Unique molecular features and cellular responses differentiate two populations of motor cortical layer 5b neurons in a preclinical model of ALS.

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease.

Cannabinoids Reduce Extracellular Vesicle Release from HIV-1 Infected Myeloid Cells and Inhibit Viral Transcription.

Of the 37.9 million individuals infected with human immunodeficiency virus type 1 (HIV-1), approximately 50% exhibit HIV-associated neurocognitive disorders (HAND). We and others previously showed that HIV-1 viral RNAs, such as trans-activating response (TAR) RNA, are incorporated into extracellular vesicles (EVs) and elicit an inflammatory response in recipient naïve cells. Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the primary cannabinoids present in cannabis, are effective in reducing inflammation. Studies show that cannabis use in people living with HIV-1 is associated with lower viral load, lower circulating CD16+ monocytes and high CD4+ T-cell counts, suggesting a potentially therapeutic application. Here, HIV-1 infected U1 monocytes and primary macrophages were used to assess the effects of CBD. Post-CBD treatment, EV concentrations were analyzed using nanoparticle tracking analysis. Changes in intracellular and EV-associated viral RNA were quantified using RT-qPCR, and changes in viral proteins, EV markers, and autophagy proteins were assessed by Western blot. Our data suggest that CBD significantly reduces the number of EVs released from infected cells and that this may be mediated by reducing viral transcription and autophagy activation. Therefore, CBD may exert a protective effect by alleviating the pathogenic effects of EVs in HIV-1 and CNS-related infections.

Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells.

HIV-1 Tat is known to be released by HIV infected non-neuronal cells in the brain, and after entering neurons, compromises brain homeostasis by impairing pro-survival pathways, thus contributing to the development of HIV-associated CNS disorders commonly observed in individuals living with HIV. Here, we demonstrate that synapsins, phosphoproteins that are predominantly expressed in neuronal cells and play a vital role in modulating neurotransmitter release at the pre-synaptic terminal, and neuronal differentiation become targets for Tat through autophagy and protein quality control pathways. We demonstrate that the presence of Tat in neurons results in downregulation of BAG3, a co-chaperone for heat shock proteins (Hsp70/Hsc70) that is implicated in protein quality control (PQC) processes by eliminating mis-folded and damaged proteins, and selective macroautophagy. Our results show that treatment of cells with Tat or suppression of BAG3 expression by siRNA in neuronal cells disturbs subcellular distribution of synapsins and synaptotagmin 1 (Syt1) leading to their accumulation in the neuronal soma and along axons in a punctate pattern, rather than being properly distributed at axon-terminals. Further, our results revealed that synapsins partially lost their stability and their removal via lysosomal autophagy was noticeably impaired in cells with low levels of BAG3. The observed impairment of lysosomal autophagy, under this condition, is likely caused by cells losing their ability to process LC3-I to LC3-II, in part due to a decrease in the ATG5 levels upon BAG3 knockdown. These observations ascribe a new function for BAG3 in controlling synaptic communications and illuminate a new downstream target for Tat to elicit its pathogenic effect in impacting neuronal cell function and behavior.

HIV Infection Induces Extracellular Cathepsin B Uptake and Damage to Neurons.

HIV-associated neurocognitive disorders prevail in 20-50 percent of infected individuals. Macrophages transmigrate through the blood brain barrier during HIV-1 infection, triggering neuronal dysfunction. HIV-infected macrophages secrete cathepsin B (CATB), and serum amyloid p component (SAPC), inducing neuronal apoptosis by an unknown mechanism. We hypothesized that HIV infection facilitates CATB/SAPC secretion from macrophages followed by neuronal internalization, promoting dysfunction. SK-N-SH neuronal cells were exposed to active recombinant histidine-tagged cathepsin B (His-CATB). His-CATB entry was tracked by intracellular flow cytometry, and neuronal dysfunction was verified by western blot. Macrophage-derived extracellular vesicles (EVs) were tested for the presence of CATB and SAPC. Neurons internalized His-CATB, an effect that was partially decreased by pre-treatment with anti-CATB antibody. Pre-treatment with CATB and SAPC antibodies decreased cleavage of caspase-3 and restored synaptophysin in neurons. Neurons exposed to macrophage-conditioned media differentially internalized His-CATB, dependent on the HIV replication levels. Finally, CATB and SAPC were secreted in EVs. We report for the first time that CATB is secreted from macrophages both free and in EVs, and is internalized by neurons. Moreover, HIV-replication levels modulate the amount of CATB neuronal uptake, and neuronal dysfunction can be decreased with CATB antibodies. In conclusion, the CATB/SAPC complex represents a novel target against HIV-associated neurocognitive disorders.

HIV-1 infection alters energy metabolism in the brain: Contributions to HIV-associated neurocognitive disorders.

