HIV-1 Tat-mediated microglial ferroptosis involves the miR-204-ACSL4 signaling axis.

This study was focused on exploring the role of the HIV-1 Tat protein in mediating microglial ferroptosis. Exposure of mouse primary microglial cells (mPMs) to HIV-1 Tat protein resulted in induction of ferroptosis, which was characterized by increased expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4), in turn, leading to increased generation of oxidized phosphatidylethanolamine, elevated levels of lipid peroxidation, upregulated labile iron pool (LIP) and ferritin heavy chain-1 (FTH1), decreased glutathione peroxidase-4 and mitochondrial outer membrane rupture. Also, inhibition of ferroptosis by ferrostatin-1 (Fer-1) or deferoxamine (DFO) treatment suppressed ferroptosis-related changes in mPMs. Similarly, the knockdown of ACSL4 by gene silencing also inhibited ferroptosis induced by HIV-1 Tat. Furthermore, increased lipid peroxidation resulted in increased release of proinflammatory cytokines, such as TNFα, IL6, and IL1ß and microglial activation. Pretreatment of mPMs with Fer-1 or DFO further blocked HIV-1 Tat-mediated microglial activation in vitro and reduced the expression and release of proinflammatory cytokines. We identified miR-204 as an upstream modulator of ACSL4, which was downregulated in mPMs exposed to HIV-1 Tat. Transient transfection of mPMs with miR-204 mimics reduced the expression of ACSL4 while inhibiting HIV-1 Tat-mediated ferroptosis and the release of proinflammatory cytokines. These in vitro findings were further validated in HIV-1 transgenic rats as well as HIV + ve human brain samples. Overall, this study underscores a novel mechanism(s) underlying HIV-1 Tat-mediated ferroptosis and microglial activation involving miR-204-ACSL4 signaling.

Involvement of lncRNA TUG1 in HIV-1 Tat-Induced Astrocyte Senescence.

HIV-1 infection in the era of combined antiretroviral therapy has been associated with premature aging. Among the various features of HIV-1 associated neurocognitive disorders, astrocyte senescence has been surmised as a potential cause contributing to HIV-1-induced brain aging and neurocognitive impairments. Recently, lncRNAs have also been implicated to play essential roles in the onset of cellular senescence. Herein, using human primary astrocytes (HPAs), we investigated the role of lncRNA TUG1 in HIV-1 Tat-mediated onset of astrocyte senescence. We found that HPAs exposed to HIV-1 Tat resulted in significant upregulation of lncRNA TUG1 expression that was accompanied by elevated expression of p16 and p21, respectively. Additionally, HIV-1 Tat-exposed HPAs demonstrated increased expression of senescence-associated (SA) markers-SA-ß-galactosidase (SA-ß-gal) activity and SA-heterochromatin foci-cell-cycle arrest, and increased production of reactive oxygen species and proinflammatory cytokines. Intriguingly, gene silencing of lncRNA TUG1 in HPAs also reversed HIV-1 Tat-induced upregulation of p21, p16, SA-ß gal activity, cellular activation, and proinflammatory cytokines. Furthermore, increased expression of astrocytic p16 and p21, lncRNA TUG1, and proinflammatory cytokines were observed in the prefrontal cortices of HIV-1 transgenic rats, thereby suggesting the occurrence of senescence activation in vivo. Overall, our data indicate that HIV-1 Tat-induced astrocyte senescence involves the lncRNA TUG1 and could serve as a potential therapeutic target for dampening accelerated aging associated with HIV-1/HIV-1 proteins.

Role of the gut-brain axis in HIV and drug abuse-mediated neuroinflammation.

Drug abuse and related disorders are a global public health crisis affecting millions, but to date, limited treatment options are available. Abused drugs include but are not limited to opioids, cocaine, nicotine, methamphetamine, and alcohol. Drug abuse and human immunodeficiency virus-1/acquired immune deficiency syndrome (HIV-1/AIDS) are inextricably linked. Extensive research has been done to understand the effect of prolonged drug use on neuronal signaling networks and gut microbiota. Recently, there has been rising interest in exploring the interactions between the central nervous system and the gut microbiome. This review summarizes the existing research that points toward the potential role of the gut microbiome in the pathogenesis of HIV-1-linked drug abuse and subsequent neuroinflammation and neurodegenerative disorders. Preclinical data about gut dysbiosis as a consequence of drug abuse in the context of HIV-1 has been discussed in detail, along with its implications in various neurodegenerative disorders. Understanding this interplay will help elucidate the etiology and progression of drug abuse-induced neurodegenerative disorders. This will consequently be beneficial in developing possible interventions and therapeutic options for these drug abuse-related disorders.

