Interferon-ß deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV.

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 ß. 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 IFNß (IFNßKO) 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 IFNß but in a sex-dependent fashion. Notably, in cerebral cortex of IFNßKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNßKO 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 IFNß-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNß-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNßKO 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 IFNß on multiple components with more pronounced changes in IFNßKO females. In contrast, the effects of IFNßKO 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 IFNß 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 IFNß plays a vital role in maintaining neuronal homeostasis and memory function.

A critical role for Macrophage-derived Cysteinyl-Leukotrienes in HIV-1 induced neuronal injury.

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.

Health disparities in COVID-19: immune and vascular changes are linked to disease severity and persist in a high-risk population in Riverside County, California.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

HIV Protein Tat Induces Macrophage Dysfunction and Atherosclerosis Development in Low-Density Lipoprotein Receptor-Deficient Mice.

PURPOSE: HIV infection is consistently associated with an increased risk of atherosclerotic cardiovascular disease, but the underlying mechanisms remain elusive. HIV protein Tat, a transcriptional activator of HIV, has been shown to activate NF-κB signaling and promote inflammation in vitro. However, the atherogenic effects of HIV Tat have not been investigated in vivo. Macrophages are one of the major cell types involved in the initiation and progression of atherosclerosis. We and others have previously revealed the important role of IκB kinase ß (IKKß), a central inflammatory coordinator through activating NF-κB, in the regulation of macrophage functions and atherogenesis. This study investigated the impact of HIV Tat exposure on macrophage functions and atherogenesis. METHODS: To investigate the effects of Tat on macrophage IKKß activation and atherosclerosis development in vivo, myeloid-specific IKKß-deficient LDLR-deficient (IKKßΔMyeLDLR-/-) mice and their control littermates (IKKßF/FLDLR-/-) were exposed to recombinant HIV protein Tat. RESULTS: Exposure to Tat significantly increased atherosclerotic lesion size and plaque vulnerability in IKKßF/FLDLR-/- but not IKKßΔMyeLDLR-/- mice. Deficiency of myeloid IKKß attenuated Tat-elicited macrophage inflammatory responses and atherosclerotic lesional inflammation in IKKßΔMyeLDLR-/- mice. Further, RNAseq analysis demonstrated that HIV protein Tat affects the expression of many atherosclerosis-related genes in vitro in an IKKß-dependent manner. CONCLUSIONS: Our findings reveal atherogenic effects of HIV protein Tat in vivo and demonstrate a pivotal role of myeloid IKKß in Tat-driven atherogenesis.

Comprehensive review of lipocalin 2-mediated effects in lung inflammation.

Lipocalin-2 (LCN2) is an inflammatory mediator best known for its role as an innate acute-phase protein. LCN2 mediates the innate immune response to pathogens by sequestering iron, thereby inhibiting pathogen growth. Although LCN2 and its bacteriostatic properties are well studied, other LCN2 functions in the immune response to inflammatory stimuli are less well understood, such as its role as a chemoattractant and involvement in the regulation of cell migration and apoptosis. In the lungs, most studies thus far investigating the role of LCN2 in the immune response have looked at pathogenic inflammatory stimuli. Here, we compile data that explore the role of LCN2 in the immune response to various inflammatory stimuli in an effort to differentiate between protective versus detrimental roles of LCN2.

A pivotal role for Interferon-α receptor-1 in neuronal injury induced by HIV-1.

