{"paper_id":"0e62cf9a-ef88-4f29-b0ec-0c169d910141","body_text":"Original Research article 1 \nHIV and Cocaine exposure promote Tau phosphorylation 2 \nthrough RSK-1 in a GSK3β-independent manner. 3 \nAdhikarimayum Lakhikumar Sharma 1, Ilker K. Sariyer 2, Ulhas P. Naik 1, and Mudit Tyagi 1* 4 \n1 Department of Medicine, Center for Translational Medicine, Sidney Kimmel Medical College, Thomas Jefferson 5 \nUniversity, 1020 Locust Street, Jefferson Alumni Hall, Philadelphia, PA 19107, USA 6 \n2 Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing, Temple 7 \nUniversity Lewis Katz School of Medicine, Philadelphia, PA 19140, USA. 8 \n*Correspondence: mudit.tyagi@jefferson.edu (M.T); Tel.: +1 215-503-5157, +1 609-509-6709 9 \nAbstract 10 \nHIV and cocaine are known to disrupt neuronal signaling and contribute to neurocognitive dysfunction, yet 11 \nthe underlying molecular mechanisms are not clear . In this study, we delineate the underlying molecular 12 \nmechanism by which HIV and/or cocaine enhance Tau phosphorylation (p -Tau S396), a marker of Tau -13 \nmediated neuropathies. Furthermore, we elucidate how these two independent neuropathogenic factors , 14 \ncocaine and HIV , exploit distinct yet convergent signaling pathways to drive this pathological event. We 15 \ndemonstrate that HIV robustly activates and upregulates RSK1, which functions upstream of AKT and 16 \npromotes Tau phosphorylation through an AKT -independent mechanism while simultaneously inactivating 17 \nGSK3β via serine -9 phosphorylation (p -GSK3β S9). However, cocaine not only activates RSK1 but also 18 \nstrongly stimulates AKT1, resulting in sustained GSK3 β inhibition and persistent Tau phosphorylation. 19 \nNotably, Tau phosphorylation persists even under conditions of GSK3β inactivation in both HIV and cocaine 20 \nexposure, revealing a previously unrecognized GSK3 β-independent mechanism of Tau modification. 21 \nCollectively, these findings identify RSK1 as the primary mediator of Tau phosphorylation upon HIV and/or 22 \ncocaine exposure,  and uncover a novel RSK1 -driven, GSK3 β-independent pathway contributing to 23 \nTauopathy. Through a combination of immunofluorescence, immunoblotting, genetic knockout, and 24 \noverexpression approaches, we establish RSK1 as a central signaling hub linking the AKT-GSK3β pathway 25 \nto Tau phosphorylation. We demonstrate that RSK1 operates as a critical upstream regulator of AKT and 26 \nGSK3β signaling, playing dual roles, both activating AKT and suppressing GSK3β, thereby uncovering a novel 27 \nlayer of pathways that regulates Tau phosphorylation. The reproducibility of these main signaling pathways 28 \nacross SH-SY5Y neurons, mixed cell 3D spheroids, and human brain organoids underscores the robustness 29 \nand biological relevance of this mechanism.  Collectively, these findings reveal mechanistic convergence of 30 \nHIV and cocaine on RSK1-dependent signaling and provide critical insight into how diverse neuropathic / 31 \nneuropathological factors remodel neuronal signaling to drive Tau-associated dysfunction. These findings 32 \nprovide novel mechanistic insight into the molecular underpinnings of neuro -HIV and substance abuse 33 \nassociated Tauopathy. By identifying RSK1 as a master regulator and demonstrating that Tau phosphorylation 34 \ncan bypass GSK3β inhibition, our study advances understanding of signaling complexity and highlights new 35 \nopportunities for therapeutic intervention. Targeting RSK1 may represent a promising strategy to mitigate Tau 36 \npathology, induced due to insoluble aggregates of phosphorylated Tau, a common factor promoting cognitive 37 \ndecline not only in individuals with Alzheimer’s disease but also in those exposed to cocaine or/and infected 38 \nwith HIV. 39 \nSignificances 40 \nThis study demonstrates that exposure to HIV and/or cocaine induces Tau phosphorylation at serine 396 41 \n(S396), a well -established marker of Tau pathology, and delineates how these two independent 42 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nneuropathogenic factors engage distinct yet convergent signaling pathways to drive this pathogenic event. 43 \nWe show that HIV exposure drives robust RSK1 activation, positioning it upstream of AKT to promote Tau 44 \nphosphorylation via an AKT-independent mechanism, while concurrently suppressing GSK3β activity through 45 \nserine-9 phosphorylation. In contrast, cocaine, while only moderately activating RSK1, primarily enhances 46 \nAKT signaling, leading to sustained GSK3 β inhibition and increased Tau phosphorylation. Notably, Tau 47 \nphosphorylation persists even under conditions of GSK3β inactivation in both settings, revealing a previously 48 \nunrecognized, RSK1 -centered, GSK3 β-independent pathway of Tau modification.  Overall, our  findings 49 \ndemonstrate that Tau phosphorylation in the context of HIV infection and cocaine exposure is a complex, 50 \nmulti-layered regulatory process involving multiple signaling nodes. Importantly, we identify RSK1 as a central 51 \nintegrative hub linking viral and substance -induced signaling to downstream Tau pathology. This work 52 \nadvances our understanding of the molecular mechanisms underlying neuroHIV and substance abuse –53 \nassociated neurodegeneration. Furthermore, it highlights RSK1 as a novel and promising therapeutic target 54 \nfor mitigating Tauopathy in both cocaine-using and non-using people with HIV (PWH). 55 \nHighlighted points 56 \n• RSK1 acts as a central regulator of Tau phosphorylation, capable of driving this process through a GSK3β-57 \nindependent mechanism.  58 \n• HIV promotes Tau phosphorylation primarily via robust upregulation and activation of RSK1, operating 59 \nlargely independent of AKT1, while concurrently inducing GSK3β inactivation.  60 \n• Drugs of abuse, such as cocaine induces Tau phosphorylation through dual activation of AKT1 and RSK1, 61 \nalongside sustained inactivation of GSK3β.  62 \n• Tau phosphorylation persists despite GSK3β inhibition, revealing a complex AKT1 -RSK1 signaling axis 63 \nand underscoring the dominant role of GSK3β-independent mechanisms in Tau pathology following HIV 64 \nand cocaine exposure.  65 \n• HIV and cocaine engage distinct yet convergent signaling pathways that disrupt neuronal homeostasis 66 \nand drive tauopathy, providing mechanistic insight into neuroHIV and substance abuse -associated 67 \nneurodegeneration.  68 \n• RSK1 functions as a key upstream modulator of AKT and GSK3β pathways, positively regulating AKT 69 \nsignaling while negatively regulating GSK3β activity.  70 \n• RSK1 emerges as a potential therapeutic target, offering new opportunities for intervention in HIV -71 \nassociated neurocognitive disorders (HAND) and drug-induced neurodegeneration. 72 \n• Established and characterized H80 cells as a novel neuronal cell model and demonstrated their suitability 73 \nfor studying neuron-specific signaling pathways, including Tau phosphorylation. 74 \n• The conserved and widespread nature of the signaling cascade driving Tau phosphorylation in response 75 \nto HIV and/or cocaine exposure was validated across multiple model systems, including both 2D neuronal 76 \ncell cultures and 3D systems such as human brain organoids and spheroids. 