HIV Vpr induces demethylation of the SNCA antisense promoter leading to neurocognitive impairment

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Kirby, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6405901/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Alpha-synuclein (α-Syn) aggregation is a hallmark of neurodegenerative diseases. In individuals with HIV-1, cognitive impairments are associated with α-Syn accumulation and aggregation. The direct mechanistic link between α-Syn dysregulation and HIV-associated neurocognitive disorders (HAND) remains unclear. Emerging evidence suggests epigenetic changes, particularly DNA demethylation, play a role in α-Syn regulation. We show that the HIV-1 protein Vpr demethylates the antisense promoter (AS-1) within intron 1 of the SNCA gene, leading to increased α-Syn expression. Elevated α-Syn levels promote its aggregation, resulting in synaptic dysfunction and impaired mitochondrial transport. These processes contribute to the development of HAND. Additionally, we find that Vpr's activation of AS-1 depends on demethylation; DMOG, a Tet inhibitor, reverses this demethylation and reduces Vpr-induced activation of AS-1. Our results indicate that α-Syn dysregulation contributes to cognitive decline in people living with HIV and imply that targeting α-Syn regulatory pathways could mitigate HIV-related neurodegeneration. To our knowledge, this is the first study to demonstrate that an HIV protein epigenetically activates the SNCA antisense promoter, linking viral infection to α-synuclein deregulation. Future research should explore how AS-1 demethylation causes neuronal dysfunction and examine the broader effects of α-Syn dysregulation on neuronal health. Biological sciences/Neuroscience Biological sciences/Neuroscience/Diseases of the nervous system Biological sciences/Neuroscience/Epigenetics in the nervous system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION HIV-associated neurocognitive disorders (HAND) continue to present a significant clinical challenge despite the widespread use of combination antiretroviral therapy (cART). HAND encompasses a spectrum of cognitive, motor, and behavioral impairments that arise from persistent inflammation and neurodegeneration due to HIV infection in the brain [ 1 ]. A subset of individuals with HAND exhibit Parkinson-like motor symptoms, including tremors, bradykinesia, rigidity, and postural instability, which closely resemble features observed in Parkinson’s disease (PD) [ 2 – 7 ]. Four interrelated mechanistic pathways drive HAND-related neurodegeneration. First, HIV infects glial cells, resulting in neuronal damage within motor control regions such as the basal ganglia and promoting local chronic inflammation. Second, persistent immune activation induces widespread chronic inflammation, even in the presence of cART. Third, toxic viral proteins, including HIV-1 Tat and Vpr, disrupt cellular homeostasis and exacerbate neurotoxicity, partly by modulating inflammatory processes. Fourth, these viral proteins facilitate the accumulation of alpha-synuclein (α-Syn), thereby linking HAND-related degeneration to Parkinson-like symptoms through neuronal dysfunction [ 8 ]. While cART alleviates some symptoms, neurodegeneration frequently persists, underscoring the necessity of targeting these interconnected mechanisms beyond viral suppression [ 9 , 10 ]. HIV-1 viral protein R (Vpr) is a 15-kDa multifunctional protein that facilitates viral nuclear import, induces G2-phase arrest, and modulates the HIV-1 promoter [ 11 ]. Vpr also interacts with host epigenetic machinery and promotes DNA demethylation [ 12 , 13 ]. Previous findings have demonstrated Vpr's effects on cellular pathways; however, the mechanisms by which Vpr interacts with host epigenetic machinery to regulate SNCA expression remain poorly understood. The direct relationship between Vpr-induced epigenetic changes, α-Syn accumulation, and their impact on neuronal function represents a critical knowledge gap. The present study addresses this gap. Recent data indicate that Vpr reactivates the SNCA gene's antisense promoter in intron 1, leading to increased α-Syn transcription. This accumulation alters lysosomal pH, impairs autophagy, and promotes α-Syn aggregation, thereby contributing to neuronal dysfunction and HAND pathogenesis [ 14 , 15 ]. Alpha-synuclein (α-Syn) is a 14-kDa protein localized at presynaptic terminals and is essential for neurotransmitter release and synaptic plasticity [ 16 , 17 ]. Genetic and epigenetic alterations, such as SNCA antisense promoter demethylation, increase α-Syn expression and promote its aggregation [ 18 , 19 ]. Aggregated α-Syn disrupts mitochondrial and lysosomal function, impairs synaptic communication, and elevates oxidative stress, ultimately resulting in neuronal loss. Additionally, α-Syn activates microglia and astrocytes, inducing the release of pro-inflammatory cytokines that exacerbate neuroinflammation and neuronal injury [ 14 , 15 ]. The antisense promoter located in intron 1 of the SNCA gene plays a critical role in regulating α-Syn expression. It modulates transcriptional activity via antisense RNA transcripts, which influence overall SNCA mRNA and protein levels. Epigenetic modifications, such as promoter DNA methylation, can alter α-Syn expression, thereby linking this promoter to HAND and other neurodegenerative diseases [ 20 ]. This study demonstrates that HIV-1 Vpr reactivates the SNCA antisense promoter, leading to α-Syn aggregation and neurotoxicity through its effects on inflammation, autophagy, and synaptic function. These findings elucidate the mechanisms by which HIV-1 contributes to HAND-related neurodegeneration and underscore the potential of targeting α-Syn regulation as a therapeutic strategy for people living with HIV (PLWH). METHODS Cell Culture. SH-SY5Y neuroblastoma cells were purchased from ATCC (CRL-2266) and were maintained as previously described [ 21 ]. Cells were differentiated with 10µM retinoic acid for at least four days before treatment and subsequent experiments. Primary neuronal cultures were prepared as previously described with minor modifications. Briefly, hippocampus tissues were collected from 17–18-day-old embryonic C57/BL6 mice (Taconic farm), digested in 0.125% trypsin for 30 minutes, and then rinsed twice in HBSS solution (Corning cellgro). Digested tissue was then triturated and dissociated into single cells in seeding medium (DMEM containing 4.5 g/L glucose, 10% FBS, 1 × glutamax, 1 × non-essential amino acids, 100 IU/ml penicillin and streptomycin, Invitrogen). Cells in suspension were centrifuged at 150 g for 10 minutes, and the pellet was gently resuspended in the seeding medium. The pellet was passed through mesh #400 to remove the non-dispersed tissue before being seeded to plates. Recombinant Proteins and Treatment. HIV-1 Vpr (100ng/ml), produced in SF9 insect cells (BioBasic Canada Inc.), was purified to > 95% homogeneity as confirmed by SDS-PAGE and validated for biological activity through cell cycle arrest assays prior to use. The recombinant protein was added to the cells at concentrations of 7nM (100ng/ml) for all experiments unless otherwise specified. Chemical Reagents. Cells were treated with decitabine (5-Aza-2'-deoxycytidine, 5-Aza-dC) (5 µM, purchased from Sigma-Aldrich) (an FDA-approved drug that inhibits DNA methylation) for 24 and 48 hours before the addition of Vpr protein. Cells were also treated with 0.5 and 1.0 mM of DMOG (dimethyloxaloylglycine) for 48 hours before the addition of Vpr protein. RNA Extraction. Total RNA was extracted from the sample using the SurePrep™ TrueTotal™ RNA purification kit from Fisher Bioreagents (BP280050). Nanodrop was used to determine the purity and concentration of the RNA extracted. Stereotaxic Surgery and Spatial Memory Testing . C57B1/6J male mice (8–10 weeks old) (purchased from the Jackson Lab) were singly housed with ad libitum access to food and water before and after stereotaxic surgery. Under isoflurane anesthesia, mice received bilateral intrahippocampal injections (1 µl per site) of either saline or Vpr (100 ng/µl) at rostral and caudal coordinates, for a total of four injections per mouse. Intraperitoneal saline was administered immediately and 36 hours post-surgery. Seventy-two hours after injection, spatial memory was evaluated using the object location memory test. In this test, mice explored two identical objects in an arena during three training trials, with their exploration time recorded. After 24 hours, one object was relocated, and mice were tested for 3 minutes, with exploration time recorded to assess spatial memory. Increased time spent exploring the displaced object indicated intact spatial memory. All procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Real-time PCR. Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer’s protocol and then reverse transcribed into cDNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed using SYBR Premix Real-time PCR kit (Roche) per the manufacturer’s instructions. The mRNA levels were normalized against β-actin and presented as 2 − ddCT . The primer sequences are listed below: SNCA forward: 5′-ggctttgtcaagaaggaccag-3′, reverse: 5′-cctctgaaggcatttcat aagcc-3′. β-actin forward: 5′-ggctgtattcccctccatcg-3′, reverse: 5′-cgtcccagttggtaacaatgcc-3′. For experiments with DMOG, we used the following primers: SNCA forward: 5'-ccagttgggcaagaatgaaga a-3', reverse: 5'-cttgatacccttcctcagaaggc-3; SNCA-AS forward: 5'-gcccaagaaataacacgcaac-3', and reverse: 5'-caatgctccttgagctttcc-3'. GAPDH forward: 5'-tcgacagtcagccgcatcttcttt-3', and reverse: 5’-accaaatccgttgactccgacctt-3'. Bisulfite treatment of genomic DNA was conducted with a Zymo bisulfite conversion kit. Genomic DNA was extracted from the brains of HIV patients or age-matched normal counterparts. Bisulfite-converted DNA was amplified with primers of interest in genomic regions. PCR products were subcloned into the pCR4 TOPO vector following the manufacturer’s instructions. Colony PCR was carried out with a FailSafe colony PCR kit, and plasmids were extracted with the Promega Wizard Plus SV Minipreps kit. At least 20 colonies were sent for sequencing. Sequencing results were analyzed using the QUMA bioinformatics online tool with the following parameters: a minimum sequence identity of 90%, a minimum conversion rate of 95%, and CpG sites were considered methylated when methylation was detected in more than 50% of reads. Case-level metadata for all human samples (de-identified ID, age, sex, HIV/ART group, and repository neuropathology summary) are provided in Supplementary Table S1 ; no additional comorbidity, ART regimen, or viral load data were available from the repository. Slice Preparation, Electrophysiology, and Data Analysis. Hippocampal slices were prepared from adult mice (males and females) (purchased from the Jackson Lab), and electrophysiology was conducted as described previously [ 22 ]. Animals were housed under standard conditions with free access to food and water. All procedures followed Temple University IACUC animal care guidelines in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals . Briefly, brains were removed and placed in artificial cerebrospinal fluid (aCSF) prepared with sucrose (248 mM) instead of NaCl. Then, using a vibratome (Vibratome 3000 plus; Vibratome, Bannockburn, IL), transverse hippocampal sections (400 µm thick) were sliced. The slices were incubated for three hours in aCSF (in mM: 124 NaCl, 2.5 KCl, 2 NaH 2 PO 4 , CaCl 2 , 2 MgSO 4 , 10 dextrose, and 26 NaHCO 3 ) or aCSF containing 200 ng/ml viral protein R at room temperature and bubbled with a gas mixture containing 95% O 2 and 5% CO 2 . Then the slices were transferred to the recording chambers (Warner Instruments, Hamden, CT) with a constant flow of aCSF at 1.5 to 2.0 mL/min