People with HIV (PWH) develop neurocognitive impairment despite control of viral infection. Key pathological features of NeuroHIV patients, including activated microglia, neuronal damage and behavioral impairment, are present in transgenic mice expressing HIV-1 envelope glycoprotein gp120 in their brain (HIVgp120tg). Here we show that microglial mitogen-activated protein kinase p38α plays a crucial in vivo role in neuronal injury triggered by viral gp120. Cre-expression driven by the Cx3cr1 promotor in HIVgp120tg mice with floxed p38α alleles results in deletion of microglial p38α and protection from neuronal injury and behavioral impairment. Moreover, the expression patterns of neurotransmission-related genes differ between gp120-transgenic brains protected from neuronal injury and non-transgenic controls, suggesting that microglial p38α deficiency permits a non-toxic modulation of neurons in the presence of the viral protein. Bulk RNA-seq analysis of murine microglia and follow-up in human microglial cells in which p38α has been knocked out using CRISPR/Cas9 technology using qRT-PCR reveals that deficiency of the kinase leads to a distinct gene expression pattern of reduced inflammatory but preserved anti-viral responses.

Abstract Introduction HIV-associated neurocognitive disorders (HAND) remain a major complication in AIDS patients and are thought to arise from chronic neuroinflammation. Our previous work established that Δ9-tetrahydrocannabinol (THC) exerts neuroprotective effects in HIV gp120 transgenic (Tg) mice by downregulating gp120 expression and reducing astrocytic activation. Methods In this study, we employed spatial transcriptomics to map the neuroimmune landscape of the gp120 Tg brain and to define the regional effects of THC treatment. Results Soluble gp120 protein was distributed throughout the hippocampus, corpus callosum, cortex, and thalamus, coinciding with increased reactive astrocytes–an effect markedly diminished by THC. While reactive microglia were evenly distributed across brain regions, THC treatment enhanced oligodendrocyte populations and induced IL-33 expression in the corpus callosum and stria. Notably, THC increased excitatory neurons in the cortex and thalamus but decreased inhibitory neurons primarily in the hypothalamus, suggesting restoration of neural balance underlying cognition and memory. T cells and dendritic cells localized around the subfornical organ and lateral ventricles decreased after THC exposure. Conclusion These spatial data align with our prior single-cell RNA-seq findings, collectively revealing that THC modulates the neuroimmune and cellular architecture of the HIV-compromised brain. Our findings highlight spatial transcriptomics as a powerful approach to uncover region-specific neuroimmune remodeling and support the therapeutic potential of cannabinoids in HAND. Funding Source Supported by NIH grants P20GM103641, P30GM154631, R01ES030144, and R01AI160896 Topic Categories Neuroimmunology (NEUR)

The COVID-19 pandemic resulted in over 7 million reported deaths and over 700.4 million reported infections to-date. Many individuals who recover from COVID-19 report prolonged dyspnea, sometimes persisting for months. Furthermore, COVID-19 has been linked to systemic and neuronal inflammation which may have downstream impacts on the neural control of breathing. Therefore, we hypothesized that individuals recovered from COVID-19 may exhibit changes in their ventilatory chemosensitivity to carbon dioxide and hypoxia, and that these changes may be linked to systemic inflammation. To test this hypothesis, we measured baseline ventilatory patterns and chemoreflex sensitivity in individuals recovered from COVID-19 (n = 77) and individuals with no prior COVID-19 infection (n = 41). Peripheral venous blood samples were also collected for inflammatory biomarker expression and profiling. Recovered participants demonstrated a small but progressive decrease in the hypercapnic ventilatory response under a co-stimulus with hypoxia (control vs. 24-month post-recovery; p = 0.023). Additionally, we identified several significant correlations between plasma inflammatory markers and ventilatory chemoreflex characteristics, including a positive correlation between SAA and CRP and the ventilatory response to hypoxia (p < 0.05 within recovered and control cohorts). Finally, expression of six vascular inflammatory markers (Myoglobin, NGAL, MMP-2, OPN, IGFBP-4, and Cystatin C) was unexpectedly decreased in recovered participants compared to the control cohort for up to one-year post recovery. Overall, this data indicates that COVID-19 and other acute viral infections may have a modest impact on the chemoreflex control of breathing as well as systemic inflammatory profiles, and that these changes may be linked to each other. These findings may strengthen our understanding of the pathology of long-COVID symptoms.

