perturbing the ERß/BDNF axis and ultimately instigating anxiety and chronic stress.

BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines.

Synaptic structural plasticity, the expansion of dendritic spines in response to synaptic stimulation, is essential for experience-dependent plasticity and is driven by branched actin polymerization. The WAVE regulatory complex (WRC) is confined to nanodomains at the postsynaptic membrane where it catalyzes actin polymerization. As the netrin/RGM receptor Neogenin is a critical regulator of the WRC, its nanoscale organization may be an important determinant of WRC nanoarchitecture and function. Using super-resolution microscopy, we reveal that Neogenin is highly organized on the spine membrane at the nanoscale level. We show that Neogenin binding to the WRC promotes co-clustering into nanodomains in response to brain-derived neurotrophic factor (BDNF), indicating that nanoclustering occurs in response to synaptic stimulation. Disruption of Neogenin/WRC binding not only prevents BDNF-mediated actin remodeling but also inhibits BDNF-induced calcium signaling. We conclude that the assembly of Neogenin/WRC nanodomains is a prerequisite for BDNF-mediated structural and synaptic plasticity.

Neuropeptidergic regulation of neuromuscular signaling in larval zebrafish alters swimming behavior and synaptic transmission.

Chemical synaptic transmission is modulated to accommodate different activity levels, thus enabling homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cholinergic neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase evoked locomotion. We generated mutants lacking the neuropeptide-processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. Recording excitatory postsynaptic currents demonstrated overall larger amplitudes in the wild type. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. Both mutant muscles expressed more nicotinic acetylcholine receptors (nAChRs) on their surface; thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input.

Unveiling differential gene co-expression networks and its effects on levodopa-induced dyskinesia.

Levodopa-induced dyskinesia (LID) refers to involuntary motor movements of chronic use of levodopa in Parkinson's disease (PD) that negatively impact the overall well-being of people with this disease. The molecular mechanisms involved in LID were investigated through whole-blood transcriptomic analysis for differential gene expression and identification of new co-expression and differential co-expression networks. We found six differentially expressed genes in patients with LID, and 13 in patients without LID. We also identified 12 co-expressed genes exclusive to LID, and six exclusive hub genes involved in 23 gene-gene interactions in patients with LID. Convergently, we identified novel genes associated with PD and LID that play roles in mitochondrial dysfunction, dysregulation of lipid metabolism, and neuroinflammation. We observed significant changes in disease progression, consistent with previous findings of maladaptive plastic changes in the basal ganglia leading to the development of LID, including a chronic pro-inflammatory state in the brain.

Divergent input patterns to the central lateral amygdala play a duet in fear memory formation.

Somatostatin (SOM)-expressing neurons in the central lateral amygdala (CeL) are responsible for fear memory learning, but the circuit and molecular mechanisms underlying this biology remain elusive. Here, we found that glutamatergic neurons in the lateral parabrachial nucleus (LPB) directly dominated the activity of CeLSOM neurons, and that selectively inhibiting the LPBGlu→CeLSOM pathway suppressed fear memory acquisition. By contrast, inhibiting CeL-projecting glutamatergic neurons in the paraventricular thalamic nucleus (PVT) interfered with consolidation-related processes. Notably, CeLSOM-innervating neurons in the LPB were modulated by presynaptic cannabinoid receptor 1 (CB1R), and knock down of CB1Rs in LPB glutamatergic neurons enhanced excitatory transmission to the CeL and partially rescued the impairment in fear memory induced by CB1R activation in the CeL. Overall, our study reveals the mechanisms by which CeLSOM neurons mediate the formation of fear memories during fear conditioning in mice, which may provide a new direction for the clinical research of fear-related disorders.

Branch-specific clustered parallel fiber input controls dendritic computation in Purkinje cells.

Most central neurons have intricately branched dendritic trees that integrate massive numbers of synaptic inputs. Intrinsic active mechanisms in dendrites can be heterogeneous and be modulated in a branch-specific way. However, it remains poorly understood how heterogeneous intrinsic properties contribute to processing of synaptic input. We propose the first computational model of the cerebellar Purkinje cell with dendritic heterogeneity, in which each branch is an individual unit and is characterized by its own set of ion channel conductance densities. When simultaneously activating a cluster of parallel fiber synapses, we measure the peak amplitude of a response and observe how changes in P-type calcium channel conductance density shift the dendritic responses from a linear one to a bimodal one including dendritic calcium spikes and vice-versa. These changes relate to the morphology of each branch. We show how dendritic calcium spikes propagate and how Kv4.3 channels block spreading depolarization to nearby branches.