bubbled with 95% O 2 /5% CO 2 and maintained at 32°C to 34°C by an inline solution heater (TC-324; Warner Instruments). Field excitatory postsynaptic potentials (fEPSPs) were recorded from the dorsal hippocampus using an extracellular glass pipette (3–5 MΩ) filled with aCSF. Recording electrodes were placed in the stratum radiatum of area CA1, and bipolar tungsten stimulating electrodes were placed 200 to 300 µm from the recording pipette in the Schaffer collateral fibers of the stratum radiatum. First, an input-output curve was generated (0 to 300 µA stimulation in increments of 20 µA), and a stimulus intensity was selected that produced one-third of the maximum fEPSP amplitude. Baseline fEPSPs were then recorded in response to stimulus pulses delivered every 30 sec for 20 min before tetanization. Slices with unstable baseline were eliminated if the normalized rise/slope dropped 20 to 50 mV/msec in an approximately 10-minute period. LTP was then induced by four trains of 100-Hz stimulation delivered in 20-second intervals. Post-tetanus fEPSPs were recorded every 30 sec for 90 min following tetanization. The fEPSP rise/slope (mV/msec) between 30% and 90% was measured with Clampfit 10.3 (Molecular Devices, Sunnyvale, CA) and normalized to the mean rise/slope of the baseline. Data are presented as the mean percent of the baseline fEPSP slope. Recordings and data analysis were performed by investigators blind to the treatment conditions. Fluorescence intensity was measured using ImageJ software (NIH) with background subtraction and normalized to cell count (DAPI-positive nuclei). Immunofluorescence SH-SY5Y with SNCA . Following 3 weeks of treatment with 100 ng/ml of recombinant HIV-1 Vpr protein under retinoic acid differentiation, the medium was removed, and cells were fixed in 4% formaldehyde (Sigma-Aldrich, 252,549) in PBS. SH-SY5Y cells (ATCC® CRL-2266™) were stained for SNCA/α-synuclein. Cells were permeabilized with 0.2% Triton X-100 (Sigma, T8787)/PBS for 5 min. Primary rabbit SNCA/α-synuclein (1:500; Santa Cruz Biotechnology, sc-7011) antibodies were diluted in 3% (w/v) bovine serum albumin (Sigma-Aldrich, A3059) in PBS and incubated at 4°C overnight. Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, A-31,573) was used at 1:1000 dilutions. Cells were mounted on glass slides and observed using the 60x oil objective lens of the confocal microscope (Leica DMI 4000B, Leica Microsystems). For immunofluorescence images, quantifications were performed using ImageJ to measure the mean fluorescence intensity per cell (arbitrary units) from ≥ 5 randomly selected fields per condition (~ 100 cells total), and the results were displayed as bar graphs . Statistical Analysis. All experiments were performed at least in triplicate with independent batches of cell cultures. Data were expressed as means ± SEM. Student’s t-test analyzed two-group comparisons. Multiple comparisons were analyzed by one-way ANOVA followed by the LSD test. *p < 0.05, **p < 0.01 versus mock group; *p < 0.05, **p < 0.01 versus rVpr-treated group. Human Ethical Statement. All experiments involving human brain tissue samples were conducted following the guidelines set by the National Institutes of Health (NIH). De-identified human tissue samples - limited to information on gender, age, and HIV status - were obtained from the National NeuroAIDS Tissue Consortium (NNTC) and approved by the Institutional Biosafety Committee (IBC) and the Institutional Review Board (IRB) at Temple University. For details on the samples used, see Table S1 . Consent from guardians was not required, as the samples were de-identified before receipt. Animal Ethical Statement . All experiments involving mouse samples were conducted in accordance with the guidelines established by the National Institutes of Health (NIH). All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at either Temple University or Baylor College of Medicine. Animal studies were conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure ethical standards and research reproducibility. RESULTS Alpha-Synuclein Accumulation: A Shared Mechanism Across Aging and Neurodegeneration Models. Previous studies demonstrated increased α-Syn levels in human brains infected with HIV, monkey brains infected with SIV, and in cell line models [ 14 ]. In the present study, endogenous α-Syn levels were assessed in mice aged 2, 9, and 33 months. These results revealed age-dependent accumulation (see Fig. 1 A for representative staining and panel 1B for quantification ). Quantification of fluorescence intensity indicated a significant increase in α-Syn levels with age. Mice aged 33 months exhibited approximately 3.5-fold higher expression compared to 2-month-old mice (p < 0.01). These results support the hypothesis that α-Syn accumulation adversely affects aging, memory, and movement. To further investigate these observations, Vpr-treated SH-SY5Y cells were analyzed. A similar increase in α-Syn levels was observed (see Fig. 1 C for representative images and panel D for quantification). This result is consistent with previous findings in human and monkey models. It enables exploration of the molecular mechanisms underlying α-Syn accumulation. Although the models differ, they are complementary and collectively support the role of α-Syn in aging and neurodegeneration. HIV-1 Vpr Alters Epigenetic Regulation at the SNCA Antisense Promoter. Previous studies demonstrat ed the aggregation of α-Syn in Vpr-treated cells [ 14 , 15 ]. Data from Fig. 1 support further investigation into whether Vpr influences α-Syn at the transcriptional level through epigenetic regulation of the SNCA antisense promoter. Several transcription factors regulate the SNCA gene. A unique regulatory element is the antisense promoter, which is located within intron 1 of the gene (Fig. 2 A). This 450-nucleotide region contains 23 CpG sites. Some studies describe this region as a long non-coding RNA (lncRNA) that influences SNCA gene expression. To further investigate the underlying mechanisms, the study focused on the epigenetic regulation of α-Syn expression. The SNCA antisense promoter is a critical regulatory region that becomes active upon demethylation. This activation influences SNCA gene expression. Significant demethylation was observed at CpG sites 8 and 12 within the promoter, suggesting a crucial role in epigenetic regulation of SNCA expression in response to Vpr treatment. CpG sites 3, 7, 8, and 12 also showed demethylation, although to a lesser extent, and may still contribute to changes in gene expression (Fig. 2 B, *p < 0.05, **p < 0.01). These findings highlight the importance of these CpG sites in modulating SNCA expression. This prompted further exploration of Vpr's impact on α-synuclein levels. The results suggest that Vpr directly induces epigenetic modifications within the SNCA antisense promoter. This finding indicates a potential mechanism by which Vpr regulates α-Syn expression and contributes to its pathological aggregation. Differential Expression of α-Synuclein and Its Antisense Promoter in Response to HIV-1 Vpr . Building on the methylation analysis presented in Fig. 2 , the present study evaluated the functional consequences of Vpr-induced demethylation on α-Syn expression and SNCA antisense promoter activity. As shown in Figs. 3 A and 3 B, Vpr treatment resulted in a significant, time-dependent increase in α-Syn expression in both SH-SY5Y cells and primary mouse neurons. This confirms that Vpr induces α-Syn overexpression at the mRNA level. T his increase in α-Syn expression was closely coordinated with upregulation of the SNCA antisense promoter, as illustrated in Figs. 3 C and 3 D. This indicates linked transcriptional regulation in response to Vpr. The findings suggest that Vpr-induced demethylation at CpG sites 8 and 12 (observed in Fig. 2 ) likely contributes to enhanced transcriptional activation of the SNCA gene. This further supports the hypothesis that Vpr plays a critical role in regulating α-Syn expression and its associated pathological aggregation. Epigenetic Mechanisms Drive α-Syn Overexpression. To examine the link between DNA methylation and α-Syn accumulation, the FDA-approved DNA methylation inhibitor 5-Aza-dC (Aza) was used to induce DNA demethylation. Treatment with 5-Aza-dC was compared to a negative control using heat-inactivated Vpr. Notably, α-Syn accumulation increased in cells treated with 5-Aza-dC. Conversely, cells exposed to active Vpr showed significantly higher α-Syn levels than both control groups and cells treated with heat-inactivated Vpr (Fig. 3 E; quantification in panel F ). These findings highlight the importance of epigenetic regulation, especially CpG demethylation, in Vpr-mediated α-Syn overexpression. Demethylation of SNCA Intron 1 in Human Brain Tissues. To extend these findings to human samples, the methylation status of SNCA intron 1 was analyzed in brain tissues from HIV-positive individuals both before and after cART. Comparisons were made to HIV-negative controls. Both HIV-positive groups demonstrated significantly reduced methylation levels compared to controls (Fig. 4 A). The reduction in methylation was consistent across pre- and post-cART HIV-positive groups. This indicates that HIV infection, rather than cART treatment, is a primary factor in these epigenetic changes. Bisulfite sequencing profiles showed pronounced demethylation in HIV-positive samples, with a marked loss of methylation marks relative to the control group (Fig. 4 B). Quantitative analysis confirmed these reductions, with statistical significance supporting the presence of HIV-mediated changes in the DNA methylation landscape of SNCA intron 1 (Fig. 4 C). These results indicate a persistent HIV-mediated epigenetic modification of SNCA intron 1 that is independent of cART. This modification may contribute to the pathogenesis of HIV-associated neurodegenerative disorders. To determine whether these epigenetic changes are associated with alterations in gene expression, α-Syn and SNCA antisense promoter mRNA expression were measured in brain tissues from people living with HIV (PLWH). The analysis was stratified by HIV-associated conditions such as HIV encephalitis (HIVE) and HIV-associated dementia (HAD). α-Syn (see Fig. 4 D ) and SNCA antisense promoter (see Fig. 4 E ) levels were significantly elevated in individuals with HIVE and HAD compared to HIV-negative controls. These results suggest that HIV infection, together with persistent epigenetic modifications such as demethylation of SNCA intron 1, leads to increased expression of α-Syn . This increase may contribute to the neuropathology observed in HIV-related neurodegenerative conditions. The observed correlation between elevated α-Syn expression and HIV-related neuropathology further supports the role of HIV-mediated gene regulation alterations in the development of cognitive and motor impairments in PLWH. Confirmation of Vpr-Mediated Demethylation Pathway To further validate involvement of the demethylation pathway in Vpr-mediated enhancement of α-Syn accumulation and aggregation, cells were treated with DMOG (0.5 and 1.0 mM) for 48 hours. DMOG is a non-specific inhibitor of 2-oxoglutarate-dependent dioxygenases and inhibits Tet enzymes, preventing DNA demethylation. Vpr is proposed to promote demethylation of CG sites within the antisense promoter. Therefore, DMOG treatment is expected to inhibit this process. As a result, DMOG prevented demethylation of the antisense promoter and reduced Vpr-induced activation of the antisense transcript, as shown in Fig. 4 F (antisense transcript levels) and Fig. 4 G (promoter methylation levels). HIV-1 Vpr Induces Synaptic Plasticity and Memory Impairments in an Animal Model . To evaluate the functional impact of HIV-1 Vpr, hippocampal synaptic plasticity and memory performance were