AIDS patients develop HIV-associated neurocognitive disorders (HAND), believed to result from neuroinflammation. In this study, we examined the effect of Delta-9-Tetrahydrocannabinol (THC) in HIV gp120 transgenic (Tg) mice and the possible role of the microbiome in causing neurogenesis. For this purpose, wild-type (WT) and gp120Tg mice were treated with either vehicle or THC, and brains were examined for neurogenesis. In gp120 Tg mice, THC treatment reduced reactive gliosis by downregulating the synthesis of gp120 and overexpression of gfap. scRNA sequencing of brain-derived cells demonstrated changes in the microglial subsets, and T cells, with gp120Tg mice expressing low levels of CCR5+ cells, which increased following THC treatment. Treatment of gp120Tg mice with THC led to cAMP-dependent increase in PKA expression. The gut microbiome in gp120Tg mice showed microbial dysbiosis with an increase in pro-inflammatory microbiota while THC-treatment reversed gut dysbiosis. When C57BL/6 wt germ-free (B6gf) mice were transplanted with fecal microbiota from gp120Tg mice (gp120Tg FMT à B6gf mice), they showed neuronal injury by elevating number of CD4+ T cells producing IL-17A and IFN-γ. Our findings demonstrate the critical role of THC in regulating gut microbial dysbiosis and attenuation of neuroinflammation through the gut-brain axis in gp120 Tg mice. This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896 Neuroimmunology (NEUR)

In all eukaryotes, the mitogen activated protein kinase (MAPK) cascade, a multilayered interconnected network of enzymes, connects external stimuli to gene regulation, dictating cellular fate. However, mechanisms for encoding information in this complex, fluctuating network to activate specific responses remain elusive. Here, we demonstrate that the central human stress regulator protein p38 MAPK encodes information regarding experienced stresses as different frequency oscillations of its activation state. These oscillations are used to drive specific responses through frequency-dependent resonance of oscillating biochemical phosphorylation reactions between p38 and downstream targets. These interactions closely mirror those of electronic alternating current (AC) circuits and their components, providing a unique framework through which to understand signal transduction in the MAPK cascade. Finally, we demonstrate how this understanding of bioresonance allows us to induce specific genetic responses simply by exposing cells to sugar to force activation state oscillations of p38 at predetermined frequencies.

In eukaryotes, the mitogen activated protein kinase (MAPK) cascade, a multilayered interconnected network of enzymes, connects external stimuli to gene regulation, determining cellular fate 1 . Environmental stress sensed by a cell starts a complex chain of reactions between MAPK enzymes that ultimately activates the master stress response regulator protein p38 MAPK 2,3 . Thus activated, p38 must then selectively activate targets from a pool of hundreds to initiate appropriate cellular responses 3 . Mechanisms for how p38 performs this selection remain unclear 4,5 . Here we show that human p38 target selectivity is based on the same principles as modern electronic telecommunications systems, except using waves of chemicals rather than electromagnetic fields or electric currents. p38 encodes information about stimuli as different frequency oscillations of its activation state, and targets are selected through frequency-dependent resonance of oscillating biochemical reactions between p38 and its targets. We demonstrate this mechanism by activating various genetic responses in human cells by applying only sugar at different frequencies. These results unify observations of oscillating signaling components and altered responses 6–19 into a coherent framework to understand and control human gene expression. As failures of this mechanism may contribute to some p38-associated diseases 2,20–28 , these findings may have implications for pharmaceutical development and therapeutic strategies.