77 \n 78 \nStrength of the Study 79 \nThis original study provides novel mechanistic insight into how HIV and cocaine, two independent 80 \nneuropathological factors, converge and diverge on intracellular signaling pathways to regulate Tau 81 \nphosphorylation. By integrating immunofluorescence, immunoblotting, genetic knockout, and overexpression 82 \napproaches, we identif ied RSK1 as a master regulator of Tau phosphorylation. Importantly, we discovered 83 \nthat HIV robustly upregulates and activates RSK1 to promote Tau phosphorylation through an AKT -84 \nindependent route while simultaneously inactivating GSK3β. On the other hand, cocaine exerts a moderate 85 \neffect on RSK1 but strongly stimulates AKT to induce GSK3β inactivation and drive Tau phosphorylation. A 86 \nkey strength of this work is the discovery that Tau phosphorylation persists despite GSK3 β inactivation, 87 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nrevealing a complex, GSK3β-independent mechanism, involving RSK1 in Tau pathology. Moreover, our study, 88 \nfor the first time, identify RSK1 as an upstream regulator of AKT-GSK3β signaling cascade, enhancing AKT 89 \nsignaling while simultaneously inhibiting GSK3β activity, thereby underscoring the critical role of RSK1 in Tau 90 \nphosphorylation and associated illnesses, such as HAND and Alzheimer’s disease. Together, these findings 91 \nnot only advance our understanding of the molecular underpinnings of neuroHIV and substance abuse  92 \nassociated tauopathy but also highlight RSK1 as a promising therapeutic target for not only HIV and cocaine 93 \ninduced neurotoxicity but also other neurodegenerative diseases, such as Alzheimer’s disease. Another key 94 \nstrength of this study is the establishment and characterization of H80 cells as a novel neuronal model, 95 \ndemonstrating their suitability for investigating neuron -specific signaling pathways, including Tau 96 \nphosphorylation. The combination of comparative signaling analysis, genetic perturbations, and integrative 97 \nmechanistic modeling makes this study both conceptually and technically novel, besides broadly relevant to 98 \nthe fields of neurovirology, addiction neuroscience, neurodegeneration, and cognitive impairments. 99 \nIntroduction 100 \nHuman immunodeficiency virus (HIV) infection remains a significant global health concern, with an estimated 101 \n38 million people currently living with the virus worldwide  [1]. Although the introduction of combination 102 \nantiretroviral therapy (ART) has markedly improved life expectancy and viral suppression in people with HIV 103 \n(PWH), the burden of HIV -associated neurocognitive disorders (HAND) persists  [2, 3] . HAND affects 104 \napproximately 30-50% of PWH, even in those achieving robust viral suppression on ART [4]. The etiology of 105 \nHAND is multifactorial and complex, involving persistent neuroinflammation,  the activity of  neurotoxic viral 106 \nproteins (e.g., Tat, gp120), and dysregulation of host signaling pathways that collectively disrupt synaptic 107 \nintegrity and neuronal function [3]. Importantly, while neurons are the primary cells affected in HAND, glial 108 \ncell population in the central nervous system (CNS), are increasingly recognized as key mediators of HAND-109 \nrelated neuropathology [5, 6]. 110 \nThe microtubule-associated protein Tau is a central regulator of neuronal function, ensuring the stability of 111 \naxonal microtubules and supporting efficient transport of cargo essential for synaptic activity and neuronal 112 \nsurvival [7, 8] . Under physiological conditions, Tau protein undergoes tightly regulated cycles of 113 \nphosphorylation and dephosphorylation that allow dynamic modulation of cytoskeletal structure. However, 114 \ndisruption of this phosphorylation event (Tau hyperphosphorylation) gives rise to neurodegenerative disease 115 \n[7, 9, 10]. Aberrantly phosphorylated Tau exhibits diminished binding to microtubules, misfolds into abnormal 116 \nconformations, and progressively accumulate into insoluble neurofibrillary tangles [11, 12]. These inclusions 117 \nnot only serve as histopathological hallmarks of Alzheimer’s disease (AD) and related tauopathies but also 118 \ncorrelate strongly with synaptic dysfunction, neuronal loss, and the severity of cognitive decline  [13, 14]. 119 \nMultiple factors contribute to the pathological transformation of Tau, ultimately driving its involvement in AD 120 \nand related dementias [15, 16]. Emerging evidence suggests that HIV infection, even in individuals receiving 121 \nsuppressive antiretroviral therapy, can disrupt the physiological regulation of Tau phosphorylation. Since 122 \nneurons do not express canonical HIV entry receptors such as CD4 and co-receptors CCR5 or CXCR4, they 123 \nare not infected by the virus  [17, 18] . Nevertheless, neuron cells remain profoundly susceptible to the 124 \ndownstream consequences of viral exposure. Instead of direct infection, neuronal injury arises predominantly 125 \nthrough indirect mechanisms, most notably the actions of soluble viral proteins , mainly Tat and gp120, and 126 \nreleased cytokines from infected glial or immune cells  [19, 20] . These cytotoxic factors dysrupt cellular 127 \nhomeostasis and interfere with host kinase -phosphatase signaling cascades, leading to dysregulated Tau 128 \nphosphorylation events that compromise cytoskeletal integrity, axonal transport, and synaptic stability  129 \nultimately leading to HAND, and related tauopathies.  130 \nCocaine, one of the most prevalent substances abused among PWH, is a well -established cofactor in the 131 \nprogression of HAND  [21, 22] . Cocaine abuse independently exacerbates neurodegenerative processes, 132 \naccelerates cognitive decline, and has been associated with increased susceptibility to HIV infection and 133 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nreplication within the CNS [22-24]. Mechanistic studies suggest that cocaine induces oxidative stress, disrupts 134 \nblood-brain barrier integrity, activates glial cells, and modulates multiple signaling pathways, including those 135 \ngoverning inflammation and cell survival.  Our recent studies further reveal that cocaine use enhances HIV 136 \ntranscription by activating transcription factors such as NF-κB and MSK1, altering epigenetic modifications at 137 \nthe long terminal HIV repeat (LTR) promoter [24, 25].  138 \nBeyond transcriptional activation, we have shown that cocaine increases the susceptibility of CD4⁺ T cells to 139 \nHIV infection by augmenting key co-stimulatory signaling pathways , involving, NF -kB, NFAT and AP -1, 140 \nthereby creating a cellular state more favorable to viral entry and replication  [25-27]. Additionally, we have 141 \ndemonstrated that cocaine activates DNA-dependent protein kinase (DNA-PK) in both T cells and microglial 142 \ncells, which alleviates RNA polymerase II pausing at the LTR. This effect is mediated through phosphorylation 143 \nof TRIM28, a chromatin -associated repressor, thus enabling more efficient transcriptional elongation and 144 \nsustained viral gene expression  [28, 29]. These molecular changes establish a favorable environment for 145 \npersistent HIV activity and may synergize with host signaling dysregulation to exacerbate neuropathology, 146 \nparticularly within the central nervous system. 