assessed in animals treated with or injected with Vpr. The effects of recombinant HIV-1 Vpr protein on hippocampal synaptic function were examined by applying the protein to acute mouse brain slices. Field excitatory postsynaptic potential (fEPSP) recordings across various stimulus intensities showed no significant impact of Vpr (Fig. 5 A). This finding indicates normal basal synaptic transmission. In contrast, Vpr impaired hippocampal long-term potentiation (LTP) (Fig. 5 B), indicating disrupted synaptic plasticity, which is essential for memory formation. These results indicate that Vpr directly disrupts synaptic plasticity, likely through postsynaptic mechanisms. Next, we assessed the impact of Vpr on memory in these animals. Mice received stereotaxic injections of Vpr protein (or saline control) bilaterally into the hippocampi, followed by IP injections of saline or Vpr protein. Memory was tested using the object location memory test for spatial memory, 72 hours after stereotaxic injection. During the object location test, mice were trained to recognize the spatial location of two identical objects in an arena. After three training trials, mice spent about 50% of their time with each object because the objects were identical. During the test phase, one object (the "displaced" object or "DO") was moved to a new location. If the mouse recalls the original spatial pattern and recognizes that one object has been moved, it will spend more time exploring the displaced object. A significant increase in time spent with the displaced object during the test phase, relative to the training phase, indicates good spatial memory. Animals injected with Vpr protein spent significantly less time with the DO compared to controls during the testing phase (Fig. 5 C), confirming that Vpr impairs spatial memory. Given the established role of α-Syn in synaptic function, its aggregation in the hippocampus may contribute to the observed memory impairments. α-Syn aggregation is implicated in both cognitive and motor dysfunctions. In Parkinson's disease, motor deficits are prominent initially, and spatial memory impairments develop over time. These findings suggest that Vpr-induced α-Syn aggregation may produce a range of effects. The impact depends on the brain region affected and may include both spatial memory and motor functions. DISCUSSION Our research shows that HIV-1 Vpr triggers epigenetic changes at the SNCA antisense promoter, increasing α-synuclein (α-Syn) expression and aggregation in neuronal cells. Vpr demethylates specific CpG sites in this promoter, activating SNCA transcription, which raises α-Syn levels and drives its aggregation. We observed these results in cell cultures and human brain tissues from people with HIV-associated neurocognitive disorders (HAND), highlighting Vpr’s key role in HIV-related neurodegeneration [ 14 , 15 ]. Our data also show that α-Syn dysregulation contributes to memory problems and synaptic dysfunction, which may explain the high rate of neurocognitive disorders in people living with HIV (PLWH). A recent study shows that α-synuclein increases HIV-1 entry and replication in CNS-resident cells, which may help maintain viral reservoirs [ 23 ]. These findings suggest a feed-forward loop where Vpr-driven α-Syn aggregation both worsens neurodegeneration and encourages HIV-1 infection. Targeting this interaction could lead to new therapies for HAND and related disorders. Our findings align with previous studies showing Vpr targets epigenetic machinery, such as chromatin-modifying proteins and DNA methylation regulators, to counteract host silencing mechanisms [ 12 ]. Vpr induces histone gene demethylation, promotes degradation of histone deacetylases like HDAC1, HDAC3, and SIRT7, and facilitates chromatin accessibility and gene activation [ 13 ]. In our ChIP-Seq assay for CTCF, we observed increased expression of TET2, involved in gene demethylation, and DNMT3A and DNMT3B, responsible for de novo methylation. These result s further support that Vpr may dysregulate methylation and demethylation machinery, contributing to α-Syn accumulation. The precise mechanism by which Vpr promotes α-Syn accumulation remains to be fully elucidated. Vpr may directly activate SNCA transcription by demethylating its promoter, or alternatively, Vpr could indirectly promote α-Syn buildup by modulating the methylation and demethylation machinery that controls α-Syn expression. In addition, Vpr may induce cellular stress, mitochondrial dysfunction, or autophagy inhibition, which also drive α-Syn aggregation, as indicated by our previous work [ 14 , 15 ]. We are investigating the relative contributions of these pathways. Clarifying these mechanistic links will establish how Vpr-induced α-Syn aggregation leads to cognitive impairments, such as spatial memory deficits, and motor dysfunction in HAND and related conditions . Antisense promoters, especially those in introns, control gene expression in neurodegeneration [ 24 , 25 ]. These promoters initiate long non-coding RNAs (lncRNAs) that regulate sense genes through chromatin remodeling, DNA methylation, and histone modification [ 26 ]. Disruption of these processes contributes to neurodegenerative diseases like Parkinson’s and Alzheimer’s. In our study, we identify the SNCA antisense promoter as essential for regulating α-Syn expression through CpG methylation, linking methylation loss to elevated α-Syn and aggregation (18–20). We confirmed that inhibiting Tet with DMOG blocks SNCA antisense promoter demethylation and prevents α-Syn upregulation (Fig. 4 F). This finding clarifies that Tet activity is required for Vpr-induced demethylation, which in turn increases SNCA-AS expression, suggesting a mechanistic link between Vpr action and α-Syn buildup. However, it remains unclear whether activating SNCA-AS alone can cause α-Syn aggregation; future experiments will directly test this by increasing SNCA-AS independently of Vpr using antisense oligonucleotides or CRISPR editing. We will also determine if activation of the SNCA sense promoter occurs simultaneously, directly linking SNCA-AS activity to α-Syn expression. Unresolved questions persist regarding how Vpr influences α-Syn accumulation, specifically at which level of gene regulation this occurs. It remains unclear whether Vpr-induced epigenetic changes primarily impact α-Syn gene transcription or protein translation and processing. Additionally, chromatin structural changes, such as R-loop formation, may play a role in the regulation of α-Syn by Vpr. Clarification of these mechanistic links is needed to fully understand Vpr’s impact. Studying the SNCA antisense promoter reveals how DNA and RNA interact. The antisense promoter in the first SNCA intron may produce LncRNAs that control the sense promoter. Chromatin remodeling, RNA-RNA interactions, and methylation work together to regulate gene expression [ 27 ]. This type of control affects other neurodegenerative genes, such as TP53 [ 28 ] and BRCA1 [ 29 ], and viral genomes, including HIV-1 [ 30 ]. DNA demethylation of antisense promoters can reactivate them, causing abnormal gene expression and contributing to diseases like cancer, neurodegeneration, and chronic viral infections. Consistent with this analysis, in brain tissues from controls, PLWH, and people with Parkinson’s (PD) or Alzheimer’s disease (AD), we see increased α-Syn and more senescence-associated heterochromatic foci (SAHF), linking α-Syn aggregation to cellular aging [31, and studies in progress]. Brain tissue analyses show more SAHF-positive and distort ed nuclei in HIV cases compared to age-matched controls, matching levels seen in AD and PD (Table S2). This supports the idea that HIV promotes early chromatin condensation and nuclear aging in neurons. α-Syn buildup may make neurons senescent and lead to cognitive decline in HAND. Increased mTOR activity in PLWH may boost cell stress, further increase α-Syn, and link Vpr-triggered epigenetic changes to aging. Vpr thus causes direct viral damage and drives cellular aging, helping to explain lasting motor and cognitive problems in PLWH. Vpr causes DNA damage and then activates epigenetic changes that drive disease. Even in non-dividing neurons, Vpr triggers DNA damage responses (DDR) [ 11 , 32 ], which change chromatin and DNA methylation [ 12 , 13 ]. Our data show that Vpr may interact with TET enzymes or their regulators. Blocking TET with DMOG stops Vpr-induced demethylation and antisense activation. We need to learn more about how Vpr interacts with these regulators. Though DDR pathways are best known in dividing cells, they also reshape methylation in neurons , such as at the SNCA antisense promoter. This matches patterns in other neurodegenerative diseases, where DNA repair causes reprogramming of disease genes [ 33 ]. So, CpG demethylation in the SNCA antisense promoter may follow DNA damage, raise α-Syn, and encourage aggregation. Several studies support the idea that CpG demethylation, such as at CpG-2 (intron 1, especially CpG sites 8 and 12) in SNCA, contributes to α-Syn overexpression, a key factor in neurodegenerative diseases. Matsumoto et al. (2010) emphasized the role of CpG demethylation in regulating α-synuclein expression and aggregation [ 18 ]. Similarly, Yang et al. (2017) [ 19 ] showed that CpG demethylation in SNCA intron-1 leads to elevated SNCA expression in regions of Parkinson’s disease (PD) brain, including the substantia nigra pars compacta (SNpc), putamen, and cortex. Jowaed et al. (2010) [ 20 ] and Miranda-Morales et al. (2017) [ 34 ] reported significant demethylation of intron 1 in PD patient brain samples, which may explain the increased expression of SNCA. However, Guhathakurta et al. (2017) suggested that CpG demethylation and α-Syn accumulation are independent of PD development [ 35 ]. Despite this, data from Young et al. (2019) and Henderson (2021) confirm significant hypomethylation in genes from the substantia nigra region and blood samples from PD patients, reinforcing the association between methylation loss and PD pathology [ 36 , 37 ]. To assess the relevance of our findings to human neuropathology, we analyzed brain tissues from both PLWH and SIV-infected monkeys [ 14 ]. We observed upregulation of α-Syn and demethylation of the antisense promoter, supporting the translational significance of our in vitro results. Additionally, we identified mitochondrial dysfunction, reduced ATP production, impaired mitophagy, and increased reactive oxygen species (ROS) levels in Vpr-treated neurons [ 15 ]. These disturbances in cellular energy homeostasis and mitochondrial dynamics, along with ER stress and β-amyloid accumulation, indicate that Vpr-induced α-Syn accumulation impairs cellular function and activates stress pathways characteristic of neurodegenerative diseases. Deficits in mitochondrial axonal transport and microtubule stability further implicate α-Syn dysregulation as a central factor in neuronal damage and potential cognitive and motor dysfunction [ 15 ]. In vivo , Vpr-treated mice displayed cognitive impairments, including deficits in spatial memory and long-term potentiation (LTP), which align with the established role of α-Syn in cognitive decline associated with HAND. Structural changes such as brain atrophy were also observed in PLWH [ 38 ], further connecting molecular alterations in SNCA regulation to neurodegeneration. These findings from animal models, together with human brain tissue data, emphasize the importance of Vpr-induced epigenetic modulation of the SNCA antisense promoter in driving neurodegenerative processes in HAND. This research elucidates the complex relationship between viral factors, epigenetic modifications, and neurodegenerative disease progression, providing a basis for developing novel therapeutic strategies for HAND and related disorders. In