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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.

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.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Alcohol abuse remains one of the primary preventable sources of mortality in the United States. Model species can be used to evaluate behavioral and other biological changes associated with alcohol and to identify novel treatments. This report describes methods for evaluating the behavioral effects of ethanol (EtOH) in crayfish. Crayfish (Procambarus clarkii) were immersed in ethanol concentrations ranging from 0.1 to 1.0 molar, for 10-30 min. Studies evaluated hemolymph alcohol concentration, locomotion in an open field and anxiety-like behavior using a Light/Dark transfer approach. EtOH immersion produced dose-dependent increases in hemolymph EtOH (up to 249 mg/dL) and reductions in open field locomotion that depended on EtOH concentration or exposure duration. Untreated crayfish exhibit avoidance of the open parts of the locomotor arena and a preference for a covered portion. Acute EtOH immersion decreased time spent in the covered portion of the Light/Dark arena, consistent with a decrease in anxiety-like behavior. Daily EtOH immersion for 5 days did not alter locomotor responses, however, activity was increased 3 days after the repeated EtOH regimen. Overall, this study shows that this inexpensive, easily maintained species can be used for behavioral pharmacological experiments designed to assess the acute and repeated effects of EtOH.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer's disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-β-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

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.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c +/-(Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention.

Treatment options for alcohol use disorders (AUDs) have minimally advanced since 2004, while the annual deaths and economic toll have increased alarmingly. Phosphodiesterase type 4 (PDE4) is associated with alcohol and nicotine dependence. PDE4 inhibitors were identified as a potential AUD treatment using a bioinformatics approach. We prioritized a newer PDE4 inhibitor, apremilast, as ideal for repurposing (i.e., FDA approved for psoriasis, low incidence of adverse events, excellent safety profile) and tested it using multiple animal strains and models, as well as in a human phase IIa study. We found that apremilast reduced binge-like alcohol intake and behavioral measures of alcohol motivation in mouse models of genetic risk for drinking to intoxication. Apremilast also reduced excessive alcohol drinking in models of stress- facilitated drinking and alcohol dependence. Using site-directed drug infusions and electrophysiology, we uncovered that apremilast may act to lessen drinking in mice by increasing neural activity in the nucleus accumbens, a key brain region in the regulation of alcohol intake. Importantly, apremilast (90 mg/d) reduced excessive drinking in non-treatment-seeking individuals with AUD in a double-blind, placebo-controlled study. These results demonstrate that apremilast suppresses excessive alcohol drinking across the spectrum of AUD severity., Introduction Alcohol use disorder (AUD) is a complex psychiatric disease with far reaching impacts on society, including more than 95,000 associated deaths annually in the United States and a net [...]

Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target.Significance statementToll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets.

Alcoholic liver disease (ALD) progresses from a normal liver, to steatosis, steatohepatitis, fibrosis and hepatocellular carcinoma (HCC). Despite intensive studies, the pathogenesis of ALD is poorly understood, in part due to a lack of suitable animal models which mimic the stages of ALD progression. Furthermore, the role of IL-17 in ALD has not been evaluated. We and others have recently demonstrated that IL-17 signaling plays a critical role in development of liver fibrosis and cancer. Here we summarize the most recent evidence supporting the role of IL-17 in ALD. As a result of a collaborative effort of Drs. Karin, Gao, Tsukamoto and Kisseleva, we developed several improved models of ALD in mice: 1) chronic-plus-binge model that mimics early stages of steatohepatitis, 2) intragastric ethanol feeding model that mimics alcoholic steatohepatitis and fibrosis, and 3) diethylnitrosamine (DEN)+alcohol model that mimics alcoholic liver cancer. These models might provide new insights into the mechanism of IL-17 signaling in ALD and help identify novel therapeutic targets.

Methamphetamine (METH) abuse is frequent in individuals infected with human immunodeficiency virus type-1 (HIV-1) and is suspected to aggravate HIV-associated neurocognitive disorders (HAND). METH is a psychostimulant that compromises several neurotransmitter systems and HIV proteins trigger neuronal injury but the combined effects of viral infection and METH abuse are incompletely understood. In this study we treated transgenic mice expressing the HIV envelope protein gp120 in the brain (HIV-1 gp120tg) at 3-4 months of age with an escalating-dose, multiple-binge METH regimen. The long-term effects were analyzed after 6-7 months of drug abstinence employing behavioral tests and analysis of neuropathology, electrophysiology and gene expression. Behavioral testing showed that both HIV-1 gp120tg and WT animals treated with METH displayed impaired learning and memory. Neuropathological analysis revealed that METH similar to HIV-1 gp120 caused a significant loss of neuronal dendrites and pre-synaptic terminals in hippocampus and cerebral cortex of WT animals. Electrophysiological studies in hippocampal slices showed that METH exposed HIV-1 gp120tg animals displayed reduced post-tetanic potentiation, whereas both gp120 expression and METH lead to reduced long-term potentiation. A quantitative reverse transcription-polymerase chain reaction array showed that gp120 expression, METH and their combination each caused a significant dysregulation of specific components of GABAergic and glutamatergic neurotransmission systems, providing a possible mechanism for synaptic dysfunction and behavioral impairment. In conclusion, both HIV-1 gp120 and METH caused lasting behavioral impairment in association with neuropathology and altered gene expression. However, combined METH exposure and HIV-1 gp120 expression resulted in the most pronounced, long lasting pre- and post-synaptic alterations coinciding with impaired learning and memory.

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.

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

This article describes the integration of neurobiological and molecular genetic approaches in the study of the function of the neuropeptide corticotropin-releasing factor (CRF). CRF is a particularly relevant subject for targeted genetic mutation because of its hypothesized critical role in hormonal and behavioral responses to stressors, its potential role in psychopathology, and the limited neuropharmacological agents available to increase or decrease function in this system. We review the strategy of targeted genetic mutation in neuropharmacology using research directed at understanding the function of CRF and related neuropeptides through molecular genetic manipulation of the levels of the endogenous agonist, its different receptors, and the binding protein. Genetically engineered approaches to functional analysis cross-validate experiments to promote the neurobiological elucidation of complex systems and provide critical information about constituent differences that contribute to individual differences in brain function.