The Epigenetic Role of miR-124 in HIV-1 Tat- and Cocaine-Mediated Microglial Activation.

HIV-1 and drug abuse have been indissolubly allied as entwined epidemics. It is well-known that drug abuse can hasten the progression of HIV-1 and its consequences, especially in the brain, causing neuroinflammation. This study reports the combined effects of HIV-1 Transactivator of Transcription (Tat) protein and cocaine on miR-124 promoter DNA methylation and its role in microglial activation and neuroinflammation. The exposure of mouse primary microglial cells to HIV-1 Tat (25 ng/mL) and/or cocaine (10 µM) resulted in the significantly decreased expression of primary (pri)-miR-124-1, pri-miR-124-2, and mature miR-124 with a concomitant upregulation in DNMT1 expression as well as global DNA methylation. Our bisulfite-converted genomic DNA sequencing also revealed significant promoter DNA methylation in the pri-miR-124-1 and pri-miR-124-2 in HIV-1 Tat- and cocaine-exposed mouse primary microglial cells. We also found the increased expression of proinflammatory cytokines such as IL1ß, IL6 and TNF in the mouse primary microglia exposed to HIV-1 Tat and cocaine correlated with microglial activation. Overall, our findings demonstrate that the exposure of mouse primary microglia to both HIV-1 Tat and cocaine could result in intensified microglial activation via the promoter DNA hypermethylation of miR-124, leading to the exacerbated release of proinflammatory cytokines, ultimately culminating in neuroinflammation.

HIV-1 Tat induced microglial EVs leads to neuronal synaptodendritic injury: microglia-neuron cross-talk in NeuroHIV.

Aim: Activation of microglial NLRP3 inflammasome is an essential contributor to neuroinflammation underlying HIV-associated neurological disorders (HAND). Under pathological conditions, microglia-derived-EVs (MDEVs) can affect neuronal functions by delivering neurotoxic mediators to recipient cells. However, the role of microglial NLRP3 in mediating neuronal synaptodendritic injury has remained unexplored to date. In the present study, we sought to assess the regulatory role of HIV-1 Tat induced microglial NLRP3 in neuronal synaptodendritic injury. We hypothesized that HIV-1 Tat mediated microglia EVs carrying significant levels of NLRP3 contribute to the synaptodendritic injury, thereby affecting the maturation of neurons. Methods: To understand the cross-talk between microglia and neuron, we isolated EVs from BV2 and human primary microglia (HPM) cells with or without NLRP3 depletion using siNLRP3 RNA. EVs were isolated by differential centrifugation, characterized by ZetaView nanoparticle tracking analysis, electron microscopy, and western blot analysis for exosome markers. Purified EVs were exposed to primary rat neurons isolated from E18 rats. Along with green fluorescent protein (GFP) plasmid transfection, immunocytochemistry was performed to visualize neuronal synaptodendritic injury. Western blotting was employed to measure siRNA transfection efficiency and the extent of neuronal synaptodegeneration. Images were captured in confocal microscopy, and subsequently, Sholl analysis was performed for analyzing dendritic spines using neuronal reconstruction software Neurolucida 360. Electrophysiology was performed on hippocampal neurons for functional assessment. Results: Our findings demonstrated that HIV-1 Tat induced expression of microglial NLRP3 and IL1ß, and further that these were packaged in microglial exosomes (MDEV) and were also taken up by the neurons. Exposure of rat primary neurons to microglial Tat-MDEVs resulted in downregulation of synaptic proteins- PSD95, synaptophysin, excitatory vGLUT1, as well as upregulation of inhibitory proteins- Gephyrin, GAD65, thereby implicating impaired neuronal transmissibility. Our findings also showed that Tat-MDEVs not only caused loss of dendritic spines but also affected numbers of spine sub-types- mushroom and stubby. Synaptodendritic injury further affected functional impairment as evidenced by the decrease in miniature excitatory postsynaptic currents (mEPSCs). To assess the regulatory role of NLRP3 in this process, neurons were also exposed to Tat-MDEVs from NLRP3 silenced microglia. Tat-MDEVs from NLRP3 silenced microglia exerted a protective role on neuronal synaptic proteins, spine density as well as mEPSCs. Conclusion: In summary, our study underscores the role of microglial NLRP3 as an important contributor to Tat-MDEV mediated synaptodendritic injury. While the role of NLRP3 in inflammation is well-described, its role in EV-mediated neuronal damage is an interesting finding, implicating it as a target for therapeutics in HAND.