BACKGROUND: HIV-1 infection remains a major public health concern despite effective combination antiretroviral therapy (cART). The virus enters the central nervous system (CNS) early in infection and continues to cause HIV-associated neurocognitive disorders (HAND). The pathogenic mechanisms of HIV-associated brain injury remain incompletely understood. Since HIV-1 activates the type I interferon system, which signals via interferon-α receptor (IFNAR) 1 and 2, this study investigated the potential role of IFNAR1 in HIV-induced neurotoxicity. METHODS: We cross-bred HIVgp120-transgenic (tg) and IFNAR1 knockout (IFNAR1KO) mice. At 11-14 months of age, we performed a behavioral assessment and subsequently analyzed neuropathological alterations using deconvolution and quantitative immunofluorescence microscopy, quantitative RT-PCR, and bioinformatics. Western blotting of brain lysates and an in vitro neurotoxicity assay were employed for analysis of cellular signaling pathways. RESULTS: We show that IFNAR1KO results in partial, sex-dependent protection from neuronal injury and behavioral deficits in a transgenic model of HIV-induced brain injury. The IFNAR1KO rescues spatial memory and ameliorates loss of presynaptic terminals preferentially in female HIVgp120tg mice. Similarly, expression of genes involved in neurotransmission reveals sex-dependent effects of IFNAR1KO and HIVgp120. In contrast, IFNAR1-deficiency, independent of sex, limits damage to neuronal dendrites, microgliosis, and activation of p38 MAPK and restores ERK activity in the HIVgp120tg brain. In vitro, inhibition of p38 MAPK abrogates neurotoxicity caused similarly by blockade of ERK kinase and HIVgp120. CONCLUSION: Our findings indicate that IFNAR1 plays a pivotal role in both sex-dependent and independent processes of neuronal injury and behavioral impairment triggered by HIV-1.

Lipocalin-2 mediates HIV-1 induced neuronal injury and behavioral deficits by overriding CCR5-dependent protection.

People living with HIV (PLWH) continue to develop HIV-associated neurocognitive disorders despite combination anti-retroviral therapy. Lipocalin-2 (LCN2) is an acute phase protein that has been implicated in neurodegeneration and is upregulated in a transgenic mouse model of HIV-associated brain injury. Here we show that LCN2 is significantly upregulated in neocortex of a subset of HIV-infected individuals with brain pathology and correlates with viral load in CSF and pro-viral DNA in neocortex. However, the question if LCN2 contributes to HIV-associated neurotoxicity or is part of a protective host response required further investigation. We found that the knockout of LCN2 in transgenic mice expressing HIVgp120 in the brain (HIVgp120tg) abrogates behavioral impairment, ameliorates neuronal damage, and reduces microglial activation in association with an increase of the neuroprotective CCR5 ligand CCL4. In vitro experiments show that LCN2 neurotoxicity also depends on microglia and p38 MAPK activity. Genetic ablation of CCR5 in LCN2-deficient HIVgp120tg mice restores neuropathology, suggesting that LCN2 overrides neuroprotection mediated by CCR5 and its chemokine ligands. RNA expression of 168 genes involved in neurotransmission reveals that neuronal injury and protection are each associated with genotype- and sex-specific patterns affecting common neural gene networks. In conclusion, our study identifies LCN2 as a novel factor in HIV-associated brain injury involving CCR5, p38 MAPK and microglia. Furthermore, the mechanistic interaction between LCN2 and CCR5 may serve as a diagnostic and therapeutic target in HIV patients at risk of developing brain pathology and neurocognitive impairment.

Transgenic mice expressing HIV-1 envelope protein gp120 in the brain as an animal model in neuroAIDS research.

HIV-1 infection causes injury to the central nervous system (CNS) and is often associated with neurocognitive disorders. One model for brain damage seen in AIDS patients is the transgenic (tg) mouse expressing a soluble envelope protein gp120 of HIV-1 LAV in the brain in astrocytes under the control of the promoter of glial fibrillary acidic protein. These GFAP-gp120tg mice manifest several key neuropathological features observed in AIDS brains, such as decreased synaptic and dendritic density, increased numbers of activated microglia, and pronounced astrocytosis. Several recent studies show that brains of GFAP-gp120tg mice and neurocognitively impaired HIV patients share also a significant number of differentially regulated genes, activation of innate immunity and other cellular signaling pathways, disturbed neurogenesis, and learning deficits. These findings support the continued relevance of the GFAP-gp120tg mouse as a useful model to investigate neurodegenerative mechanisms and develop therapeutic strategies to mitigate the consequences associated with HIV infection of the CNS, neuroAIDS, and HAND.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+ channels upstream of p38 MAPK.

BACKGROUND: The chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined. METHODS: Primary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK. RESULTS: Here, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12. CONCLUSIONS: Our findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+ channels upstream of p38 MAPK activation.

CCR5 knockout prevents neuronal injury and behavioral impairment induced in a transgenic mouse model by a CXCR4-using HIV-1 glycoprotein 120.