The brain is particularly sensitive to changes in energy supply. Defects in glucose utilization and mitochondrial dysfunction are hallmarks of nearly all neurodegenerative diseases and are also associated with the cognitive decline that occurs as the brain ages. Chronic neuroinflammation driven by glial activation is commonly implicated as a contributing factor to neurodegeneration and cognitive impairment. Human immunodeficiency virus-1 (HIV-1) disrupts normal brain homeostasis and leads to a spectrum of HIV-associated neurocognitive disorders (HAND). HIV-1 activates stress responses in the brain and triggers a state of chronic neuroinflammation. Growing evidence suggests that inflammatory processes and bioenergetics are interconnected in the propagation of neuronal dysfunction. Clinical studies of people living with HIV and basic research support the notion that HIV-1 creates an environment in the CNS that interrupts normal metabolic processes at the cellular level to collectively alter whole brain metabolism. In this review, we highlight reports of abnormal brain metabolism from clinical studies and animal models of HIV-1. We also describe diverse CNS cell-specific changes in bioenergetics associated with HIV-1. Moreover, we propose that attention should be given to adjunctive therapies that combat sources of metabolic dysfunction as a mean to improve and/or prevent neurocognitive impairments.

Astrocyte activation and altered metabolism in normal aging, age-related CNS diseases, and HAND.

Astrocytes regulate local cerebral blood flow, maintain ion and neurotransmitter homeostasis, provide metabolic support, regulate synaptic activity, and respond to brain injury, insults, and infection. Because of their abundance, extensive connectivity, and multiple roles in the brain, astrocytes are intimately involved in normal functioning of the CNS and their dysregulation can lead to neuronal dysfunction. In normal aging, decreased biological functioning and reduced cognitive abilities are commonly experienced in individuals free of overt neurological disease. Moreover, in several age-related CNS diseases, chronic inflammation and altered metabolism have been reported. Since people with HIV (PWH) are reported to experience rapid aging with chronic inflammation, altered brain metabolism is likely to be exacerbated. In fact, many studies report altered metabolism in astrocytes in diseases such as Alzheimer's, Parkinson's, and HIV. This review will address the roles of astrocyte activation and altered metabolism in normal aging, in age-related CNS disease, and in HIV-associated neurocognitive disorders.

Cocaine-mediated activation of microglia and microglial MeCP2 and BDNF production.

The molecular substrates underlying cocaine reinforcement and addiction have been studied for decades, with a primary focus on signaling molecules involved in modulation of neuronal communication. Brain-derived neurotrophic factor (BDNF) is an important signaling molecule involved in neuronal dendrite and spine modulation. Methyl CpG binding protein 2 (MeCP2) binds to the promoter region of BDNF to negatively regulate its expression and cocaine can recruit MeCP2 to alter the expression of genes such as BDNF that are involved in synaptic plasticity. For several decades, BDNF has been implicated in mediating synaptic plasticity associated with cocaine abuse, and most studies report that neurons are the primary source for BDNF production in the brain. The current study assessed the effects of intravenous cocaine self-administration on microglial activation, and MeCP2 and BDNF expression in reward regions of the brain in vivo, as well as determined specific effects of cocaine exposure on MeCP2 and BDNF expression in human primary neurons and microglia. The results from this study highlight a distinct molecular pathway in microglia through which cocaine increases BDNF, including the phosphorylation of MeCP2 its subsequent translocation from the nucleus to the cytosol, which frees the BDNF promoter and permits its transcriptional activation. Results from these studies show for the first time that cocaine self-administration increases microglial activation, and that microglial MeCP2 is a sensitive target of cocaine resulting in increased release of BDNF from microglia, and possibly contributing to cocaine-induced synaptic plasticity.

Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection.

The emergence of >300 serovars of Leptospira confounded the use of generalized bacterin, the whole cell lysate, as vaccines to control leptospirosis. Because of substantial genetic and geographic heterogeneity among circulating serovars, one vaccine strain per serovar cannot be efficacious against all the serovars. We have performed heterologous DNA prime-protein boost vaccination challenge studies in hamsters using in vivo expressed, leptospiral recombinase A (RecA) and flagellar hook associated protein (FliD). We prepared the monovalent recombinant protein, plasmid DNA, and DNA prime protein boost adjuvant vaccines. The whole cell bacterin served as a control. Our data show that (i) RecA and FliD have multiple immunogenic B and T-cell epitopes with highly conserved domains among most prevalent pathogenic Leptospira spp., (ii) humoral and cell mediated immune responses were induced remarkably, (iii) provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infection for the heterologous prime-protein boost (∼91-100%) and, the DNA vaccine (∼75-83%). Recombinant protein vaccine shows only partial protection (∼58-66%), (iv) RecA prime-protein boost vaccine shows sterilizing immunity, with heterologous protection. This RecA/FliD prime-protein boost strategy holds potential for vaccination against animal leptospirosis and for a better control of zoonotic transmission.