Alzheimer's-Like Pathology at the Crossroads of HIV-Associated Neurological Disorders.

Despite the widespread success of combined antiretroviral therapy (cART) in suppressing viremia, the prevalence of human immunodeficiency virus (HIV)-associated neurological disorders (HAND) and associated comorbidities such as Alzheimer's disease (AD)-like symptomatology is higher among people living with HIV. The pathophysiology of observed deficits in HAND is well understood. However, it has been suggested that it is exacerbated by aging. Epidemiological studies have suggested comparable concentrations of the toxic amyloid protein, amyloid-ß42 (Aß42), in the cerebrospinal fluid (CSF) of HAND patients and in the brains of patients with dementia of the Alzheimer's type. Apart from abnormal amyloid-ß (Aß) metabolism in AD, a better understanding of the role of similar pathophysiologic processes in HAND could be of substantial value. The pathogenesis of HAND involves either the direct effects of the virus or the effect of viral proteins, such as Tat, Gp120, or Nef, as well as the effects of antiretrovirals on amyloid metabolism and tauopathy, leading, in turn, to synaptodendritic alterations and neuroinflammatory milieu in the brain. Additionally, there is a lack of knowledge regarding the causative or bystander role of Alzheimer's-like pathology in HAND, which is a barrier to the development of therapeutics for HAND. This review attempts to highlight the cause-effect relationship of Alzheimer's-like pathology with HAND, attempting to dissect the role of HIV-1, HIV viral proteins, and antiretrovirals in patient samples, animal models, and cell culture model systems. Biomarkers associated with Alzheimer's-like pathology can serve as a tool to assess the neuronal injury in the brain and the associated cognitive deficits. Understanding the factors contributing to the AD-like pathology associated with HAND could set the stage for the future development of therapeutics aimed at abrogating the disease process.

HIV-1 and drug abuse comorbidity: Lessons learned from the animal models of NeuroHIV.

Various research studies that have investigated the association between HIV infection and addiction underpin the role of various drugs of abuse in impairing immunological and non-immunological pathways of the host system, ultimately leading to augmentation of HIV infection and disease progression. These studies have included both in vitro and in vivo animal models wherein investigators have assessed the effects of various drugs on several disease parameters to decipher the impact of drugs on both HIV infection and progression of HIV-associated neurocognitive disorders (HAND). However, given the inherent limitations in the existing animal models of HAND, these investigations only recapitulated specific aspects of the disease but not the complex human syndrome. Despite the inability of HIV to infect rodents over the last 30 years, multiple strategies have been employed to develop several rodent models of HAND. While none of these models can accurately mimic the overall pathophysiology of HAND, they serve the purpose of modeling some unique aspects of HAND. This review provides an overview of various animal models used in the field and a careful evaluation of methodological strengths and limitations inherent in both the model systems and study designs to understand better how the various animal models complement one another.

Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity.

Serine palmitoyltranferase (SPT) is a pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.

FSH3 mediated cell death is dependent on NUC1 in Saccharomyces cerevisiae.