addition to HIV-1 Vpr, other viral proteins, such as those from SARS-CoV-2, have been shown to interact with α-Syn, inducing Lewy body-like pathology in vitro [ 39 ]. This suggests that viral proteins can influence α-Syn dynamics, potentially contributing to neurodegenerative processes. These findings have important clinical implications for managing HAND in PLWH. Identifying specific epigenetic mechanisms underlying α-Syn accumulation presents new opportunities for therapeutic intervention. Targeting the Tet-mediated demethylation pathway or antisense promoter activity may reduce α-Syn aggregation and related neurodegeneration. Furthermore, monitoring α-Syn levels or the methylation status of the SNCA antisense promoter could serve as biomarkers for early detection of neurocognitive decline in PLWH, facilitating timely intervention. Future studies involving larger cohorts of PLWH will be necessary to validate these potential biomarkers and therapeutic targets. In summary, this study provides evidence that HIV-1 Vpr causes epigenetic changes at the SNCA antisense promoter, leading to α-Syn dysregulation and aggregation. These are key factors in HIV-associated neurodegeneration. The similarities between these findings and the known role of epigenetic modifications in synucleinopathies suggest that HIV-1 infection may share mechanisms with diseases like Parkinson’s disease. This research sheds light on how Vpr-induced epigenetic changes could impact hippocampal synaptic plasticity, spatial memory, and motor function in PLWH, possibly explaining motor problems such as gait disturbances and balance issues. Further research on these epigenetic changes in larger PLWH groups may help find new biomarkers for early detection and new targets for treatment, leading to better management of HIV-related cognitive issues and neurodegeneration. Finally, studying the SNCA antisense promoter reveals how viral proteins exploit host epigenetic machinery to drive neurodegeneration. Defining these interactions will uncover therapeutic targets for mitigating HAND and potentially other α-synucleinopathies. Declarations CONFLICT OF INTEREST The authors declare that the research was performed in the absence of any financial relationships and has no potential conflict of interest. AI ASSISTANCE The manuscript was enhanced with the assistance of AI, specifically ChatGPT, to improve clarity, flow, and readability. AI was used exclusively for text editing and refinement, with no involvement in data analysis. FUNDING This work is supported by an NIH-NIA grant AG054411 and by previous NIH grants NS076402 and MH093331 awarded to BES. Author Contribution YW, MS, DK, and JP designed and performed the studies. LK, JC, JJ, and NS provided technical assistance and reagents. JJ and LK also edited the manuscript. BES edited the manuscript, directed, and supervised the work. Acknowledgement We would like to thank the National NeuroAIDS Tissue Consortium (NNTC) for providing postmortem human brain tissues. Data Availability All processed data are included in this manuscript. 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11:45:18","extension":"xml","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114309,"visible":true,"origin":"","legend":"","description":"","filename":"5595a8366fbc434ba42c8cd05c5216e21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/b3e683752dc286b3521affef.xml"},{"id":94916180,"identity":"21a34ea5-b0bd-41a8-a163-9106e5b291b8","added_by":"auto","created_at":"2025-11-01 11:45:18","extension":"html","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130776,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/b28d121e9a80a636e8fb71d2.html"},{"id":94987743,"identity":"873bf82f-6675-49e2-a655-28333211989a","added_by":"auto","created_at":"2025-11-03 07:02:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eAccumulation of α-Synuclein in Aged Mice and Vpr-Treated SH-SY5Y Cells\u003c/u\u003e. \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003eImmunofluorescence staining shows α-Syn (green) and nuclei (DAPI, blue) in brain tissue from mice aged 2, 9, and 33 months. α-Syn accumulation increases with age. Scale bars are 50 μm. \u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Quantification of Panel A shows α-synuclein fluorescence intensity for all age groups (n=3 each). Asterisks indicate significant differences by one-way ANOVA with post-hoc testing (*p \u0026lt; 0.05; *\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep \u0026lt; 0.01\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e). \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eImmunofluorescence staining reveals α-Syn (green) and nuclei (DAPI, blue) in SH-SY5Y cells. \u003cem\u003e\u003cstrong\u003eD.\u003c/strong\u003e\u003c/em\u003eA bar graph compares α-Syn expression levels in Vpr-treated and control cells. The observed increase of α-Syn in Vpr-treated cells relative to control may indicate that Vpr influences mechanisms involved in α-Syn accumulation. Scale bars are 50 μm.\u003c/p\u003e","description":"","filename":"Slide1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/4a1bc4591e357041d9e5cd9c.jpeg"},{"id":94916141,"identity":"a5260c32-da55-4aa9-b667-af5a080eba16","added_by":"auto","created_at":"2025-11-01 11:45:16","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eHIV-1 Vpr Reduces CpG Methylation at the SNCA Antisense Promoter.\u003c/u\u003e \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e Structure of the SNCA gene, highlighting coding exons (yellow boxes) and the antisense promoter (green arrow) within intron 1. Transcription factors (GATA-1 and 2, ZSCAN21, ZNF219), regulatory elements (TATA box), and the 450 nt-long antisense RNA are indicated. The sequence of the SNCA antisense promoter, showing 23 analyzed CpG sites (red), is also shown. \u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e Methylation levels at the SNCA antisense promoter in Vpr-treated SH-SY5Y cells reveal significant demethylation at CpG sites 3, 7, 8, and 12 (*p \u0026lt; 0.05, **p \u0026lt; 0.01). Data are presented as the mean ± SD from n = 3 replicates. \u003cstrong\u003eStatistical significance was determined by one-way ANOVA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/2dd962de84181c882850c1c9.jpeg"},{"id":94916144,"identity":"eb5f9596-7c42-42f0-95ef-6a2b3744e9c3","added_by":"auto","created_at":"2025-11-01 11:45:16","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eDifferential expression of α-Syn and its antisense promoter in response to HIV or Vpr\u003c/u\u003e. \u003cem\u003e\u003cstrong\u003eA, B.\u003c/strong\u003e\u003c/em\u003eqPCR shows α-Syn mRNA in SH-SY5Y cells (A) and primary mouse neurons (B) after rVpr (7nM) for various times. \u003cem\u003e\u003cstrong\u003eC, D.\u003c/strong\u003e\u003c/em\u003e Antisense SNCA promoter activity increases in SH-SY5Y cells at 48 hours and 7 days after Vpr treatment. Data are mean ± SEM. Statistical significance was tested (e.g., ANOVA, t-test). E. Images show increased α-Syn in neuronal cells treated with 7nM Vpr. Control and heat-inactivated Vpr groups show reduced levels. The methylation inhibitor 5-Aza-dC lowers α-Syn, indicating epigenetic regulation by Vpr. The \u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Bar graph quantifies fluorescence from panel E, with mean ± SEM (n = 3). Active rVpr raises α-Syn over all groups (***p \u0026lt; 0.001). 5-Aza partially reduces α-Syn levels vs active rVpr (*p \u0026lt; 0.05). Mock and rVpr (inactivated) maintain low baseline fluorescence. One-way ANOVA with Tukey’s post hoc test was used.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/cb2a60efdac1e494e0535bf0.jpeg"},{"id":94916152,"identity":"49c80ab4-370d-49f8-96cb-29aeeaf246bb","added_by":"auto","created_at":"2025-11-01 11:45:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":319405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eDemethylation of SNCA Intron 1 in human brain tissues\u003c/u\u003e. \u003cem\u003e\u003cstrong\u003eA. \u003c/strong\u003e\u003c/em\u003eA scatter plot displays SNCA intron 1 methylation levels in HIV-positive pre- and post-cART groups and HIV-negative controls. HIV-positive groups show significantly reduced methylation (\u003cstrong\u003ep \u0026lt; 0.05, one-way ANOVA with Tukey’s post-hoc test, n = 13 per group\u003c/strong\u003e). \u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e Representative bisulfite sequencing profiles of SNCA intron 1 in HIV-negative, HIV-positive pre-, and post-cART individuals. Open circles indicate unmethylated CpG sites, while filled circles indicate methylated CpG sites. The HIV-positive sample exhibits decreased methylation. \u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003eA bar graph shows SNCA intron 1 methylation levels (relative to normal) in samples from the NNTC cohort. HIV-positive pre- and post-ART samples have significantly lower methylation than HIV-negative controls (\u003cstrong\u003ep \u0026lt; 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons, n = 13 per group\u003c/strong\u003e). \u003cem\u003e\u003cstrong\u003eD, E.\u003c/strong\u003e\u003c/em\u003e Increased α-Syn (\u003cem\u003eD\u003c/em\u003e) and SNCA antisense promoter (\u003cem\u003eE\u003c/em\u003e) mRNA expression are observed in brain tissues of people living with HIV (PLWH). Expression is higher in patients with HIVE and HAD compared to controls (\u003cstrong\u003ep \u0026lt; 0.05, one-way ANOVA with Tukey’s post-hoc, n = 3 per group\u003c/strong\u003e). \u003cem\u003e\u003cstrong\u003eF.\u003c/strong\u003e\u003c/em\u003eSNCA-AS1 expression was measured by qPCR in SH-SY5Y cells transfected with pcDNA3 or pcDNA3-Vpr plasmids and treated with DMOG (0.5 or 1.0 mM). Data are shown as mean ± SEM. Two-way ANOVA determined statistical significance with Tukey’s post-hoc test (*\u003cstrong\u003ep \u0026lt; 0.05, p \u0026lt; 0.01, n = 3 per condition). Cohort details (A-E): de-identified male donors from the NNTC. Supplementary Table S1 provides additional information, including case ID, age, HIV/ART group, and repository neuropathology summaries. Only age, sex, HIV/ART status, and brief neuropathology data were available. Broader comorbidity, ART regimen, and viral load data were not available.