Early hippocampal hyperexcitability and synaptic reorganization in mouse models of amyloidosis.

The limited success of plaque-reducing therapies in Alzheimer's disease suggests that early treatment might be more effective in delaying or reversing memory impairments. Toward this end, it is important to establish the progression of synaptic and circuit changes before onset of plaques or cognitive deficits. Here, we used quantitative, fluorescence-based methods for synapse detection in CA1 pyramidal neurons to investigate the interaction between abnormal circuit activity, measured by Fos-immunoreactivity, and synapse reorganization in mouse models of amyloidosis. Using a genetically encoded, fluorescently labeled synaptic marker in juvenile mice (prior to sexual maturity), we find both synapse gain and loss depending on dendritic location. This progresses to broad synapse loss in aged mice. Elevated hippocampal activity in both CA3 and CA1 was present at weaning and preceded this reorganization. Thus, Aß overproduction may initiate abnormal activity and subsequent input-specific synapse plasticity. These findings indicate that sustained amyloidosis drives heterogeneous and progressive circuit-wide abnormalities.

NF1-dependent disruption of the blood-nerve-barrier is improved by blockade of P2RY14.

The blood-nerve-barrier (BNB) that regulates peripheral nerve homeostasis is formed by endoneurial capillaries and perineurial cells surrounding the Schwann cell (SC)-rich endoneurium. Barrier dysfunction is common in human tumorigenesis, including in some nerve tumors. We identify barrier disruption in human NF1 deficient neurofibromas, which were characterized by reduced perineurial cell glucose transporter 1 (GLUT1) expression and increased endoneurial fibrin(ogen) deposition. Conditional Nf1 loss in murine SCs recapitulated these alterations and revealed decreased tight junctions and decreased caveolin-1 (Cav1) expression in mutant nerves and in tumors, implicating reduced Cav1-mediated transcytosis in barrier disruption and tumorigenesis. Additionally, elevated receptor tyrosine kinase activity and genetic deletion of Cav1 increased endoneurial fibrin(ogen), and promoted SC tumor formation. Finally, when SC lacked Nf1, genetic loss or pharmacological inhibition of P2RY14 rescued Cav1 expression and barrier function. Thus, loss of Nf1 in SC causes dysfunction of the BNB via P2RY14-mediated G-protein coupled receptor (GPCR) signaling.

Respiratory infection with influenza A virus delays remyelination and alters oligodendrocyte metabolism.

Peripheral viral infection disrupts oligodendrocyte (OL) homeostasis such that endogenous remyelination may be affected. Here, we demonstrate that influenza A virus infection perpetuated a demyelination- and disease-associated OL phenotype following cuprizone-induced demyelination that resulted in delayed OL maturation and remyelination in the prefrontal cortex. Furthermore, we assessed cellular metabolism ex vivo, and found that infection altered brain OL and microglia metabolism in a manner that opposed the metabolic profile induced by remyelination. Specifically, infection increased glycolytic capacity of OLs and microglia, an effect that was recapitulated by lipopolysaccharide (LPS) stimulation of mixed glia cultures. In contrast, mitochondrial dependence was increased in OLs during remyelination, which was similarly observed in OLs of myelinating P14 mice compared to adult and aged mice. Collectively, our data indicate that respiratory viral infection is capable of suppressing remyelination, and suggest that metabolic dysfunction of OLs is implicated in remyelination impairment.

Altering heparan sulfate suppresses cell abnormalities and neuron loss in Drosophila presenilin model of Alzheimer Disease.

We examined the function of heparan-sulfate-modified proteoglycans (HSPGs) in pathways affecting Alzheimer disease (AD)-related cell pathology in human cell lines and mouse astrocytes. Mechanisms of HSPG influences on presenilin-dependent cell loss were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss-of-function of sulfateless (sfl), a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in human cell lines, Drosophila, and mouse astrocytes. RNA interference (RNAi) of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. Neuron-directed knockdown of Psn in Drosophila produced apoptosis and cell loss in the brain, phenotypes suppressed by reductions in sfl expression. Abnormalities in mitochondria, liposomes, and autophagosome-derived structures in animals with Psn knockdown were also rescued by reduction of sfl. These findings support the direct involvement of HSPGs in AD pathogenesis.