Background: Pathological inflammation with a loss of synaptic integrity and function has been implicated in HIV Associated Neurocognitive Disorders (HAND). Although therapeutics exist to increase the lifespan of people living with HIV (PLWH), they are not effective at preventing neuroinflammation and HIV induced neuronal damage persists. In this study, we investigate the ephrin-B/EphB axis, which regulates inflammation, in post-mortem brain specimen of PLWH and experimental models in order to assess its potential role in HIV induced neuroinflammation. Methods: We analyze mRNA samples of post-mortem brain specimen of PLWH and uninfected controls obtained from the National NeuroAIDS Tissue Consortium (NNTC) and, for comparison, of a transgenic mouse model of neuroHIV using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Follow-up experiments employ mouse brain tissue and in vitro models, including immortalized human microglia, human induced pluripotent stem cell (iPSC)-derived mixed neuroglial cell cultures, cellular and molecular interference, functional and multiplex assays, immunofluorescence and mRNA sequencing to examine the role of the ephrin-B/EphB axis in neuroinflammation and the associated neurotoxicity. Results: Using qRT-PCR we find increased expression of EphB2 in post-mortem brain of PLWH, and detect a correlation with pro-viral DNA, viral RNA and an inverse correlation with abstract executive function and verbal fluency. Increased expression of ephrin-B/EphB at mRNA and protein level is also observed in brains of a transgenic mouse model of neuroHIV suggesting the upregulation can be driven, at least in part, by expression of viral gp120 envelope protein and a type I interferon, IFNβ. Additionally, we find induction of ephrin-B1 expression in microglia following activation by IFNβ. Given the previously reported impact of EphB2 on inflammation in the periphery, the functional role of EphB2-mediated ephrin-B reverse signaling on microglia is assessed for a pro-inflammatory and anti-viral signature. We find that EphB2 treated microglia secrete inflammatory and anti-viral factors but also exert contact-independent neurotoxicity. Finally, knockdown of microglial ephrin-B1, an EphB2 binding partner, shows a partial alleviation of the microglial pro-inflammatory signature and neurotoxicity. Conclusion: Our study suggests that elevated EphB2, and its reverse signaling through ephrin-B1 in microglia contribute to neuroinflammation and neurotoxicity in neuroHIV.

Methamphetamine (METH) use is frequent among people with HIV (PWH) and appears to increase the risk of neuronal injury and neurocognitive impairment (NCI). This study explored in vivo the effects of a 12 week (long-term), low-dose METH regimen in a transgenic animal model of neuroHIV with inducible expression of HIV-1 transactivator of transcription (Tat). Seven months after transient Tat induction and five months after METH exposure ended, we detected behavioral changes in the Barnes maze (BM) spatial memory task in the Tat and METH groups but not the combined Tat + METH group. The novel object recognition (NOR) task revealed that Tat extinguished discrimination in female animals with and without METH, although METH alone slightly improved NOR. In contrast, in males, Tat, METH, and Tat + METH all compromised NOR. Neuropathological examination detected sex-dependent and brain region-specific changes of pre-synaptic terminals, neurites, and activation of astrocytes and microglia. RNA-sequencing and quantitative reverse transcription polymerase chain reaction indicated that METH and Tat significantly altered gene expression, including factors linked to Alzheimer’s disease-like NCI. In summary, chronic low-dose METH exerts long-term effects on behavioral function, neuropathology, and mRNA expression, and modulates the effects of Tat, suggesting sex-dependent and -independent mechanisms may converge in HIV brain injury and NCI.

Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.

Abstract The GFAP-gp120Tg mice express soluble HIV-envelope protein gp120 in astrocytes and share many neuropathological features observed in the CNS of AIDS patients. Here, we investigated the effect of a cannabinoid, ∆9-THC, on gp120-mediated neuroinflammation and progression of neurodegeneration. We treated groups of GFAP-gp120Tg and C57/BL6 WT mice with ∆9-THC or the vehicle. Immunofluorescence imaging, flow cytometry, snRNA-sequencing, and shotgun sequencing were performed to understand the cellular and molecular level incidents. ∆9-THC reduced the neuroinflammation driven by gp120 by down-regulating the inflammatory chemokine receptors such as CCR5, CXCR4 on immune cells that directly access the CNS. Pronounced astrocytosis and microglial activation seen in Tg mice was significantly reduced following treatment with ∆9-THC. Neuroglial gene expression was also reversed after ∆9-THC treatment. Tg mice exhibited significant gut microbiota dysbiosis which was reversed by ∆9-THC. Members of the genera Alistipes, Prevotella, Plasmodium, Helicobacter, Desulfovibrio had higher relative abundance in Tg mice but reduced after ∆9-THC treatment. Taken together, this study demonstrates that ∆9-THC treatment significantly improves HIV-gp120-mediated pathogenesis of neurodegeneration, neuroinflammation, and helps to reshape gut microbiota (This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896).