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/4befddd9d9c8382bcdf49a91.jpg"},{"id":94987638,"identity":"58670f7c-b912-429e-a391-a76a230f15b3","added_by":"auto","created_at":"2025-11-03 07:02:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eVpr induces hippocampal synaptic plasticity and spatial memory impairments in an animal model\u003c/u\u003e. \u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003eHippocampal input/output curves and \u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e LTP with representative recordings for slices incubated for 3 hours in either 200 ng/ml viral protein R (n=3) or aCSF (control, n=2). LTP is expressed as the fEPSP slope normalized to the baseline mean pre-tetanus. Results are shown as mean ± SEM. Scale bars represent 0.5 mV (vertical) and 5 ms (horizontal). \u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e Bar graph comparing memory performance between control and Vpr-treated animals during training and testing phases, showing impaired memory in Vpr-treated animals\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/01f63c5f68ffbb9e832a9139.jpeg"},{"id":101151929,"identity":"60d2f3f1-88c2-47be-ba6a-0c394c2d8b11","added_by":"auto","created_at":"2026-01-26 16:08:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4006913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/5c4d1050-4f42-40cd-b47f-2a5c341a0a22.pdf"},{"id":94916139,"identity":"9dfc692a-b327-44c9-b3b6-308419485035","added_by":"auto","created_at":"2025-11-01 11:45:16","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54976,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6405901/v1/680b7e5c9796693fa3f313f9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"HIV Vpr induces demethylation of the SNCA antisense promoter leading to neurocognitive impairment","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHIV-associated neurocognitive disorders (HAND) continue to present a significant clinical challenge despite the widespread use of combination antiretroviral therapy (cART). HAND encompasses a spectrum of cognitive, motor, and behavioral impairments that arise from persistent inflammation and neurodegeneration due to HIV infection in the brain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A subset of individuals with HAND exhibit Parkinson-like motor symptoms, including tremors, bradykinesia, rigidity, and postural instability, which closely resemble features observed in Parkinson\u0026rsquo;s disease (PD) [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFour interrelated mechanistic pathways drive HAND-related neurodegeneration. First, HIV infects glial cells, resulting in neuronal damage within motor control regions such as the basal ganglia and promoting local chronic inflammation. Second, persistent immune activation induces widespread chronic inflammation, even in the presence of cART. Third, toxic viral proteins, including HIV-1 Tat and Vpr, disrupt cellular homeostasis and exacerbate neurotoxicity, partly by modulating inflammatory processes. Fourth, these viral proteins facilitate the accumulation of alpha-synuclein (α-Syn), thereby linking HAND-related degeneration to Parkinson-like symptoms through neuronal dysfunction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While cART alleviates some symptoms, neurodegeneration frequently persists, underscoring the necessity of targeting these interconnected mechanisms beyond viral suppression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHIV-1 viral protein R (Vpr) is a 15-kDa multifunctional protein that facilitates viral nuclear import, induces G2-phase arrest, and modulates the HIV-1 promoter [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Vpr also interacts with host epigenetic machinery and promotes DNA demethylation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous findings have demonstrated Vpr's effects on cellular pathways; however, the mechanisms by \u003cb\u003ewhich Vpr interacts with host epigenetic machinery to regulate SNCA expression remain poorly understood. The direct relationship between Vpr-induced epigenetic changes, α-Syn accumulation, and their impact on neuronal function represents a critical knowledge gap. The present study addresses this gap. Recent data indicate\u003c/b\u003e that Vpr reactivates the SNCA gene's antisense promoter in intron 1, leading to increased α-Syn transcription. This accumulation alters lysosomal pH, impairs autophagy, and promotes α-Syn aggregation, thereby contributing to neuronal dysfunction and HAND pathogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlpha-synuclein (α-Syn) is a 14-kDa protein localized at presynaptic terminals and is essential for neurotransmitter release and synaptic plasticity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Genetic and epigenetic alterations, such as SNCA antisense promoter demethylation, increase α-Syn expression and promote its aggregation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Aggregated α-Syn disrupts mitochondrial and lysosomal function, impairs synaptic communication, and elevates oxidative stress, ultimately resulting in neuronal loss. Additionally, α-Syn activates microglia and astrocytes, inducing the release of pro-inflammatory cytokines that exacerbate neuroinflammation and neuronal injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe antisense promoter located in intron 1 of the SNCA gene plays a critical role in regulating α-Syn expression. It modulates transcriptional activity via antisense RNA transcripts, which influence overall SNCA mRNA and protein levels. Epigenetic modifications, such as promoter DNA methylation, can alter α-Syn expression, thereby linking this promoter to HAND and other neurodegenerative diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study demonstrates that HIV-1 Vpr reactivates the SNCA antisense promoter, leading to α-Syn aggregation and neurotoxicity through its effects on inflammation, autophagy, and synaptic function. These findings elucidate the mechanisms by which HIV-1 contributes to HAND-related neurodegeneration and underscore the potential of targeting α-Syn regulation as a therapeutic strategy for people living with HIV (PLWH).\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eCell Culture.\u003c/b\u003e SH-SY5Y neuroblastoma cells were purchased from ATCC (CRL-2266) and were maintained as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Cells were differentiated with 10\u0026micro;M retinoic acid for at least four days before treatment and subsequent experiments.\u003c/p\u003e\u003cp\u003ePrimary neuronal cultures were prepared as previously described with minor modifications. Briefly, hippocampus tissues were collected from 17\u0026ndash;18-day-old embryonic C57/BL6 mice (Taconic farm), digested in 0.125% trypsin for 30 minutes, and then rinsed twice in HBSS solution (Corning cellgro). Digested tissue was then triturated and dissociated into single cells in seeding medium (DMEM containing 4.5 g/L glucose, 10% FBS, 1 \u0026times; glutamax, 1 \u0026times; non-essential amino acids, 100 IU/ml penicillin and streptomycin, Invitrogen). Cells in suspension were centrifuged at 150 g for 10 minutes, and the pellet was gently resuspended in the seeding medium. The pellet was passed through mesh #400 to remove the non-dispersed tissue before being seeded to plates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecombinant Proteins and Treatment. HIV-1 Vpr (100ng/ml), produced in SF9 insect cells (BioBasic Canada Inc.), was purified to \u0026gt;\u0026thinsp;95% homogeneity as confirmed by SDS-PAGE and validated for biological activity through cell cycle arrest assays prior to use. The recombinant protein was added to the cells at concentrations of 7nM (100ng/ml) for all experiments unless otherwise specified.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical Reagents.\u003c/b\u003e Cells were treated with decitabine (5-Aza-2'-deoxycytidine, 5-Aza-dC) (5 \u0026micro;M, purchased from Sigma-Aldrich) (an FDA-approved drug that inhibits DNA methylation) for 24 and 48 hours before the addition of Vpr protein. Cells were also treated with 0.5 and 1.0 mM of DMOG (dimethyloxaloylglycine) for 48 hours before the addition of Vpr protein.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA Extraction.\u003c/b\u003e Total RNA was extracted from the sample using the SurePrep\u0026trade; TrueTotal\u0026trade; RNA purification kit from Fisher Bioreagents (BP280050). Nanodrop was used to determine the purity and concentration of the RNA extracted.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStereotaxic Surgery and Spatial Memory Testing\u003c/b\u003e. C57B1/6J male mice (8\u0026ndash;10 weeks old) (purchased from the Jackson Lab) were singly housed with ad libitum access to food and water before and after stereotaxic surgery. Under isoflurane anesthesia, mice received bilateral intrahippocampal injections (1 \u0026micro;l per site) of either saline or Vpr (100 ng/\u0026micro;l) at rostral and caudal coordinates, for a total of four injections per mouse. Intraperitoneal saline was administered immediately and 36 hours post-surgery. Seventy-two hours after injection, spatial memory was evaluated using the object location memory test. In this test, mice explored two identical objects in an arena during three training trials, with their exploration time recorded. After 24 hours, one object was relocated, and mice were tested for 3 minutes, with exploration time recorded to assess spatial memory. Increased time spent exploring the displaced object indicated intact spatial memory. All procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal-time PCR.\u003c/b\u003e Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer\u0026rsquo;s protocol and then reverse transcribed into cDNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed using SYBR Premix Real-time PCR kit (Roche) per the manufacturer\u0026rsquo;s instructions. The mRNA levels were normalized against β-actin and presented as 2\u003csup\u003e\u0026minus;\u0026thinsp;ddCT\u003c/sup\u003e. The primer sequences are listed below: SNCA forward: 5\u0026prime;-ggctttgtcaagaaggaccag-3\u0026prime;, reverse: 5\u0026prime;-cctctgaaggcatttcat aagcc-3\u0026prime;. β-actin forward: 5\u0026prime;-ggctgtattcccctccatcg-3\u0026prime;, reverse: 5\u0026prime;-cgtcccagttggtaacaatgcc-3\u0026prime;.\u003c/p\u003e\u003cp\u003eFor experiments with DMOG, we used the following primers: SNCA forward: 5'-ccagttgggcaagaatgaaga a-3', reverse: 5'-cttgatacccttcctcagaaggc-3; SNCA-AS forward: 5'-gcccaagaaataacacgcaac-3', and reverse: 5'-caatgctccttgagctttcc-3'. GAPDH forward: 5'-tcgacagtcagccgcatcttcttt-3', and reverse: 5\u0026rsquo;-accaaatccgttgactccgacctt-3'.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBisulfite treatment of genomic DNA was conducted with a Zymo bisulfite conversion kit.\u003c/b\u003e Genomic DNA was extracted from the brains of HIV patients or age-matched normal counterparts. Bisulfite-converted DNA was amplified with primers of interest in genomic regions. PCR products were subcloned into the pCR4 TOPO vector following the manufacturer\u0026rsquo;s instructions. Colony PCR was carried out with a FailSafe colony PCR kit, and plasmids were extracted with the Promega Wizard Plus SV Minipreps kit. \u003cb\u003eAt least 20 colonies were sent for sequencing. Sequencing results were analyzed using the QUMA bioinformatics online tool with the following parameters: a minimum sequence identity of 90%, a minimum conversion rate of 95%, and CpG sites were considered methylated when methylation was detected in more than 50% of reads.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCase-level metadata for all human samples (de-identified ID, age, sex, HIV/ART group, and repository neuropathology summary) are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; no additional comorbidity, ART regimen, or viral load data were available from the repository.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSlice Preparation, Electrophysiology, and Data Analysis.