147 \nNeurodegeneration is the consequence of dysregulated intracellular signaling, in which kinases play a crucial 148 \nrole [30]. In the healthy normal brain, a balance between kinases and phosphatases ensures proper regulation 149 \nof cytoskeletal dynamics, synaptic activity, and stress adaptation. Disruption can cause series of  150 \nphosphorylation events that promote neuronal dysfunction  [31]. Therefore, several cellular pathways have 151 \nbeen involved in the regulation of Tau phosphorylation ultimately leading to neurodegeneration. However, 152 \nglycogen synthase kinase 3 beta (GSK3β), a serine/threonine kinase that directly phosphorylates Tau at 153 \nmultiple pathological sites remain a major kinase [32, 33]. GSK3β activity is inhibited by phosphorylation at 154 \nserine 9 (Ser9), a modification typically mediated by the upstream kinase AKT (also known as protein kinase 155 \nB), a central node in cell survival, metabolism, and growth signaling  [34]. Dysregulation of this AKT-GSK3β 156 \naxis has been consistently reported in models of tauopathy, and other neurodegenerative conditions, 157 \nunderscoring its pathogenic significance  [35, 36]. In addition to GSK3β, several other kinases  have been 158 \nshown to  phosphorylate Tau, including Cyclin-dependent kinase 5 ( CDK5), Extracellular signal-regulated 159 \nkinases ( ERK1/2), c-Jun N -terminal kinase ( JNK), p38 MAPK, Microtubule affinity -regulating kinases 160 \n(MARKs), AMP-activated protein kinase ( AMPK) [37, 38] , Protein kinase A ( PKA), Calcium/calmodulin-161 \ndependent kinase II (CaMKII) [39], and Protein kinase C (PKC), act on overlapping sets of phosphorylation 162 \nsites and often respond to cellular stress signals such as inflammation, oxidative damage, and excitotoxicity. 163 \nDysregulation of the MAPK/ERK signaling pathway has been strongly associated with neurodegenerative 164 \ndisorders, including AD, where abnormal kinase activity contributes to synaptic dysfunction, Tau 165 \nhyperphosphorylation, and neuronal loss [40, 41].  166 \nRibosomal S6 kinase 1 (RSK1, encoded by RPS6KA1) is known to be a  key downstream effector of 167 \nMAPK/ERK pathway and plays important roles in regulating cell growth, survival, and gene expression  [42, 168 \n43]. Despite its central role in MAPK/ERK signaling, RSK1 has not yet been systematically investigated in the 169 \ncontext of Tau phosphorylation or AD, and direct evidence linking its dysregulation to disease onset or 170 \nprogression remains limited. This gap in knowledge prompted us to investigate this aspect in detail and define 171 \nif RSK1 is an underexplored contributor to the molecular mechanisms underlying Tauopathy responsible for 172 \nAD and related neurodegenerative conditions. Additionally, several of the aforementioned kinases have been 173 \nextensively studied in AD and other tauopathies, their specific contributions to Tau dysregulation during HAND 174 \nremain poorly understood. Viral proteins such as Tat and gp120 are known to disrupt intracellular signaling, 175 \nyet the precise mechanisms by which they interact with the kinase -phosphatase networks that regulate Tau 176 \nremain unclear. Elucidating the signaling pathways through which Tau pathology accelerates  HAND is 177 \ntherefore a critical research priority. Addressing this knowledge gap will not only advance our mechanistic 178 \nunderstanding of HAND pathogenesis but may also reveal convergent therapeutic targets relevant to both 179 \nclassical and virally mediated neurodegenerative disorders, including AD and HAND.  180 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nSelecting an appropriate model system is critical for studying neurodegenerative diseases, as it directly 181 \ninfluences the reliability, reproducibility, and translational relevance of the findings. While primary neurons 182 \noffer high physiological relevance, they are short-lived, fragile, and technically challenging to maintain under 183 \nin vitro conditions, highlighting the need for alternative neuron -like systems that are more experimentally 184 \ntractable yet retain key neuronal features [44]. Glioma cell lines provide a robust, practical, and experimentally 185 \ntractable platform to investigate mechanisms of neurodegeneration due to their neural origin, robust growth 186 \nproperties, and retention of signaling pathways relevant to function and disease pathology [45, 46]. Unlike 187 \nprimary neurons, which are post-mitotic and difficult to maintain long term, glioma cells readily expand in vitro, 188 \nenabling reproducible experiments and large-scale molecular and pharmacological studies [47]. Importantly, 189 \nthese cells retain critical signaling pathways relevant to disease pathology, such as MAPK/ERK and PI3K/AKT 190 \nsignaling, oxidative stress responses, and glial -neuronal interactions, all of which are central to the 191 \nprogression of neurodegenerative disorders  [48-50]. Since glial dysfunction and altered kinase signaling 192 \ncontribute significantly to synaptic loss, protein aggregation, and neuronal death, glioma cells serve as a 193 \npractical surrogate model to dissect these mechanisms. Although glioma cells cannot fully replicate the 194 \ncomplexity of the CNS, especially neuronal and glial interactions in vivo, their tractability and physiological 195 \nrelevance make them a useful and valuable model for mechanistic studies of neurodegeneration, as well as 196 \ntesting the potential therapeutics. 197 \nIn this study , using both 2D and 3D neuronal model systems, we delineate the molecular mechanisms 198 \nunderlying Tau phosphorylation in response to HIV infection and/or cocaine exposure. We demonstrate that 199 \nHIV exposure robustly upregulates and activates RSK1, which in turn inactivates GSK3β through an AKT -200 \nindependent mechanism. Activated RSK1 directly promotes Tau phosphorylation. On the other hand, cocaine 201 \nexposure not only induces RSK1 but also strongly activates AKT1, leading to GSK3β inactivation through 202 \nphosphorylation at serine 9 (p -GSK3β S9) in an AKT -dependent manner, thereby further enhancing Tau 203 \nphosphorylation. Notably, Tau phosphorylation persists even under conditions of GSK3β inhibition during both 204 \nHIV and cocaine exposure, indicating that Tau modification is primarily driven through an RSK1 -centered, 205 \nGSK3β-independent pathway. These findings highlight the pivotal role of RSK1 and underscore the 206 \ncomplexity of the signaling networks regulating Tau phosphorylation. Using complementary genetic and 207 \npharmacological approaches, including CRISPR/Cas9 -mediated knockout, overexpression systems, and 208 \nselective kinase inhibition, we further establish that RSK1 functions upstream of both AKT activation and 209 \nGSK3β inactivation, exerting context -dependent effects on Tau phosphorylation.  Collectively, our findings 210 \nhighlight the kinase signaling crosstalk underlying HAND and cocaine-associated tauopathy, identifying RSK1 211 \nas a mechanistic hub and potential therapeutic target for neurotoxicity and HAND in PWH, including those 212 \nwho use illicit substances, such as cocaine.  