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

While the concept of pericyte heterogeneity in the brain microvasculature is becoming more widely accepted, little is known about how they arise, or their functional contributions to the blood-brain barrier (BBB). We therefore set out to examine the distribution of subtypes of pericytes at the BBB and sought to elucidate some of their functional characteristics by examining their unique mRNA expression patterns. We demonstrate that type-1 pericytes (PC1) that are associated with young healthy brains and BBB homeostasis, can transition into type-2 pericytes (PC2) that are associated with disease and BBB breakdown, both in vitro and in vivo, in the presence of both endogenous and disease associated ligands. We identified PC1 and PC2 in single-cell RNA-sequencing from vascular enriched mouse brain and identified transcriptional differences between PC1 and PC2. PC2 showed increased expression of genes associated with phagocytosis and peripheral immune cell infiltration. On the contrary, PC1 displayed increased expression of genes involved in hedgehog signaling, which is known to promote tight junction formation at the BBB. Our data support the PC1-to-PC2 transition as an origin of PC diversity and suggest a functional role for PC1 in maintaining BBB homeostasis and PC2 in responding to pathological conditions.

Background:Little is known about the pathogenesis of Bipolar Disorder, and even less is known about the genetic differences between its subtypes. Bipolar Disorder is classified into different subtypes, which present different symptoms and lifetime courses. While genetic studies have been conducted in Bipolar Disorder, most examined the gene expression of only Bipolar Disorder Type 1. Studies that include Bipolar Disorder Type 1 and Bipolar Disorder Type 2 often fail to differentiate them into separate conditions. Few large transcriptomic meta-analyses in Bipolar Disorder have been conducted to identify genetic pathways. Thus, using publicly available data sets we aim here to uncover significant differential gene expression that allows distinguishing Type 1 and Type 2 Bipolar Disorders, as well as find patterns in Bipolar Disorder as a whole.Methods:We analyze 17 different gene expression data sets from different tissue in Bipolar Disorder using GEO2R and manual analysis, of which 15 contained significant differential gene expression results. We use STRING and Cytoscape to examine Gene Ontology to find significantly affected genetic pathways. We identify hub genes using cytoHubba, a plugin in Cytoscape. We find genes common to data sets of the same material or subtype.Results:12 out of 15 data sets are enriched for immune system and RNA related pathways. 9 out of 15 data sets are enriched for neurocognitive and metal ion related GO terms. Analysis of Bipolar Disorder Type 1 vs Bipolar Disorder Type 2 revealed most differentially expressed genes were related to immune function, especially cytokines. Terms related to synaptic signaling and neurotransmitter secretion were found in down-regulated GO terms while terms related to neuron apoptosis and death were up-regulated. We identify the gene SNCA as a potential biomarker for overall Bipolar Disorder diagnosis due to its prevalence in our data sets.Conclusions:The immune system and RNA related pathways are significantly enriched across the Bipolar Disorder data sets. The role of these pathways is likely more critically important to the function of Bipolar Disorder than currently understood. Further studies should clearly label the subtype of Bipolar Disorder used in their research and more effort needs to be undertaken to collect samples from Cyclothymic Disorder and Bipolar Disorder Type 2.

Counting cells is a cornerstone of tracking disease progression in neuroscience. A common approach for this process is having trained researchers individually select and count cells within an image, which is not only difficult to standardize but also very time-consuming. While tools exist to automatically count cells in images, the accuracy and accessibility of such tools can be improved. Thus, we introduce a novel tool ACCT: Automatic Cell Counting with Trainable Weka Segmentation which allows for flexible automatic cell counting via object segmentation after user-driven training. ACCT is demonstrated with a comparative analysis of publicly available images of neurons and an in-house dataset of immunofluorescence-stained microglia cells. For comparison, both datasets were manually counted to demonstrate the applicability of ACCT as an accessible means to automatically quantify cells in a precise manner without the need for computing clusters or advanced data preparation.