\u003c/b\u003e Hippocampal slices were prepared from adult mice (males and females) (purchased from the Jackson Lab), and electrophysiology was conducted as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Animals were housed under standard conditions with free access to food and water. All procedures followed Temple University IACUC animal care guidelines in accordance with the National Research Council \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e. Briefly, brains were removed and placed in artificial cerebrospinal fluid (aCSF) prepared with sucrose (248 mM) instead of NaCl. Then, using a vibratome (Vibratome 3000 plus; Vibratome, Bannockburn, IL), transverse hippocampal sections (400 \u0026micro;m thick) were sliced. The slices were incubated for three hours in aCSF (in mM: 124 NaCl, 2.5 KCl, 2 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e, 2 MgSO\u003csub\u003e4\u003c/sub\u003e, 10 dextrose, and 26 NaHCO\u003csub\u003e3\u003c/sub\u003e) or aCSF containing 200 ng/ml viral protein R at room temperature and bubbled with a gas mixture containing 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e. Then the slices were transferred to the recording chambers (Warner Instruments, Hamden, CT) with a constant flow of aCSF at 1.5 to 2.0 mL/min bubbled with 95% O\u003csub\u003e2\u003c/sub\u003e/5% CO\u003csub\u003e2\u003c/sub\u003e and maintained at 32\u0026deg;C to 34\u0026deg;C by an inline solution heater (TC-324; Warner Instruments). Field excitatory postsynaptic potentials (fEPSPs) were recorded from the dorsal hippocampus using an extracellular glass pipette (3\u0026ndash;5 MΩ) filled with aCSF. Recording electrodes were placed in the stratum radiatum of area CA1, and bipolar tungsten stimulating electrodes were placed 200 to 300 \u0026micro;m from the recording pipette in the Schaffer collateral fibers of the stratum radiatum. First, an input-output curve was generated (0 to 300 \u0026micro;A stimulation in increments of 20 \u0026micro;A), and a stimulus intensity was selected that produced one-third of the maximum fEPSP amplitude. Baseline fEPSPs were then recorded in response to stimulus pulses delivered every 30 sec for 20 min before tetanization. Slices with unstable baseline were eliminated if the normalized rise/slope dropped 20 to 50 mV/msec in an approximately 10-minute period. LTP was then induced by four trains of 100-Hz stimulation delivered in 20-second intervals. Post-tetanus fEPSPs were recorded every 30 sec for 90 min following tetanization. The fEPSP rise/slope (mV/msec) between 30% and 90% was measured with Clampfit 10.3 (Molecular Devices, Sunnyvale, CA) and normalized to the mean rise/slope of the baseline. Data are presented as the mean percent of the baseline fEPSP slope. Recordings and data analysis were performed by investigators blind to the treatment conditions. \u003cb\u003eFluorescence intensity was measured using ImageJ software (NIH) with background subtraction and normalized to cell count (DAPI-positive nuclei).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence SH-SY5Y with SNCA\u003c/b\u003e. Following 3 weeks of treatment with 100 ng/ml of recombinant HIV-1 Vpr protein under retinoic acid differentiation, the medium was removed, and cells were fixed in 4% formaldehyde (Sigma-Aldrich, 252,549) in PBS. SH-SY5Y cells (ATCC\u0026reg; CRL-2266\u0026trade;) were stained for SNCA/α-synuclein. Cells were permeabilized with 0.2% Triton X-100 (Sigma, T8787)/PBS for 5 min. Primary rabbit SNCA/α-synuclein (1:500; Santa Cruz Biotechnology, sc-7011) antibodies were diluted in 3% (w/v) bovine serum albumin (Sigma-Aldrich, A3059) in PBS and incubated at 4\u0026deg;C overnight. Donkey anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, A-31,573) was used at 1:1000 dilutions. Cells were mounted on glass slides and observed using the 60x oil objective lens of the confocal microscope (Leica DMI 4000B, Leica Microsystems). \u003cb\u003eFor immunofluorescence images, quantifications were performed using ImageJ to measure the mean fluorescence intensity per cell (arbitrary units) from \u0026ge;\u0026thinsp;5 randomly selected fields per condition (~\u0026thinsp;100 cells total), and the results were displayed as bar graphs\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analysis.\u003c/b\u003e All experiments were performed at least in triplicate with independent batches of cell cultures. Data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Student\u0026rsquo;s t-test analyzed two-group comparisons. Multiple comparisons were analyzed by one-way ANOVA followed by the LSD test. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus mock group; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus rVpr-treated group.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman Ethical Statement.\u003c/b\u003e All experiments involving human brain tissue samples were conducted following the guidelines set by the National Institutes of Health (NIH). De-identified human tissue samples - limited to information on gender, age, and HIV status - were obtained from the National NeuroAIDS Tissue Consortium (NNTC) and approved by the Institutional Biosafety Committee (IBC) and the Institutional Review Board (IRB) at Temple University. For details on the samples used, see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Consent from guardians was not required, as the samples were de-identified before receipt.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal Ethical Statement\u003c/b\u003e. All experiments involving mouse samples were conducted in accordance with the guidelines established by the National Institutes of Health (NIH). All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at either Temple University or Baylor College of Medicine. Animal studies were conducted in compliance with the ARRIVE (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments) guidelines to ensure ethical standards and research reproducibility.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eAlpha-Synuclein Accumulation: A Shared Mechanism Across Aging and Neurodegeneration Models.\u003c/b\u003e Previous studies demonstrated increased α-Syn levels in human brains infected with HIV, monkey brains infected with SIV, and in cell line models [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the present study, endogenous α-Syn levels were assessed in mice aged 2, 9, and 33 months. These results revealed age-dependent accumulation (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA for representative staining and panel 1B for quantification\u003cb\u003e). Quantification of fluorescence intensity indicated a significant increase in α-Syn levels with age. Mice aged 33 months exhibited approximately 3.5-fold higher expression compared to 2-month-old mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/b\u003e These results support the hypothesis that α-Syn accumulation adversely affects aging, memory, and movement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate these observations, Vpr-treated SH-SY5Y cells were analyzed. A similar increase in α-Syn levels was observed (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC for representative images and panel D for quantification). This result is consistent with previous findings in human and monkey models. It enables exploration of the molecular mechanisms underlying α-Syn accumulation. Although the models differ, they are complementary and collectively support the role of α-Syn in aging and neurodegeneration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHIV-1 Vpr Alters Epigenetic Regulation at the SNCA Antisense Promoter. Previous studies demonstrat\u003c/b\u003eed the aggregation of α-Syn in Vpr-treated cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Data from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e support further investigation into whether Vpr influences α-Syn at the transcriptional level through epigenetic regulation of the SNCA antisense promoter. Several transcription factors regulate the SNCA gene. A unique regulatory element is the antisense promoter, which is located within intron 1 of the gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This 450-nucleotide region contains 23 CpG sites. Some studies describe this region as a long non-coding RNA (lncRNA) that influences SNCA gene expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the underlying mechanisms, the study focused on the epigenetic regulation of α-Syn expression. The SNCA antisense promoter is a critical regulatory region that becomes active upon demethylation. This activation influences SNCA gene expression. Significant demethylation was observed at CpG sites 8 and 12 within the promoter, suggesting a crucial role in epigenetic regulation of SNCA expression in response to Vpr treatment. CpG sites 3, 7, 8, and 12 also showed demethylation, although to a lesser extent, and may still contribute to changes in gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings highlight the importance of these CpG sites in modulating SNCA expression. This prompted further exploration of Vpr's impact on α-synuclein levels. The results suggest that Vpr directly induces epigenetic modifications within the SNCA antisense promoter. This finding indicates a potential mechanism by which Vpr regulates α-Syn expression and contributes to its pathological aggregation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferential Expression of α-Synuclein and Its Antisense Promoter in Response to HIV-1 Vpr\u003c/b\u003e. Building on the methylation analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the present study evaluated the functional consequences of Vpr-induced demethylation on α-Syn expression and SNCA antisense promoter activity. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Vpr treatment resulted in a significant, time-dependent increase in α-Syn expression in both SH-SY5Y cells and primary mouse neurons. This confirms that Vpr induces α-Syn overexpression at the mRNA level. T\u003cb\u003ehis increase in α-Syn expression was closely coordinated with upregulation of the SNCA antisense promoter, as illustrated in\u003c/b\u003e Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. \u003cb\u003eThis indicates linked transcriptional regulation in response to Vpr.\u003c/b\u003e The findings suggest that Vpr-induced demethylation at CpG sites 8 and 12 (observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) likely contributes to enhanced transcriptional activation of the SNCA gene. This further supports the hypothesis that Vpr plays a critical role in regulating α-Syn expression and its associated pathological aggregation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpigenetic Mechanisms Drive α-Syn Overexpression.\u003c/b\u003e To examine the link between DNA methylation and α-Syn accumulation, the FDA-approved DNA methylation inhibitor 5-Aza-dC (Aza) was used to induce DNA demethylation. Treatment with 5-Aza-dC was compared to a negative control using heat-inactivated Vpr. Notably, α-Syn accumulation increased in cells treated with 5-Aza-dC. Conversely, cells exposed to active Vpr showed significantly higher α-Syn levels than both control groups and cells treated with heat-inactivated Vpr (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; \u003cb\u003equantification in panel F\u003c/b\u003e). These findings highlight the importance of epigenetic regulation, especially CpG demethylation, in Vpr-mediated α-Syn overexpression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDemethylation of SNCA Intron 1 in Human Brain Tissues.\u003c/b\u003e To extend these findings to human samples, the methylation status of SNCA intron 1 was analyzed in brain tissues from HIV-positive individuals both before and after cART. Comparisons were made to HIV-negative controls. Both HIV-positive groups demonstrated significantly reduced methylation levels compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The reduction in methylation was consistent across pre- and post-cART HIV-positive groups. This indicates that HIV infection, rather than cART treatment, is a primary factor in these epigenetic changes. Bisulfite sequencing profiles showed pronounced demethylation in HIV-positive samples, with a marked loss of methylation marks relative to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Quantitative analysis confirmed these reductions, with statistical significance supporting the presence of HIV-mediated changes in the DNA methylation landscape of SNCA intron 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate a persistent HIV-mediated epigenetic modification of SNCA intron 1 that is independent of cART. This modification may contribute to the pathogenesis of HIV-associated neurodegenerative disorders.