The innate immune system has been implicated in several neurodegenerative diseases, including HIV-1-associated dementia. In this study, we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-using viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To characterize further the neuroprotective effect of CCR5 deficiency we performed a genome-wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and nontransgenic controls. A comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly upregulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion, whereas inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-using gp120. However, the combination of pharmacologic CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-using gp120, thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Our study provides evidence for an indirect pathologic role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury.

Immune, RNA, and Neurocognitive Genetic Networks in Bipolar Disorder Subtypes: A Transcriptomic Meta-Analysis.

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.

ACCT is a fast and accessible automatic cell counting tool using machine learning for 2D image segmentation.

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.

Mechanisms underlying HIV-associated cognitive impairment and emerging therapies for its management.

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.

HIV-1 gp120 protein promotes HAND through the calcineurin pathway activation.

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.

Activation of p38 MAPK is required in monocytic and neuronal cells for HIV glycoprotein 120-induced neurotoxicity.

HIV-1 envelope protein gp120 has been implicated in neurotoxin production by monocytic cells (i.e., macrophages and microglia), as well as in the pathogenesis of HIV-1-associated neurocognitive disorders. We previously showed in cerebrocortical cell cultures from rodents containing microglia, astrocytes, and neurons that overall inhibition of p38 MAPK signaling abrogated the neurotoxic effect of HIV-1 gp120. However, the time course of p38 MAPK activation and the contribution of this kinase in the various cell types remained unknown. In this study, we found that active p38 MAPK is required in monocytic lineage cells (i.e., macrophages and microglia) and neuronal cells for HIV gp120-induced neurotoxicity to occur. In cerebrocortical cell cultures, HIV-1 gp120 stimulated a time-dependent overall increase in active p38 MAPK, and the activated kinase was primarily detected in microglia and neurons. Interestingly, increased activation of p38 MAPK and neuronal death in response to gp120 were prevented by prior depletion of microglia or the presence of CCR5 ligand CCL4 or p38 MAPK inhibitors. In human monocytic THP-1 cells and primary monocyte-derived macrophages, HIV gp120-stimulated production of neurotoxins was abrogated by prior introduction into the cells of a dominant-negative p38 MAPK mutant or p38 MAPK small interfering RNA. In addition, the neurotoxic effects of cell-free supernatants from gp120-stimulated monocytic THP-1 cells were prevented in microglia-depleted cerebrocortical cells pretreated with a pharmacological inhibitor of p38 MAPK. Thus, p38 MAPK signaling was critical, upon exposure to HIV gp120, for the neurotoxic phenotype of monocytic cells and subsequent toxin-initiated neuronal apoptosis.

Interferon-stimulated gene 15 as a general marker for acute and chronic neuronal injuries.

Activation of interferon (IFN) signaling in the central nervous system (CNS) is usually associated with inflammation. However, a robust activation of type I IFN-stimulated genes (ISGs) at pre-symptomatic stages occurs in the spinal cord of SOD1(G93A) mice, an amyotrophic lateral sclerosis (ALS) animal model, without obvious signs of inflammation. To determine if the same signaling pathway is elevated in other types of neuronal injuries, we examined the protein expression levels of an IFN-stimulated gene, ISG15, in mouse models of acute and chronic neuronal injuries. We found that ISG15 protein was dramatically increased in the brains of mice subjected to global ischemia and traumatic brain injury, and in transgenic mice overexpressing HIV gp120 protein. These results suggest that activation of ISGs is a shared feature of neuronal injuries and that ISG15 may be a suitable biomarker for detecting neuronal injuries in the CNS.

Arachidonic Acid Cascade and Eicosanoid Production Are Elevated While LTC4 Synthase Modulates the Lipidomics Profile in the Brain of the HIVgp120-Transgenic Mouse Model of NeuroHIV.