213 \nRunning Title: - Signaling Crosstalk Underlying Tauopathy during HIV infection and Cocaine Abuse 214 \nKeywords: - HIV, Cocaine, Tau phosphorylation, RSK1, AKT, GSK3β.   215 \nMaterials and Methods 216 \nCell Culture 217 \nH80 cells (originally obtained from the Darell Bigner Laboratory, Duke University)  [51] were maintained in 218 \nDMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Jurkat 219 \nT cells (human CD4 ⁺ T lymphocyte line; ATCC TIB ‑152), MT -4 cells  and U937 cells were cultured in 220 \nRPMI‑1640 supplemented with 10% FBS, 1% penicillin–streptomycin, and 2 mM L‑glutamine. HEK293T cells, 221 \nmicroglial cells, and SH ‑SY5Y neuroblastoma cells were propagated in Dulbecco’s modified Eagle medium  222 \n(DMEM) containing 10% FBS, 1% penicillin –streptomycin, and 2 mM L ‑glutamine. All cell lines were 223 \nmaintained at 37 °C in a humidified incubator with 5% CO2 and were used between passages 3 and 8. 224 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nInhibitor Treatments 225 \nH80 cells [51] were seeded and allowed to adhere overnight prior to treatment. Cells were incubated with 226 \nselective inhibitors targeting RSK1 or GSK3β, at a final concentration of 10 µM each. The RSK1 inhibitor BI-227 \nD1870 (Selleckchem, Cat. No. S2843), and GSK3β inhibitor CHIR99021 (Tocris Bioscience, Cat. No. 4423), 228 \nwere prepared as stock solutions in Dimethyl sulfoxide (DMSO) and prepared working solutions at 10 mM. 229 \nCells were treated with inhibitors or equivalent volumes of DMSO vehicle control for 24 hours at 37°C in a 230 \nhumidified 5% CO₂ incubator. Following treatment, cells were either exposed to HIV or cocaine or both and 231 \nharvested for downstream analyses including Immunoblotting. All treatments were performed in experimental 232 \ntriplicate or biological triplicate to ensure reproducibility. 233 \nCocaine Treatment 234 \nCells were treated with 10 µM cocaine hydrochloride. For acute exposure, treatments were applied for 235 \ndurations ranging from 15 minutes up to 6 hours. Otherwise, specifically mentioned all the treatments are 236 \ndone chronically. For chronic exposure, cells received two treatments each day randomly for 48 hours and at 237 \nleast 30 min-3h prior cells harvesting. Control cells were treated with PBS or kept untreated. 238 \nHIV Virus Production and Infection 239 \nJurkat cells or MT-4 cells were infected with replication -competent Human Immunodeficiency Virus Type 1 240 \n(strain 93/TH/051, R5- and X4-tropic virus) (NIH AIDS Reagent Program) by spinoculation at 1,200 × g for 2 241 \nh at 25°C in the presence of 8 µg/mL Polybrene. Following infection, cells were incubated for 48 h -72 h. 242 \nSupernatants containing HIV virions were harvested, cleared by low-speed centrifugation (500 × g, 10 min), 243 \nfiltered through a 0.45 µm syringe filter, and stored at −80 °C. HIV production was confirmed by 244 \nimmunoblotting for the HIV p24 capsid protein.  245 \nH80 Exposure to HIV  246 \nH80 were seeded and exposed to HIV-containing supernatant or in normal medium (control) by spinfection at 247 \n1,000 rpm for 2 h at room temperature  (RT). The following day, cells underwent a second spinfection under 248 \nthe same conditions and were subsequently transferred to 100 -mm dishes. After 48 h, cells were harvested 249 \nfor protein analysis. Control H80 cells were processed identically with supernatant from uninfected Jurkat/MT-250 \n4 cells. 251 \nLentiviral production and CRISPR/Cas9-mediated RPS6KA1 knockout 252 \nLentiviral particles encoding Cas9 were produced by co‑transfecting HEK293T cells with either lentiCRISPR 253 \nv2 (Addgene #52961) or lentiCas9 ‑Blast (Addgene #52962), a gift from Feng Zhang [52], together with the 254 \npackaging plasmid psPAX2 (Addgene #12260), and the envelope plasmid pMD2.G (Addgene #12259), a gift 255 \nfrom Didier Trono using Lipofectamine 2000 (Thermo Fisher Scientific). Viral supernatants were harvested 256 \n48 h post‑transfection, clarified through a 0.45‑µm filter, and used immediately or stored at −80 °C. H80 cells 257 \nwere transduced in the presence of 8 µg/mL polybrene and selected with puromycin (1–2 µg/mL)/ blasticidin 258 \nto generate stable Cas9 ‑expressing populations. To disrupt  RPS6KA1, Cas9 ‑positive H80 cells were 259 \nsubsequently transduced with lentiviral particles encoding one of three independent RPS6KA1 ‑targeting 260 \nsgRNAs (BRDN0001148481, Addgene #75499; BRDN0001145974, Addgene #75497; BRDN0001148103, 261 \nAddgene #75498), originally developed by John Doench and David Root [53]. These sgRNA lentiviruses were 262 \nproduced in HEK293T cells using the same Lipofectamine‑based system. Following transduction, cells were 263 \nallowed to recover for 48 h and then selected with puromycin (1 –2 µg/mL) for 3 –5 days to obtain 264 \nRSK1‑knockout populations. 265 \nOverexpression in H80 Cells 266 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFor RSK1 overexpression, H80 cells were seeded at 60 –70% confluence in 60 mm plates and transiently 267 \ntransfected with 2 µg of a CMV promoter -driven full-length human RSK1 expression plasmid ( pKH3-human 268 \nRSK1, Addgene cat no #13841, a gift from John Blenis [54]) or empty vector control using Lipofectamine 2000 269 \n(Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, plasmid DNA and 270 \nLipofectamine reagent were diluted separately in Opti-MEM (Gibco), combined, and incubated for 30 minutes 271 \nat RT before adding drop by drop to cells. Cells were incubated with the transfection complexes for 6 hours, 272 \nafter which the medium was replaced with fresh growth medium. Protein lysates were harvested 48 hours 273 \npost-transfection using lysis buffer with protease and phosphatase inhibitors (Roche). Overexpression 274 \nefficiency was confirmed by immunoblotting with anti-RSK1 antibodies. 275 \nSpheroids formation 276 \nThree-dimensional spheroids were generated using 96 -well round -bottom Biofloat 3D cell culture plates 277 \n(Sarstedt, Cat. No. 83.3925.400), which provides a non -adhesive surface to promote uniform spheroid 278 \nformation. To prevent cell attachment, each well was pre -treated with 60 µL of Anti -Adherence Rinsing 279 \nSolution (AARS; Stemcell Technologies, Cat no #07010) and incubated under sterile conditions at RT for 24 h. 280 \nFollowing incubation, the AARS was aspirated and stored it for potential reuse, and wells were rinsed with 281 \n100 µL of phosphate -buffered saline (PBS) to remove residual solution. Prepared plates were either used 282 \nimmediately or stored in sterile bags at 4 °C for up to two weeks. For spheroid assembly, a mixed cell 283 \nsuspension containing H80 cells, SH-SY5Y neuroblastoma cells, and microglia (5,000 cells of each type) was 284 \nprepared in 150 µL of DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. This suspension 285 \nwas dispensed into each treated well, and plates were incubated at 37 °C in a humidified atmosphere with 286 \n5% CO₂ for 24 h to allow initial aggregation and spheroid formation. After 48 h of culture, 100 µL of medium 287 \nwas carefully removed from each well and replaced with fresh medium containing HIV virus to initiate infection 288 \nor exposure. Spheroids were incubated for 5 h under the same conditions, after which the medium was 289 \nexchanged for fresh medium containing either cocaine or no treatment. Cocaine was administered twice daily 290 \nfor 48 h to mimic repeated exposure. At the end of the treatment period, spheroids were harvested by pooling 291 \n24 spheroids into a single Falcon tube, representing one biological sample. A total of 96 spheroids were 292 \ncollected, corresponding to four experimental groups: untreated control (24 spheroids), cocaine -treated (24 293 \nspheroids), HIV -exposed/infected (24 spheroids), and HIV -exposed/infected plus cocaine -treated (24 294 \nspheroids) (spheroid figure in Supplementary). Each pooled sample was washed with 1 mL PBS to remove 295 \nresidual medium and treatment compounds, followed by addition of 80 µL of 1× passive lysis buffer (Promega 296 \nE1941) to facilitate cell lysis and protein extraction for Immunoblot analyses. 