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether these epigenetic changes are associated with alterations in gene expression, \u003cb\u003eα-Syn\u003c/b\u003e and \u003cb\u003eSNCA antisense promoter\u003c/b\u003e mRNA expression were measured in brain tissues from people living with HIV (PLWH). The analysis was stratified by HIV-associated conditions such as HIV encephalitis (HIVE) and HIV-associated dementia (HAD). \u003cb\u003eα-Syn (see\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e and \u003cb\u003eSNCA antisense promoter (see\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e levels were significantly elevated in individuals with HIVE and HAD compared to HIV-negative controls. These results suggest that HIV infection, together with persistent epigenetic modifications such as demethylation of SNCA intron 1, leads to increased expression of \u003cb\u003eα-Syn\u003c/b\u003e. This increase may contribute to the neuropathology observed in HIV-related neurodegenerative conditions. The observed correlation between elevated \u003cb\u003eα-Syn\u003c/b\u003e expression and HIV-related neuropathology further supports the role of HIV-mediated gene regulation alterations in the development of cognitive and motor impairments in PLWH.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConfirmation of Vpr-Mediated Demethylation Pathway\u003c/strong\u003e\u003cp\u003eTo further validate involvement of the demethylation pathway in Vpr-mediated enhancement of \u003cb\u003eα-Syn\u003c/b\u003e accumulation and aggregation, cells were treated with DMOG (0.5 and 1.0 mM) for 48 hours. DMOG is a non-specific inhibitor of 2-oxoglutarate-dependent dioxygenases and inhibits Tet enzymes, preventing DNA demethylation. Vpr is proposed to promote demethylation of CG sites within the antisense promoter. Therefore, DMOG treatment is expected to inhibit this process. As a result, DMOG prevented demethylation of the antisense promoter and reduced Vpr-induced activation of the antisense transcript, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF (antisense transcript levels) and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG (promoter methylation levels).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHIV-1 Vpr Induces Synaptic Plasticity and Memory Impairments in an Animal Model\u003c/b\u003e. To evaluate the functional impact of HIV-1 Vpr, hippocampal synaptic plasticity and memory performance were assessed in animals treated with or injected with Vpr. The effects of recombinant HIV-1 Vpr protein on hippocampal synaptic function were examined by applying the protein to acute mouse brain slices. Field excitatory postsynaptic potential (fEPSP) recordings across various stimulus intensities showed no significant impact of Vpr (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This finding indicates normal basal synaptic transmission. In contrast, Vpr impaired hippocampal long-term potentiation (LTP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating disrupted synaptic plasticity, which is essential for memory formation. These results indicate that Vpr directly disrupts synaptic plasticity, likely through postsynaptic mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we assessed the impact of Vpr on memory in these animals. Mice received stereotaxic injections of Vpr protein (or saline control) bilaterally into the hippocampi, followed by IP injections of saline or Vpr protein. Memory was tested using the object location memory test for spatial memory, 72 hours after stereotaxic injection. During the object location test, mice were trained to recognize the spatial location of two identical objects in an arena. After three training trials, mice spent about 50% of their time with each object because the objects were identical. During the test phase, one object (the \"displaced\" object or \"DO\") was moved to a new location. If the mouse recalls the original spatial pattern and recognizes that one object has been moved, it will spend more time exploring the displaced object. A significant increase in time spent with the displaced object during the test phase, relative to the training phase, indicates good spatial memory. Animals injected with Vpr protein \u003c/p\u003e\u003cp\u003espent significantly less time with the DO compared to controls during the testing phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), confirming that Vpr impairs spatial memory.\u003c/p\u003e\u003cp\u003eGiven the established role of α-Syn in synaptic function, its aggregation in the hippocampus may contribute to the observed memory impairments. α-Syn aggregation is implicated in both cognitive and motor dysfunctions. In Parkinson's disease, motor deficits are prominent initially, and spatial memory impairments develop over time. These findings suggest that Vpr-induced α-Syn aggregation may produce a range of effects. The impact depends on the brain region affected and may include both spatial memory and motor functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur research shows that HIV-1 Vpr triggers epigenetic changes at the SNCA antisense promoter, increasing α-synuclein (α-Syn) expression and aggregation in neuronal cells. Vpr demethylates specific CpG sites in this promoter, activating SNCA transcription, which raises α-Syn levels and drives its aggregation. We observed these results in cell cultures and human brain tissues from people with HIV-associated neurocognitive disorders (HAND), highlighting Vpr\u0026rsquo;s key role in HIV-related neurodegeneration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our data also show that α-Syn dysregulation contributes to memory problems and synaptic dysfunction, which may explain the high rate of neurocognitive disorders in people living with HIV (PLWH).\u003c/p\u003e\u003cp\u003eA recent study shows that α-synuclein increases HIV-1 entry and replication in CNS-resident cells, which may help maintain viral reservoirs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings suggest a feed-forward loop where Vpr-driven α-Syn aggregation both worsens neurodegeneration and encourages HIV-1 infection. Targeting this interaction could lead to new therapies for HAND and related disorders.\u003c/p\u003e\u003cp\u003eOur findings align with previous studies showing Vpr targets epigenetic machinery, such as chromatin-modifying proteins and DNA methylation regulators, to counteract host silencing mechanisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Vpr induces histone gene demethylation, promotes degradation of histone deacetylases like HDAC1, HDAC3, and SIRT7, and facilitates chromatin accessibility and gene activation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our ChIP-Seq assay for CTCF, we observed increased expression of \u003cb\u003eTET2, involved in gene demethylation, and DNMT3A and DNMT3B, responsible for de novo methylation. These result\u003c/b\u003es further support that Vpr may dysregulate methylation and demethylation machinery, contributing to α-Syn accumulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe precise mechanism by which Vpr promotes α-Syn accumulation remains to be fully elucidated. Vpr may directly activate SNCA transcription by demethylating its promoter, or alternatively, Vpr could indirectly promote α-Syn buildup by modulating the methylation and demethylation machinery that controls α-Syn expression. In addition, Vpr may induce cellular stress, mitochondrial dysfunction, or autophagy inhibition, which also drive α-Syn aggregation, as indicated by our previous work\u003c/b\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cb\u003eWe are investigating the relative contributions of these pathways. Clarifying these mechanistic links will establish how Vpr-induced α-Syn aggregation leads to cognitive impairments, such as spatial memory deficits, and motor dysfunction in HAND and related conditions\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAntisense promoters, especially those in introns, control gene expression in neurodegeneration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These promoters initiate long non-coding RNAs (lncRNAs) that regulate sense genes through chromatin remodeling, DNA methylation, and histone modification [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Disruption of these processes contributes to neurodegenerative diseases like Parkinson\u0026rsquo;s and Alzheimer\u0026rsquo;s. In our study, we identify the SNCA antisense promoter as essential for regulating α-Syn expression through CpG methylation, linking methylation loss to elevated α-Syn and aggregation (18\u0026ndash;20). We confirmed that inhibiting Tet with DMOG blocks SNCA antisense promoter demethylation and prevents α-Syn upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). This finding clarifies that Tet activity is required for Vpr-induced demethylation, which in turn increases SNCA-AS expression, suggesting a mechanistic link between Vpr action and α-Syn buildup. However, it remains unclear whether activating SNCA-AS alone can cause α-Syn aggregation; \u003cb\u003efuture experiments will directly test this by increasing SNCA-AS independently of Vpr using antisense oligonucleotides or CRISPR editing. We will also determine if activation of the SNCA sense promoter occurs simultaneously, directly linking SNCA-AS activity to α-Syn expression.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUnresolved questions persist regarding how Vpr influences α-Syn accumulation, specifically at which level of gene regulation this occurs. It remains unclear whether Vpr-induced epigenetic changes primarily impact α-Syn gene transcription or protein translation and processing. Additionally, chromatin structural changes, such as R-loop formation, may play a role in the regulation of α-Syn by Vpr. Clarification of these mechanistic links is needed to fully understand Vpr\u0026rsquo;s impact.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStudying the SNCA antisense promoter reveals how DNA and RNA interact. The antisense promoter in the first SNCA intron may produce LncRNAs that control the sense promoter. Chromatin remodeling, RNA-RNA interactions, and methylation work together to regulate gene expression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This type of control affects other neurodegenerative genes, such as TP53 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and BRCA1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and viral genomes, including HIV-1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. DNA demethylation of antisense promoters can reactivate them, causing abnormal gene expression and contributing to diseases like cancer, neurodegeneration, and chronic viral infections.\u003c/p\u003e\u003cp\u003eConsistent with this analysis, in brain tissues from controls, PLWH, and people with Parkinson\u0026rsquo;s (PD) or Alzheimer\u0026rsquo;s disease (AD), we see increased α-Syn and more senescence-associated heterochromatic foci (SAHF), linking α-Syn aggregation to cellular aging [31, and studies in progress]. Brain tissue analyses show more SAHF-positive and distort\u003cb\u003eed nuclei in HIV cases compared to age-matched controls, matching levels seen in AD and PD (Table S2).\u003c/b\u003e This supports the idea that HIV promotes early chromatin condensation and nuclear aging in neurons. α-Syn buildup may make neurons senescent and lead to cognitive decline in HAND. Increased mTOR activity in PLWH may boost cell stress, further increase α-Syn, and link Vpr-triggered epigenetic changes to aging. Vpr thus causes direct viral damage and drives cellular aging, helping to explain lasting motor and cognitive problems in PLWH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVpr causes DNA damage and then activates epigenetic changes that drive disease. Even in non-dividing neurons, Vpr triggers DNA damage responses (DDR)\u003c/b\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cb\u003ewhich change chromatin and DNA methylation\u003c/b\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cb\u003eOur data show that Vpr may interact with TET enzymes or their regulators. Blocking TET with DMOG stops Vpr-induced demethylation and antisense activation. We need to learn more about how Vpr interacts with these regulators. Though DDR pathways are best known in dividing cells, they also reshape methylation in neurons\u003c/b\u003e, such as at the SNCA antisense promoter. This matches patterns in other neurodegenerative diseases, where DNA repair causes reprogramming of disease genes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. So, CpG demethylation in the SNCA antisense promoter may follow DNA damage, raise α-Syn, and encourage aggregation.