Family of Serine Hydrolases (FSH) members FSH1, FSH2 and FSH3 in Saccharomyces cerevisiae share conserved sequences with the human candidate tumor suppressor OVCA2. In this study, hydrogen peroxide (H2O2) exposure increased the expression of both mRNA and protein levels of FSH3 in wild-type (WT) yeast cells. The deletion of FSH3 improved the yeast growth rate under H2O2-induction as compared to WT control cells. The overexpression of FSH3 in WT yeast cells caused an apoptotic phenotype, including accumulation of reaction oxygen species, decreased cell viability and cell death. The double deletions fsh1Δ fsh2Δ, fsh1Δ fsh3Δ and fsh2Δ fsh3Δ displayed increased growth compared to WT cells. However, the overexpression of FSH3 effectively inhibited cell growth in all double deletions. Moreover, the overexpression of FSH3 in cells lacking NUC1 did not cause any growth defect in the presence or absence of H2O2. Our results suggest that FSH3 induced apoptosis of yeast in a NUC1 dependent manner.

Orm/ORMDL proteins: Gate guardians and master regulators.

Sphingolipids comprise a diverse family of lipids that perform multiple functions in both structure of cellular membranes and intra- and inter-cellular signaling. The diversity of this family is generated by an array of enzymes that produce individual classes and molecular species of family members and enzymes which catabolize those lipids for recycling pathways. However, all of these lipids begin their lives with a single step, the condensation of an amino acid, almost always serine, and a fatty acyl-CoA, almost always the 16-carbon, saturated fatty acid, palmitate. The enzyme complex that accomplishes this condensation is serine palmitoyltransferase (SPT), a membrane-bound component of the endoplasmic reticulum. This places SPT in the unique position of regulating the production of the entire sphingolipid pool. Understanding how SPT activity is regulated is currently a central focus in the field of sphingolipid biology. In this review we examine the regulation of SPT activity by a set of small, membrane-bound proteins of the endoplasmic reticulum, the Orms (in yeast) and ORMDLs (in vertebrates). We discuss what is known about how these proteins act as homeostatic regulators by monitoring cellular levels of sphingolipid, but also how the Orms/ORMDLs regulate SPT in response to other stimuli. Finally, we discuss the intriguing connection between one of the mammalian ORMDL isoforms, ORMDL3, and the pervasive pulmonary disease, asthma, in humans.

Lipid Homeostasis Is Maintained by Dual Targeting of the Mitochondrial PE Biosynthesis Enzyme to the ER.

Spatial organization of phospholipid synthesis in eukaryotes is critical for cellular homeostasis. The synthesis of phosphatidylcholine (PC), the most abundant cellular phospholipid, occurs redundantly via the ER-localized Kennedy pathway and a pathway that traverses the ER and mitochondria via membrane contact sites. The basis of the ER-mitochondrial PC synthesis pathway is the exclusive mitochondrial localization of a key pathway enzyme, phosphatidylserine decarboxylase Psd1, which generates phosphatidylethanolamine (PE). We find that Psd1 is localized to both mitochondria and the ER. Our data indicate that Psd1-dependent PE made at mitochondria and the ER has separable cellular functions. In addition, the relative organellar localization of Psd1 is dynamically modulated based on metabolic needs. These data reveal a critical role for ER-localized Psd1 in cellular phospholipid homeostasis, question the significance of an ER-mitochondrial PC synthesis pathway to cellular phospholipid homeostasis, and establish the importance of fine spatial regulation of lipid biosynthesis for cellular functions.

Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER.

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

Endoplasmic reticulum stress affects the transport of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum in S.cerevisiae.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two of the most abundant phospholipids in cells. Although both lipids can be synthesized in the endoplasmic reticulum (ER), in S. cerevisiae PE can also be produced in mitochondria and endosomes; this PE can be transported back to the ER where it is converted to PC. In this study we found that dithiothreitol (DTT), which induces ER stress, decreases PE export from mitochondria to the ER. This results in decreased levels of total cellular PC and mitochondrial PC. These decreases were not caused by changes in levels of PC synthesizing or degrading enzymes. PE export from mitochondria to the ER during ER stress was further reduced in cells lacking Mdm10p, a component of an ER-mitochondrial tethering complex that may facilitated lipid exchange between these compartments. We also found that reducing mitochondrial PC levels induces mitophagy. In conclusion, we show that ER stress affected PE export from mitochondria to ER and the Mdm10p is important for this process.

Transport of phosphatidylserine from the endoplasmic reticulum to the site of phosphatidylserine decarboxylase2 in yeast.

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.