297 \nGeneration of human cerebral organoids (hCOs)  298 \nHuman cerebral organoids (hCOs) were generated from human induced pluripotent stem cells (hiPSCs) 299 \nfollowing our previously established protocols, as described in detail in reference [55]. Briefly, human induced 300 \npluripotent stem cells (hiPSCs), derived from dermal fibroblasts, were used to generate hCOs following 301 \nSTEMdiff™ protocols (STEMCELL Technologies). The cells were plated in ultra-low attachment 96-well plates 302 \nat 11,000 cells per well and incubated for 24 hours to form embryoid bodies (EBs). As the EBs grew to 400–303 \n600 μm over about 5 days, they were transferred to 24 -well plates and cultured in induction medium for 48 304 \nhours. The EBs were then embedded in Matrigel and moved to 6-well plates with expansion medium, where 305 \nthey developed neuroepithelial structures after 3 days. Finally, the organoids were matured on an orbital 306 \nshaker at 70 rpm in maturation medium at 37°C for an additional 40 days. 307 \nImmunoblotting 308 \nTotal cell lysates were prepared using 1X Passive Lysis Buffer (Promega E1941) supplemented with protease 309 \nand phosphatase inhibitor cocktails (Roche), following the manufacturers’ instructions. Following cell 310 \nharvesting, lysates were incubated on ice for 30 minutes with intermittent vortexing for 30 seconds every 311 \n10 minutes to facilitate complete lysis. For the Spheroids and organoids, samples were lysed by mechanical 312 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\ndisruption with passive lysis buffer through repeated passage through a 200 µL pipette tip, followed by eight 313 \ncycles of rapid freeze–thaw in liquid nitrogen and a 37 °C water bath. The lysates were then incubated on ice. 314 \nAfter incubation, samples were centrifuged at maximum speed (≥14,000 × g) for 30 minutes at 4 °C to pellet 315 \ncell debris. The resulting supernatants were collected, and protein concentrations were determined using the 316 \nPierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).  Protein concentration was normalized, and an 317 \nequal amount of protein was mixed with 5X Laemmle Sample buffer, heated to 95ºC for 10 min, and then 318 \nresolved by SDS -PAGE on a 9% or 10% or 12% gel at 120 volts until the dye reached the bottom. The 319 \nresolved proteins were then transferred to a nitrocellulose membrane (Amersham). Membranes were blocked 320 \nfor 1 h at RT in 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), 321 \nfollowed by overnight incubation at 4 °C with primary antibodies against phospho-RSK1 Ser380 (sc-136476), 322 \nphospho-RSK1 thr348 (sc-101770), phospho-p90RSK (Thr359/Ser363) (CST#9344), RSK1 (CST #9347), 323 \nRSK1/2/3  (CST #14813), phospho-AKT T308 (CST #4056), phospho-AKT S473 (CST #4060), AKT1 (CST 324 \n#2938), phospho-GSK3β S9 (CST #5558), GSK3β (CST #12456), phospho-Tau (CST #9632S), Tau (CST 325 \n#46687), MAP2 (17490-1-AP), GAPDH (sc-25778), and β-actin (Sigma-Aldrich A5316). After three washes 326 \nwith 1X TBST, the blot was detected using the Odyssey infrared imaging system application software 3.0 (Li-327 \ncor Bioscience). 328 \nRNA Extraction and Quantitative PCR (qPCR) 329 \nTotal RNA was extracted from H80 cells after 24 h of HIV exposure using the RNeasy Plus Mini Kit (Qiagen) 330 \nfollowing the manufacturer’s protocol, ensuring elimination of genomic DNA contamination. RNA purity and 331 \nconcentration were confirmed by nanodrop and RNA gel electrophoresis. Complementary DNA (cDNA) was 332 \nsynthesized from 1 µg of total RNA using the High -Capacity cDNA Reverse Transcription Kit (Applied 333 \nBiosystems). Quantitative PCR was performed using SYBR Green on a QuantStudio 5 Real -Time PCR 334 \nsystem (Applied Biosystems) with gene-specific primers for IL-1β, TNF-α, RSK1, and GAPDH as an internal 335 \ncontrol. Relative gene expression was quantified by the 2^−ΔΔCt method, normalizing target gene expression 336 \nto GAPDH and comparing HIV exposed to controls (exposed without HIV). All reactions were conducted in 337 \ntechnical triplicates across at least three biological replicates. 338 \nImmunofluorescence staining and imaging.  339 \nTo characterize the H80 cells, H80 cells were cultured on sterile coverslips , which was initially treated with 340 \nPolyD Lysine and allowed to adhere overnight. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 341 \n30 minutes at RT, followed by permeabilization with 0. 25% Triton X-100 in PBS for 10 minutes at RT. After 342 \npermeabilization, cells were washed thrice with PBS and then incubated with a blocking solution containing 343 \n10% horse serum and 2% BSA in PBS for 60 minutes at RT to reduce non -specific binding. Subsequently, 344 \ncells were washed and then incubated overnight at 4°C with directly conjugated primary antibodies: anti-NeuN 345 \nAlexa Fluor 647 (Cat. No. 608453, BioLegend), p-Tau S396 (Phospho-Tau (Ser396) (PHF13) Mouse mAb 346 \n#9632)/anti-Tau phospho ser396 (BioLegend #829001) and MAP2 Polyclonal antibody (proteintech, 17490-347 \n1-AP). The following day, samples were washed three times with PBS, incubated with secondary for 1 h and 348 \ncounterstained with Hoechst (300 nM in PBS) for 10 minutes at RT, followed by an additional three PBS 349 \nwashes. Coverslips were mounted using Aqua -Mount mounting medium (Epredia, Cat. No. 13800) and 350 \nimaged using an EVOS M7000 Imaging System (Cat no. AMF7000) equipped with 20× and 40× oil immersion 351 \nobjectives. 352 \nTo investigate the cellular effects of cocaine and HIV exposure, exposed cells were fixed with 4% PFA in PBS 353 \nfor 15 minutes at RT. After washing the cell thrice in PBS, Fixed cells were permeabilized with 0.25% Triton 354 \nX-100 in PBS for 10 minutes at RT, then incubated for 1 hour in blocking buffer composed of 10% horse serum 355 \nand 2% BSA in PBS. Primary antibodies against phosphorylated Tau (Phospho-Tau (Ser396) (PHF13) Mouse 356 \nmAb #9632), RSK-1 (CST #9333), p-GSK3β S9 (CST #5558), p-AKT S473 (CST #4060), AKT1 (CST #2938), 357 \nwere incubated overnight at 4°C. The next day, cells were washed thoroughly to remove unbound antibodies 358 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nand subsequently incubated with species -specific, fluorophore -conjugated secondary antibodies for 45 359 \nminutes at RT in the dark. Nuclei were counterstained with Hoechst for 10 minutes at RT, followed by three 360 \nPBS washes. Coverslips were mounted using Aqua Mount (Epredia, cat. no. 13800), and samples were 361 \nimaged using an EVOS imaging system equipped with 10x, 20x and 40x objectives.  