\u003c/p\u003e\u003cp\u003eSeveral studies support the idea that CpG demethylation, such as at CpG-2 (intron 1, especially CpG sites 8 and 12) in SNCA, contributes to α-Syn overexpression, a key factor in neurodegenerative diseases. Matsumoto et al. (2010) emphasized the role of CpG demethylation in regulating α-synuclein expression and aggregation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, Yang et al. (2017) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] showed that CpG demethylation in SNCA intron-1 leads to elevated SNCA expression in regions of Parkinson\u0026rsquo;s disease (PD) brain, including the substantia nigra pars compacta (SNpc), putamen, and cortex. Jowaed et al. (2010) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Miranda-Morales et al. (2017) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported significant demethylation of intron 1 in PD patient brain samples, which may explain the increased expression of SNCA. However, Guhathakurta et al. (2017) suggested that CpG demethylation and α-Syn accumulation are independent of PD development [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite this, data from Young et al. (2019) and Henderson (2021) confirm significant hypomethylation in genes from the substantia nigra region and blood samples from PD patients, reinforcing the association between methylation loss and PD pathology [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo assess the relevance of our findings to human neuropathology, we analyzed brain tissues from both PLWH and SIV-infected monkeys [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We observed upregulation of α-Syn and demethylation of the antisense promoter, supporting the translational significance of our in vitro results. Additionally, we identified mitochondrial dysfunction, reduced ATP production, impaired mitophagy, and increased reactive oxygen species (ROS) levels in Vpr-treated neurons [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These disturbances in cellular energy homeostasis and mitochondrial dynamics, along with ER stress and β-amyloid accumulation, indicate that Vpr-induced α-Syn accumulation impairs cellular function and activates stress pathways characteristic of neurodegenerative diseases. Deficits in mitochondrial axonal transport and microtubule stability further implicate α-Syn dysregulation as a central factor in neuronal damage and potential cognitive and motor dysfunction [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e, Vpr-treated mice displayed cognitive impairments, including deficits in spatial memory and long-term potentiation (LTP), which align with the established role of α-Syn in cognitive decline associated with HAND. Structural changes such as brain atrophy were also observed in PLWH [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], further connecting molecular alterations in SNCA regulation to neurodegeneration. These findings from animal models, together with human brain tissue data, emphasize the importance of Vpr-induced epigenetic modulation of the SNCA antisense promoter in driving neurodegenerative processes in HAND. This research elucidates the complex relationship between viral factors, epigenetic modifications, and neurodegenerative disease progression, providing a basis for developing novel therapeutic strategies for HAND and related disorders.\u003c/p\u003e\u003cp\u003eIn addition to HIV-1 Vpr, other viral proteins, such as those from SARS-CoV-2, have been shown to interact with α-Syn, inducing Lewy body-like pathology \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This suggests that viral proteins can influence α-Syn dynamics, potentially contributing to neurodegenerative processes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThese findings have important clinical implications for managing HAND in PLWH. Identifying specific epigenetic mechanisms underlying α-Syn accumulation presents new opportunities for therapeutic intervention. Targeting the Tet-mediated demethylation pathway or antisense promoter activity may reduce α-Syn aggregation and related neurodegeneration. Furthermore, monitoring α-Syn levels or the methylation status of the SNCA antisense promoter could serve as biomarkers for early detection of neurocognitive decline in PLWH, facilitating timely intervention. Future studies involving larger cohorts of PLWH will be necessary to validate these potential biomarkers and therapeutic targets.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn summary, this study provides evidence that HIV-1 Vpr causes epigenetic changes at the SNCA antisense promoter, leading to α-Syn dysregulation and aggregation. These are key factors in HIV-associated neurodegeneration. The similarities between these findings and the known role of epigenetic modifications in synucleinopathies suggest that HIV-1 infection may share mechanisms with diseases like Parkinson\u0026rsquo;s disease. This research sheds light on how Vpr-induced epigenetic changes could impact hippocampal synaptic plasticity, spatial memory, and motor function in PLWH, possibly explaining motor problems such as gait disturbances and balance issues. Further research on these epigenetic changes in larger PLWH groups may help find new biomarkers for early detection and new targets for treatment, leading to better management of HIV-related cognitive issues and neurodegeneration.\u003c/p\u003e\u003cp\u003eFinally, studying the SNCA antisense promoter reveals how viral proteins exploit host epigenetic machinery to drive neurodegeneration. Defining these interactions will uncover therapeutic targets for mitigating HAND and potentially other α-synucleinopathies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare that the research was performed in the absence of any financial relationships and has no potential conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAI ASSISTANCE\u003c/h2\u003e\u003cp\u003eThe manuscript was enhanced with the assistance of AI, specifically ChatGPT, to improve clarity, flow, and readability. AI was used exclusively for text editing and refinement, with no involvement in data analysis.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis work is supported by an NIH-NIA grant AG054411 and by previous NIH grants NS076402 and MH093331 awarded to BES.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYW, MS, DK, and JP designed and performed the studies. LK, JC, JJ, and NS provided technical assistance and reagents. JJ and LK also edited the manuscript. BES edited the manuscript, directed, and supervised the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the National NeuroAIDS Tissue Consortium (NNTC) for providing postmortem human brain tissues.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll processed data are included in this manuscript. Raw data, further information, or reagents contained within the manuscript are available upon request from the corresponding author, Maryline Santerre, [[email protected]] (mailto:[email protected]) .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThompson, L. J., Genovese, J., Hong, Z., Singh, M. V. \u0026amp; Singh, V. B. HIV-associated neurocognitive disorder: a look into cellular and molecular pathology. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 4697 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoutsilieri, E., Sopper, S., Scheller, C., Meulen, V. \u0026amp; Riederer, P. ter Parkinsonism in HIV dementia. \u003cem\u003eJ. Neural Transm. (Vienna)\u003c/em\u003e 109, 767\u0026ndash;775 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTisch, S. \u0026amp; Brew, B. 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SARS-CoV-2 spike protein 1 causes aggregation of α-synuclein via microglia-induced inflammation and mitochondrial ROS: Potential therapeutic applications of metformin. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1223 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6405901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6405901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlpha-synuclein (α-Syn) aggregation is a hallmark of neurodegenerative diseases. In individuals with HIV-1, cognitive impairments are associated with α-Syn accumulation and aggregation. The direct mechanistic link between α-Syn dysregulation and HIV-associated neurocognitive disorders (HAND) remains unclear. Emerging evidence suggests epigenetic changes, particularly DNA demethylation, play a role in α-Syn regulation. \u003cb\u003eWe show that the HIV-1 protein Vpr demethylates the antisense promoter (AS-1) within intron 1 of the SNCA gene, leading to increased α-Syn expression.\u003c/b\u003e Elevated α-Syn levels promote its aggregation, resulting in synaptic dysfunction and impaired mitochondrial transport. These processes contribute to the development of HAND. Additionally, we find that Vpr's activation of AS-1 depends on demethylation; DMOG, a Tet inhibitor, reverses this demethylation and reduces Vpr-induced activation of AS-1. Our results indicate that α-Syn dysregulation contributes to cognitive decline in people living with HIV and imply that targeting α-Syn regulatory pathways could mitigate HIV-related neurodegeneration. To our knowledge, this is the first study to demonstrate that an HIV protein epigenetically activates the SNCA antisense promoter, linking viral infection to α-synuclein deregulation. Future research should explore how AS-1 demethylation causes neuronal dysfunction and examine the broader effects of α-Syn dysregulation on neuronal health.\u003c/p\u003e","manuscriptTitle":"HIV Vpr induces demethylation of the SNCA antisense promoter leading to neurocognitive impairment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-01 11:45:12","doi":"10.21203/rs.3.rs-6405901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-10T08:25:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"189874345477069121920738665936651354414","date":"2025-11-18T20:12:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T18:58:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154642888233576264835886724912669933882","date":"2025-11-02T18:00:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T09:58:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-29T06:32:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-17T03:12:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a813e079-161c-4d51-86f8-b3c2ed730aa6","owner":[],"postedDate":"November 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57151343,"name":"Biological sciences/Neuroscience"},{"id":57151344,"name":"Biological sciences/Neuroscience/Diseases of the nervous system"},{"id":57151345,"name":"Biological sciences/Neuroscience/Epigenetics in the nervous system"}],"tags":[],"updatedAt":"2026-01-26T16:04:36+00:00","versionOfRecord":{"articleIdentity":"rs-6405901","link":"https://doi.org/10.1038/s41598-026-35691-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-23 15:58:15","publishedOnDateReadable":"January 23rd, 2026"},"versionCreatedAt":"2025-11-01 11:45:12","video":"","vorDoi":"10.1038/s41598-026-35691-3","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35691-3","workflowStages":[]},"version":"v1","identity":"rs-6405901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6405901","identity":"rs-6405901","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}