Serum protein biomarker profile distinguishes acetylcholine receptor antibody seropositive myasthenia gravis patients from healthy controls.

There is an unmet need for objective disease-specific biomarkers in the heterogeneous autoimmune neuromuscular disorder myasthenia gravis (MG). This cross-sectional study identified a signature of 23 inflammatory serum proteins with proximity extension assay (PEA) that distinguishes acetylcholine receptor antibody seropositive (AChR+) MG patients from healthy controls (HCs). CCL28, TNFSF14, 4E-BP1, transforming growth factor alpha (TGF-α), and ST1A1 ranked top biomarkers. TGF-ß1 and osteoprotegerin (OPG) differed between early- and late-onset MG, whereas CXCL10, TNFSF14, CCL11, interleukin-17C (IL-17C), and TGF-α differed significantly with immunosuppressive treatment. MG patients with moderate to high disease severity had lower uPA. Previously defined MG-associated microRNAs, miR-150-5p, miR-30e-5p, and miR-21-5p, correlated inversely with ST1A1 and TNFSF14. The presented inflammatory proteins that distinguish AChR+ MG are promising serum biomarkers for validation in prospective studies to allow for molecular signatures for patient subgroup stratification and monitoring of treatment response.

Reorganization of H3K9me heterochromatin leads to neuronal impairment via the cascading destruction of the KDM3B-centered epigenomic network.

Histone H3K9 methylated heterochromatin silences repetitive non-coding sequences and lineage-specific genes during development, but how tissue-specific genes escape from heterochromatin in differentiated cells is unclear. Here, we examine age-dependent transcriptomic profiling of terminally differentiated mouse retina to identify epigenetic regulators involved in heterochromatin reorganization. The single-cell RNA sequencing analysis reveals a gradual downregulation of Kdm3b in cone photoreceptors during aging. Disruption of Kdm3b (Kdm3b +/- ) of 12-month-old mouse retina leads to the decreasing number of cones via apoptosis, and it changes the morphology of cone ribbon synapses. Integration of the transcriptome with epigenomic analysis in Kdm3b +/- retinas demonstrates gains of heterochromatin features in synapse assembly and vesicle transport genes that are downregulated via the accumulation of H3K9me1/2. Contrarily, losses of heterochromatin in apoptotic genes exacerbated retinal neurodegeneration. We propose that the KDM3B-centered epigenomic network is crucial for balancing of cone photoreceptor homeostasis via the modulation of gene set-specific heterochromatin features during aging.

Oleic acid released by sensory neurons inhibits TRPV1-mediated thermal hypersensitivity via GPR40.

Noxious stimuli activate nociceptive sensory neurons, causing action potential firing and the release of diverse signaling molecules. Several peptides have already been identified to be released by sensory neurons and shown to modulate inflammatory responses and inflammatory pain. However, it is still unclear whether lipid mediators can be released upon sensory neuron activation to modulate intercellular communication. Here, we analyzed the lipid secretome of capsaicin-stimulated nociceptive neurons with LC-HRMS, revealing that oleic acid is strongly released from sensory neurons by capsaicin. We further demonstrated that oleic acid inhibits capsaicin-induced calcium transients in sensory neurons and reverses bradykinin-induced TRPV1 sensitization by a calcineurin (CaN) and GPR40 (FFAR1) dependent pathway. Additionally, oleic acid alleviated zymosan-mediated thermal hypersensitivity via the GPR40, suggesting that the capsaicin-mediated oleic acid release from sensory neurons acts as a protective and feedback mechanism, preventing sensory neurons from nociceptive overstimulation via the GPR40/CaN/TRPV1-axis.

Neuromodulatory co-expression in cardiac vagal motor neurons of the dorsal motor nucleus of the vagus.

Vagal innervation is well known to be crucial to the maintenance of cardiac health, and to protect and recover the heart from injury. Only recently has this role been shown to depend on the activity of the underappreciated dorsal motor nucleus of the vagus (DMV). By combining neural tracing, transcriptomics, and anatomical mapping in male and female Sprague-Dawley rats, we characterize cardiac-specific neuronal phenotypes in the DMV. We find that the DMV cardiac-projecting neurons differentially express pituitary adenylate cyclase-activating polypeptide (PACAP), cocaine- and amphetamine-regulated transcript (CART), and synucleins, as well as evidence that they participate in neuromodulatory co-expression involving catecholamines. The significance of these findings is enhanced by previous knowledge of the role of PACAP at the heart and of the other neuromodulators in peripheral vagal targets.