362 \nFlowcytometry 363 \nH80 cells were analyzed for surface expression of the HIV entry receptors CD4, CCR5, and CXCR4 using 364 \nmulticolor flow cytometry. Cells were harvested, washed with PBS containing 2% FBS, and incubated with 365 \nfluorochrome‑conjugated monoclonal antibodies for 30 minutes at 4 °C in the dark. To assess co‑expression 366 \nof CD4 and CXCR4, cells were stained with APC anti ‑human CD4 (BioLegend, cat. no. 317416) and PE 367 \nanti‑human CD184 (CXCR4) (BioLegend, cat. no. 306505). For CD4 and CCR5 co ‑staining, cells were 368 \nincubated with PE anti ‑human CD4 (BioLegend, cat. no. 357403) and APC/Cyanine7 anti ‑human CD195 369 \n(CCR5) (BioLegend, cat. no. 359110). Following staining, cells were washed, resuspended in PBS, and 370 \nanalyzed on a flow cytometer. Data acquisition and compensation were performed using standard instrument 371 \nsettings, and analysis was conducted with FlowJo software (BD Biosciences).  372 \nDensitometry and Statistical Analysis 373 \nAll experiments were performed with a minimum of three independent biological replicates  and/or 374 \nexperimental triplicates. Immunoblots were quantified using ImageJ (NIH , Version 1.53e). Band intensities 375 \nwere normalized to β-actin or GAPDH or corresponding total protein and expressed as fold change relative 376 \nto controls. Data are shown as mean ± standard deviation (SD) from ≥3 independent experiments. Statistical 377 \nanalyses were performed using GraphPad Prism v9  (Version 9.1.2). For comparisons between two groups 378 \n(e.g., control vs. RSK1 knockout or Ctrl vs. RSK1O/E), unpaired two-tailed Student’s T-tests were employed. 379 \nFor experiments involving multiple conditions or time points, one -way or two -way analysis of variance 380 \n(ANOVA) followed by Dunnett’s multiple comparisons test was used to assess significance, with p < 0.05 381 \nconsidered statistically significant. 382 \nResults  383 \nH80 Cells Exhibit Neuronal Characteristics as Evidenced by NeuN, MAP2, and Tau Expression 384 \nGiven the considerable variability , as well as the growth and maintenance challenges associated with 385 \ncommonly used neuronal cell lines such as SH-SY5Y [56], we sought to evaluate whether a glioma cell line, 386 \nH80 retains key neuronal characteristics, particularly relevant signaling pathways and susceptibility to 387 \nneurotoxicity. Our findings indicate that H80 cells exhibit features suitable for modeling neuron -specific 388 \nsignaling events, including pathways involved in Tau phosphorylation. Importantly, H80 cells offer several 389 \npractical advantages over conventional neuronal models, including robust and reproducible growth, rapid 390 \nproliferation, low baseline cytotoxicity, and stable culture behavior, making them a reliable and efficient system 391 \nfor mechanistic studies of neuronal signaling and neurotoxicity. The neuronal characteristics of the H80 glioma 392 \ncells [51] were confirmed by performing immunofluorescence staining using NeuN, a nuclear marker widely 393 \nrecognized for its specificity to post -mitotic neurons. Both unstained and secondary -only controls were 394 \nincluded in parallel to validate antibody specificity and to exclude background artifacts. Our analysis revealed 395 \nrobust nuclear NeuN immunoreactivity across the majority of H80 cells, providing clear evidence that H80 is 396 \na neuronal cell line (Figure 1A). The expression of Tau, a microtubule-associated protein that plays a critical 397 \nrole in axonal stability and is centrally implicated in tauopathies and other neurodegenerative processes , 398 \nfurther substantiates the neuronal identity of H80 cells (Supplementary Figure S1). The presence of Tau not 399 \nonly reinforces the neuronal -like characteristics of H80 cells but also highlights their relevance as a model 400 \nsystem for studying Tau-associated signaling pathways and neurotoxicity.  401 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFurthermore, to demonstrate that H80 cells possess the molecular characteristics of differentiated neurons, 402 \nwe focused on the expression of microtubule -associated protein 2 (MAP2). MAP2 is a neuron -specific 403 \ncytoskeletal protein that is predominantly localized to dendrites, where it plays a pivotal role in stabilizing 404 \nmicrotubules and maintaining neuronal morphology. MAP2 expression is therefore widely recognized as a 405 \nhallmark of neuronal differentiation and identity. We initially assessed MAP2 expressions in H80 cells using 406 \nimmunofluorescence microscopy, with HEK293T cells serving as a non -neuronal reference  (control), 407 \nMicroglial cells (unknown or known MAP2 negative cell line) and SH-SY5Y cells serving as neuronal reference 408 \n(positive control or known to express MAP2). Notably, the expression of MAP2 was exclusively observed in 409 \nH80 cells (Figure 1B and supplementary  S1) and also in positive control  (SH-SY5Y), where the protein 410 \ndisplayed a distinct filamentous distribution throughout the cytoplasm, consistent with the structural 411 \norganization seen in neurons. As anticipated, HEK293T cells lacked detectable MAP2 signal whereas SHS5Y 412 \nhas a strong MAP2 expression  under identical staining conditions ( Figure 1B).  The expression of MAP2  413 \nindicates that H80 cells, but not HEK293T cells and microglial cells, belongs to neuronal lineage. To further 414 \ncorroborate these findings, we performed immunoblotting using total cellular lysates from H80 cells, HEK293T 415 \ncells, and microglial cells. Consistent with the immunofluorescence results, MAP2 protein was robustly 416 \ndetected in H80 cell lysates, whereas it was undetectable in both HEK293T and microglial samples ( Figure 417 \n1C). Importantly, the absence of MAP2 expression in microglial cells, another brain-resident glial cells of non-418 \nneuronal lineage, underscores the neuronal specificity of this marker. Together, these complementary assays 419 \nprovide convergent evidence that H80 cells exclusively express MAP2. The presence of MAP2 exclusively in 420 \nH80 cells, but not in two distinct non-neuronal cell types, strongly supports the conclusion that H80 cells is a 421 \nneuronal cell line that  exhibits cytoskeletal and molecular features consistent with neuronal identity and 422 \ndifferentiation.  423 \nTherefore, the co-expression of NeuN, MAP2, and Tau provides convergent and robust evidence that H80 424 \ncells exhibit hallmark neuronal features (Figures 1A to C, and Supplementary Figure S1). Collectively, these 425 \nfindings support the classification of H80 cells as a neuronal-like cell model. The presence of these canonical 426 \nneuronal markers not only confirms their neuronal identity but also highlights their suitability as a versatile 427 \nplatform for investigating neuron -specific molecular mechanisms. In particular, H80 cells offer a valuable 428 \nsystem for studying signaling pathways involved in the regulation of Tau protein phosphorylation and activity, 429 \nas well as broader processes underlying neuronal function and neurotoxicity. 