BACKGROUND: Combination antiretroviral therapy (cART) has transformed HIV infection from a terminal disease to a manageable chronic health condition, extending patients' life expectancy to that of the general population. However, the incidence of HIV-associated neurocognitive disorders (HANDs) has persisted despite virological suppression. Patients with HIV display persistent signs of immune activation and inflammation despite cART. The arachidonic acid (AA) cascade is an important immune response system responsible for both pro- and anti-inflammatory processes. METHODS: Lipidomics, mRNA and Western blotting analysis provide valuable insights into the molecular mechanisms surrounding arachidonic acid metabolism and the resulting inflammation caused by perturbations thereof. RESULTS: Here, we report the presence of inflammatory eicosanoids in the brains of a transgenic mouse model of NeuroHIV that expresses soluble HIV-1 envelope glycoprotein in glial cells (HIVgp120tg mice). Additionally, we report that the effect of LTC4S knockout in HIVgp120tg mice resulted in the sexually dimorphic transcription of COX- and 5-LOX-related genes. Furthermore, the absence of LTC4S suppressed ERK1/2 and p38 MAPK signaling activity in female mice only. The mass spectrometry-based lipidomic profiling of these mice reveals beneficial alterations to lipids in the brain. CONCLUSION: Targeting the AA cascade may hold potential in the treatment of neuroinflammation observed in NeuroHIV and HANDs.

HIV-1 gp120 Impairs Spatial Memory Through Cyclic AMP Response Element-Binding Protein.

HIV-associated neurocognitive disorders (HAND) remain an unsolved problem that persists despite using antiretroviral therapy. We have obtained data showing that HIV-gp120 protein contributes to neurodegeneration through metabolic reprogramming. This led to decreased ATP levels, lower mitochondrial DNA copy numbers, and loss of mitochondria cristae, all-important for mitochondrial biogenesis. gp120 protein also disrupted mitochondrial movement and synaptic plasticity. Searching for the mechanisms involved, we found that gp120 alters the cyclic AMP response element-binding protein (CREB) phosphorylation on serine residue 133 necessary for its function as a transcription factor. Since CREB regulates the promoters of PGC1α and BDNF genes, we found that CREB dephosphorylation causes PGC1α and BDNF loss of functions. The data was validated in vitro and in vivo. The negative effect of gp120 was alleviated in cells and animals in the presence of rolipram, an inhibitor of phosphodiesterase protein 4 (PDE4), restoring CREB phosphorylation. We concluded that HIV-gp120 protein contributes to HAND via inhibition of CREB protein function.

Chemokines in cerebrospinal fluid correlate with cerebral metabolite patterns in HIV-infected individuals.

Chemokines influence HIV neuropathogenesis by affecting the HIV life cycle, trafficking of macrophages into the nervous system, glial activation, and neuronal signaling and repair processes; however, knowledge of their relationship to in vivo measures of cerebral injury is limited. The primary objective of this study was to determine the relationship between a panel of chemokines in cerebrospinal fluid (CSF) and cerebral metabolites measured by proton magnetic resonance spectroscopy (MRS) in a cohort of HIV-infected individuals. One hundred seventy-one stored CSF specimens were assayed from HIV-infected individuals who were enrolled in two ACTG studies that evaluated the relationship between neuropsychological performance and cerebral metabolites. Concentrations of six chemokines (fractalkine, IL-8, IP-10, MCP-1, MIP-1ß, and SDF-1) were measured and compared with cerebral metabolites individually and as composite neuronal, basal ganglia, and inflammatory patterns. IP-10 and MCP-1 were the chemokines most strongly associated with individual cerebral metabolites. Specifically, (1) higher IP-10 levels correlated with lower N-acetyl aspartate (NAA)/creatine (Cr) ratios in the frontal white matter and higher MI/Cr ratios in all three brain regions considered and (2) higher MCP-1 levels correlated with lower NAA/Cr ratios in frontal white matter and the parietal cortex. IP-10, MCP-1, and IL-8 had the strongest associations with patterns of cerebral metabolites. In particular, higher levels of IP-10 correlated with lower neuronal pattern scores and higher basal ganglia and inflammatory pattern scores, the same pattern which has been associated with HIV-associated neurocognitive disorders (HAND). Subgroup analysis indicated that the effects of IP-10 and IL-8 were influenced by effective antiretroviral therapy and that memantine treatment may mitigate the neuronal effects of IP-10. This study supports the role of chemokines in HAND and the validity of MRS as an assessment tool. In particular, the findings identify relationships between the immune response-particularly an interferon-inducible chemokine, IP-10-and cerebral metabolites and suggest that antiretroviral therapy and memantine modify the impact of the immune response on neurons.