Health disparities in underserved communities, such as inadequate healthcare access, impact COVID-19 disease outcomes. These disparities are evident in Hispanic populations nationwide, with disproportionately high infection and mortality rates. Furthermore, infected individuals can develop long COVID with sustained impacts on quality of life. The goal of this study was to identify immune and endothelial factors that are associated with COVID-19 outcomes in Riverside County, a high-risk and predominantly Hispanic community, and investigate the long-term impacts of COVID-19 infection. 112 participants in Riverside County, California, were recruited according to the following criteria: healthy control (n = 23), outpatients with moderate infection (outpatient, n = 33), ICU patients with severe infection (hospitalized, n = 33), and individuals recovered from moderate infection (n = 23). Differences in outcomes between Hispanic and non-Hispanic individuals and presence/absence of co-morbidities were evaluated. Circulating immune and vascular biomarkers were measured by ELISA, multiplex analyte assays, and flow cytometry. Follow-up assessments for long COVID, lung health, and immune and vascular changes were conducted after recovery (n = 23) including paired analyses of the same participants. Compared to uninfected controls, the severe infection group had a higher proportion of Hispanic individuals (n = 23, p = 0.012) than moderate infection (n = 8, p = 0.550). Disease severity was associated with changes in innate monocytes and neutrophils, lymphopenia, disrupted cytokine production (increased IL-8 and IP-10/CXCL10 but reduced IFNλ2/3 and IFNγ), and increased endothelial injury (myoglobin, VCAM-1). In the severe infection group, a machine learning model identified LCN2/NGAL, IL-6, and monocyte activation as parameters associated with fatality while anti-coagulant therapy was associated with survival. Recovery from moderate COVID infection resulted in long-term immune changes including increased monocytes/lymphocytes and decreased neutrophils and endothelial markers. This group had a lower proportion of co-morbidities (n = 8, p = 1.0) but still reported symptoms associated with long COVID despite recovered pulmonary function. This study indicates increased severity of COVID-19 infection in Hispanic individuals of Riverside County, California. Infection resulted in immunological and vascular changes and long COVID symptoms that were sustained for up to 11 months, however, lung volume and airflow resistance was recovered. Given the immune and behavioral impacts of long COVID, the potential for increased susceptibility to infections and decreased quality of life in high-risk populations warrants further investigation.

For over two decades, highly active antiretroviral therapy (HAART) was able to help prolong the life expectancy of people living with HIV-1 (PLWH) and eliminate the virus to an undetectable level. However, an increased prevalence of HIV- associated neurocognitive disorders (HAND) was observed. These symptoms range from neuronal dysfunction to cell death. Among the markers of neuronal deregulation, we cite the alteration of synaptic plasticity and neuronal communications. Clinically, these dysfunctions led to neurocognitive disorders such as learning alteration and loss of spatial memory, which promote premature brain aging even in HAART-treated patients. In support of these observations, we showed that the gp120 protein deregulates miR-499-5p and its downstream target, the calcineurin (CaN) protein. The gp120 protein also promotes the accumulation of calcium (Ca2+) and reactive oxygen species (ROS) inside the neurons leading to the activation of CaN and the inhibition of miR-499-5p. gp120 protein also caused mitochondrial fragmentation and changes in shape and size. The use of mimic miR-499 restored mitochondrial functions, appearance, and size. These results demonstrated the additional effect of the gp120 protein on neurons through the miR-499-5p/calcineurin pathway.

People living with HIV are affected by the chronic consequences of neurocognitive impairment (NCI) despite antiretroviral therapies that suppress viral replication, improve health and extend life. Furthermore, viral suppression does not eliminate the virus, and remaining infected cells may continue to produce viral proteins that trigger neurodegeneration. Comorbidities such as diabetes mellitus are likely to contribute substantially to CNS injury in people living with HIV, and some components of antiretroviral therapy exert undesirable side effects on the nervous system. No treatment for HIV-associated NCI has been approved by the European Medicines Agency or the US Food and Drug Administration. Historically, roadblocks to developing effective treatments have included a limited understanding of the pathophysiology of HIV-associated NCI and heterogeneity in its clinical manifestations. This heterogeneity might reflect multiple underlying causes that differ among individuals, rather than a single unifying neuropathogenesis. Despite these complexities, accelerating discoveries in HIV neuropathogenesis are yielding potentially druggable targets, including excessive immune activation, metabolic alterations culminating in mitochondrial dysfunction, dysregulation of metal ion homeostasis and lysosomal function, and microbiome alterations. In addition to drug treatments, we also highlight the importance of non-pharmacological interventions. By revisiting mechanisms implicated in NCI and potential interventions addressing these mechanisms, we hope to supply reasons for optimism in people living with HIV affected by NCI and their care providers.