430 \nSince our study focuses on HIV induced neurotoxicity and neurons are not directly infected by HIV [17], we 431 \nnext  examined the expression of the key HIV entry receptors and co-receptors  in H80 cells (Figure 1D). To 432 \ndetermine the expression profile of HIV entry receptors on H80 cells, we performed flow cytometric analysis 433 \nfor CD4, CCR5, and CXCR4. Cells were co-stained with either CD4 (APC anti-human CD4 from BioLegend 434 \ncat no 317416) and CXCR4 (PE anti-human CD184 (CXCR4) Antibody from BioLegend cat no. 30 6505) or 435 \nCD4 (PE anti-human CD4 Antibody from BioLegend cat no. 357403 and CCR5 (APC/Cyanine7 anti-human 436 \nCD195 (CCR5) Antibody from BioLegend cat no. 359110). Our results demonstrated that H80 cells lack 437 \ndetectable CD4 expression under basal conditions, indicating the absence of the primary receptor required 438 \nfor productive HIV entry. Notably, approximately 20% of H80 cells expressed surface CXCR4, whereas CCR5 439 \nexpression was undetectable. These findings suggest that while H80 cells are unlikely to support productive 440 \nHIV infection due to the absence of CD4, the presence of CXCR4 on a subset of cells may render them 441 \nresponsive to HIV -associated proteins or signaling pathways, thereby contributing to HIV -induced 442 \nneurotoxicity. To ensure assay specificity and reliability, HEK293T cells were included as a negative control 443 \nand showed no detectable expression of CD4, CCR5, or CXCR4. In contrast, U937 cells served as a positive 444 \ncontrol and exhibited robust basal expression of CD4 and both co -receptors. Collectively, these data further 445 \nsupport the suitability of H80 cells as a model to study HIV-mediated neurotoxic effects independent of direct 446 \nviral infection.  447 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nAltogether, our findings demonstrate that H80 cells exhibit key neuronal characteristics, as evidenced by the 448 \nexpression of canonical neuronal markers, including NeuN, MAP2, and Tau. In addition, receptor profiling 449 \nrevealed that H80 cells lack detectable expression of CD4 and CCR5 but express the chemokine receptor 450 \nCXCR4 on a subset of cells. Previous studies, including those by Kaul et al., have shown that both CXCR4 451 \nand CCR5 can mediate HIV -associated neuronal injury, while CCR5 may also engage neuroprotective 452 \nsignaling pathways [57]. The selective expression of CXCR4 in H80 cells is particularly noteworthy, as it 453 \nsuggests that analogous to neurons, these cells may be responsive to HIV-associated proteins and signaling 454 \nevents linked to CXCR4 engagement, despite the absence of productive viral entry. This receptor profile 455 \nclosely aligns with current understanding that neuronal damage in NeuroHIV is largely mediated indirectly 456 \nthrough viral proteins and host signaling pathways rather than direct infection.  457 \nBased on these observations, we next sought to determine how HIV exposure influences neuronal stress 458 \nresponses and neurotoxicity in this model, with a particular focus on Tau phosphorylation, a well-established 459 \nmarker of tauopathy. Given the confirmed neuronal phenotype of H80 cells and their expression of CXCR4, 460 \nthese cells provide a biologically relevant system to study HIV-induced neuronal dysfunction independent of 461 \nproductive infection. Accordingly, we directly exposed H80 cells to HIV to investigate the underlying molecular 462 \nmechanisms driving HIV -associated neurotoxicity and Tau dysregulation, enabling us to dissect signaling 463 \npathways that contribute to neurodegenerative processes in the context of NeuroHIV.  464 \nHIV exposure upregulates RSK1 expression 465 \nTo investigate the signaling pathways underlying HIV -induced tauopathy, H80 cells were exposed to HIV 466 \nvirions (strain 93/TH/051, R5- and X4-tropic virus, dual-tropic HIV-1). Because neurons lack the primary HIV 467 \nreceptor (CD4), they are resistant to productive infection; thus, analogous to neurons, this model allows us to 468 \nspecifically examine HIV -mediated signaling and neurotoxic effects independent of viral replication. HIV 469 \nvirions were generated by infecting Jurkat T cells, and successful infection was confirmed by immunoblot 470 \ndetection of the viral capsid protein p24 in infected cell lysates ( Figure 2A). Virus-containing supernatants 471 \nfrom either Jurkat or MT -4 cells were then collected and used to expose H80 cells using a two -round 472 \nspinfection (spinoculation) protocol, which enhances viral contact and ensures efficient exposure of neuronal 473 \ncells to viral particles. As a negative control, H80 cells were exposed to supernatants from uninfected 474 \nJurkat/MT-4 cells (Figure 2A). Following exposure, H80 cells were harvested at 24 and 48 hours after the 475 \nsecond spinfection for downstream analyses. Total RNA and protein lysates were collected to assess changes 476 \nin intracellular signaling pathways and Tau phosphorylation status, enabling us to define the molecular 477 \nmechanisms by which HIV exposure induces neuronal stress and Tau dysregulation in this system. 478 \nTo assess the impact of HIV exposure on the cellular transcriptional machinery and to confirm successful viral 479 \nexposure on H80 cells, we quantified mRNA expression levels of representative inflammatory and signaling 480 \ngenes using qRT-PCR at 24 hours post-exposure. HIV-exposed H80 cells expressed a robust upregulation 481 \nof interleukin -1β (IL -1β) and tumor necrosis factor -alpha (TNF -α) transcripts, both of which are central 482 \nmediators of proinflammatory signaling cascades ( Figure 2 B). Consistent with previous reports 483 \ndemonstrating that neurons can produce cytokines in response to HIV-associated stress [58], this pronounced 484 \ninduction provides strong evidence of effective HIV exposure and activation of innate immune signaling in 485 \nH80 cells. In addition, we observed a modest but reproducible increase in RSK1 mRNA expression. Given 486 \nthe established role of RSK1 as a downstream effector of MAPK signaling, this finding suggests early 487 \nactivation of stress-responsive and pathogen-associated signaling pathways, complementing the observed 488 \ninflammatory response. Collectively, these transcriptional changes not only confirm ed the upregulation of 489 \nRSK1 upon HIV exposure but also underscore the functional reactivation of the cells, thereby validating the 490 \nfunctional responsiveness of H80 cells to HIV exposure. These results further strengthen the biological 491 \nrelevance of our H80 neuronal model for dissecting the molecular mechanisms underlying HIV -induced 492 \nneuroinflammation, stress signaling, and Tau-associated pathology. 