Astrocytic metabolic switch is a novel etiology for Cocaine and HIV-1 Tat-mediated neurotoxicity.

Calcium (Ca2+) dynamics and oxidative signaling control mitochondrial bioenergetics in the central nervous system, where astrocytes are a major energy source for neurons. Cocaine use exacerbates HIV-associated neurocognitive disorders, but little is known about disruptions in astrocyte metabolism in this context. Our data show that the HIV protein Tat and cocaine induce a metabolic switch from glucose to fatty acid oxidation in astrocytes, thereby limiting lactate transport to neurons. Mechanistic analyses revealed increased Mitochondrial Ca2+ Uniporter (MCU)-mediated Ca2+ uptake in astrocytes exposed to Tat and cocaine due to oxidation of MCU. Since our data suggest that mitochondrial oxidation is dependent in part on MCU-mediated Ca2+ uptake, we targeted MCU to restore glycolysis in astrocytes to normalize extracellular lactate levels. Knocking down MCU in astrocytes prior to Tat and cocaine exposure prevented metabolic switching and protected neurons. These findings identify a novel molecular mechanism underlying neuropathogenesis in HIV and cocaine use.

Cocaine and HIV-1 Tat disrupt cholesterol homeostasis in astrocytes: Implications for HIV-associated neurocognitive disorders in cocaine user patients.

Cholesterol synthesis and clearance by astrocytes are tightly regulated to maintain constant levels within the brain. In this context, liver X receptors (LXRs) are the master regulators of cholesterol homeostasis in the central nervous system (CNS). Increasing levels of cholesterol in astrocytes trigger LXR activation leading to the transcription of target genes involved in cholesterol trafficking and efflux, including apolipoprotein E, cytochrome P450 enzymes, sterol regulatory binding protein, and several ATP-binding cassette transporter proteins. The disturbance of LXR signaling in the brain can lead to significant dysfunctions in cholesterol homeostasis, and disruptions in this pathway have been implicated in numerous neurological diseases including Alzheimer's disease and Huntington's disease. HIV infection of the CNS in combination with cocaine use is associated with astrocyte and neuronal energy deficit and damage. We propose that dysregulation in CNS cholesterol metabolism may be involved in the progression of HIV-associated neurocognitive disorders (HAND) and in cocaine-mediated neurocognitive impairments. We hypothesize that exposure of astrocytes to cocaine and the HIV protein Tat will disrupt LXR signaling. Alterations in these pathways will in turn, affect cholesterol bioavailability for neurons. Our data show that exposure of astrocytes to cocaine and HIV-Tat significantly decreases LXRß levels, downstream signaling and bioavailability of cholesterol. Taken together, these data uncover novel alterations in a bioenergetic pathway in astrocytes exposed to cocaine and the HIV protein Tat. Results from these studies point to a new pathway in the CNS that may contribute to HAND in HIV+ cocaine user individuals.

Expression of Signaling Molecules in Progressive Multifocal Leukoencephalopathy.

INTRODUCTION: Progressive multifocal leukoencephalopathy (PML) is a debilitating demyelinating disease of the CNS caused by the infection and destruction of glial cells by JC virus (JCV) and is an AIDS-defining disease. Infection with JCV is common and most people acquire antibodies early in life. After initial infection, JCV remains in an asymptomatic persistent state and can be detected by PCR in many tissues including brain. A major question in PML pathogenesis is how the virus reactivates from persistence in HIV-1/AIDS. Our studies with primary cultures of glial cells have implicated transcription factors NF-κB and NFAT4, which bind to a unique site in the JCV noncoding control region and stimulate viral gene expression. Furthermore, these transcription factors are controlled by pathways downstream of proinflammatory cytokines, e.g., TNF-α activates NF-κB and stimulates JCV transcription. OBJECTIVES: We hypothesize that HIV-1/PML initiation may involve reactivation of JCV by cytokine disturbances in the brain such as occur in HIV-1/AIDS. In this study, the objective was to evaluate HIV-1/PML clinical samples for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 compared to non-PML controls. METHODS: We evaluated HIV-1/PML clinical samples and non-PML controls for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 using Western blot and immunohistochemistry. RESULTS: Consistent with our hypothesis, compared to non-PML controls, HIV-1/PML tissue has high levels of TNF-α and TNFR1 expression and NF-κB and NFAT4 were preferentially localized to the nucleus. CONCLUSION: The involvement of TNF-α/NF-κB/NFAT4 signaling in JCV regulation that we reported from experiments in cultured human glial cells may be clinically relevant in PML.