493 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nHIV exposure-driven RSK1 upregulation induces Tau phosphorylation 494 \nTo investigate whether HIV exposure promotes pathological Tau modification, we first performed 495 \nimmunofluorescence staining using an antibody specific for the phosphorylated Tau protein at Ser396 (p-Tau-496 \nSer396), a site commonly associated with neurotoxicity and neurodegeneration. Compared with controls  497 \n(exposing without the virus) , HIV-exposed H80 cells displayed a marked increase in p -Tau-Ser396 signal, 498 \nsuggesting that HIV exposure drives Tau phosphorylation  (Figure 2 C). Notably, in addition to the 499 \nphosphorylation of Tau, HIV exposure markedly upregulates RSK1 (Supplementary Figure S 2). The 500 \nconcurrent induction of RSK1 suggested a role of this kinase  in promoting Tau phosphorylation. These 501 \nfindings indicate that the effect of HIV exposure on Tau phosphorylation facilitated through the regulation of 502 \nRSK1, thereby highlighting a mechanistic link between RSK1 expression and Tau phosphorylation.  503 \nTo substantiate these observations, we conducted immunoblot analysis of whole -cell lysates following HIV 504 \nexposure (under the same condition s). H80 cells were cultured in four independent dishes (two biological 505 \nreplicates per condition), and whole cell lysates were collected 48 h after HIV exposure. Protein lysates from 506 \neach dish were prepared and quantified individually. Equivalent amounts of protein were resolved by 507 \nimmunoblotting to assess RSK1, RSK1/2/3, phosphorylated Tau (p Tau S396), and Tau, using actin or total 508 \nprotein as loading controls. Consistent with the immunofluorescence imaging results, immunoblotting 509 \nrevealed a robust induction of p-Tau-Ser396 in HIV-exposed cells (lanes 3-4) compared to control (exposing 510 \nwith supernatant from uninfected cells, lanes 1-2). In contrast, total Tau protein levels exhibited only a modest 511 \nincrease, indicating that HIV exposure predominantly induces post -translational modification of Tau, rather 512 \nthan significantly altering its overall expression.  Notably, this increase in Tau phosphorylation was 513 \naccompanied by a pronounced upregulation of RSK1, as well as elevated levels of the RSK1/2/3 isoforms , 514 \nsuggesting activation of the RSK signaling pathway in response to HIV exposure. These findings indicate that 515 \nRSK1 activation occurs in parallel with HIV-induced Tau phosphorylation and may serve as a critical molecular 516 \nlink between viral exposure and Tau dysregulation in H80 cells ( Figure 2D ). Quantitative densitometric 517 \nanalysis of the immunoblot signals, normalized to β actin and/or total protein, revealed a statistically significant 518 \nincrease in the levels of both RSK1 and p -Tau Ser396 in HIV exposed H80 cells relative to controls/No HIV 519 \n(Figure 2E). To further assess whether H80 cells support productive HIV infection, lysates from HIV-exposed 520 \nand control (no HIV) conditions were analyzed by immunoblotting using an anti-HIV p24 antibody, with Jurkat 521 \nT cells included as positive (HIV -exposed) and negative (uninfected) controls ( Figure 2F). Consistent with 522 \nthe absence of CD4 expression, p24 was not detected in H80 cell lysates, indicating that these cells do not 523 \nsupport productive HIV infection and are instead exposed to HIV virions without undergoing viral replication. 524 \nImportantly, despite the lack of productive infection, exposure to HIV virions was sufficient to induce robust 525 \nactivation of RSK1 signaling in H80 cells, which correlated with increased Tau phosphorylation. Given the 526 \nwell-established roles of RSK1 in nuclear gene regulation and cytoplasmic signaling crosstalk, these findings 527 \nsuggest that HIV-induced activation of RSK1 serves as a key mediator linking viral exposure to downstream 528 \nneuronal stress responses.  Collectively, our results support a model in which HIV virion exposure, 529 \nindependent of productive infection, triggers RSK1 activation, leading to pathological Tau phosphorylation 530 \nand tauopathy-associated signaling, thereby establishing a mechanistic connection between HIV exposure 531 \nand neuronal dysfunction mediated through the RSK1 pathway.  532 \nCocaine enhances Tau phosphorylation by upregulating and activating RSK1  533 \nTo investigate whether cocaine contributes to Tau pathology, we chronically exposed H80 cells to cocaine 534 \ntwice daily for 2 days and subsequently assessed Tau phosphorylation by immunofluorescence (Figure 3A). 535 \nCocaine-exposed H80 cells exhibited a pronounced increase in phosphorylated Tau (p -Tau-Ser396) 536 \ncompared with untreated controls, while the total abundance of Tau protein remained unchanged (Figure 3B). 537 \nThese findings indicate that cocaine primarily promotes post -translational modification of Tau, rather than 538 \nsignificantly altering its overall expression. Given that our previous findings demonstrated that HIV exposure 539 \nupregulates RSK1 (Figure 2), we next examined whether cocaine -induced Tau phosphorylation is similarly 540 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nassociated with upregulation in RSK1 expression. Immunofluorescence analysis revealed a significant 541 \nupregulation of RSK1 following chronic cocaine exposure ( Supplementary Figure S 2). Thus, parallel 542 \nincrement in RSK1 expression and Tau phosphorylation suggested crucial  role for RSK1 signaling in 543 \nmediating cocaine -induced Tau modification . These findings were further validated in an independent 544 \nexperiment, confirming the reproducibility of RSK1 upregulation and enhanced Tau phosphorylation in 545 \nresponse to cocaine exposure in H80 cells (Figure 3C–3F). 546 \nTo further solidify the data obtained through immunofluorescence analysis, we chronically treated H80 cells 547 \nalone or in combination with  cocaine and HIV as shown in Figure 3A. The cell lysate was analyzed  by 548 \nImmunoblotting. The i mmunoblotting of whole -cell lysates revealed that cocaine produced a modest but 549 \nreproducible increase in total RSK1 protein, accompanied by a n elevation in its phosphorylation at Thr 348, 550 \nThr 359, S363 and S380, which marks functionally active form of RSK1. Given that specific posttranslational 551 \nmodification of RSK1 was quantitatively correlated with the increase in p-Tau-Ser396, implicating RSK1 as a 552 \nmediator of cocaine-driven Tau modification (Figure 3C and 3D). Both cocaine and HIV significantly increased 553 \np-Tau-Ser396 relative to controls, although the magnitude of the effect differed: HIV produced a robust 554 \nelevation in Tau phosphorylation, whereas cocaine induced a more modest increase. Notably, co-exposure to 555 \ncocaine and HIV did not result in strictly additive effects (but it is more on higher side), suggesting that these 556 \nstimuli converge on overlapping molecular pathways.  Analysis of RSK1 activation under these conditions 557 \nrevealed that HIV strongly enhanced both total RSK1 expression and its phosphorylation, including at S380, 558 \nThr348, Thr359/ and S363 far exceeding the effects of cocaine alone. HIV exposure also led to a moderate 559 \nincrease in Thr348 phosphorylation, further distinguishing its mode of RSK1 regulation from cocaine. 560 \nImportantly, the degree of RSK1 activation in each condition closely aligned with the extent of Tau 561 \nphosphorylation, strengthening the link between RSK1 signaling and Tau modification. Together, these results 562 \nestablish that both cocaine and HIV drive activation of the RSK1 signaling axis in H80 cells, albeit with distinct 563 \nmagnitudes and mechanistic profiles (Figure 3C and 3D). Cocaine modestly increases RSK1 expression and 564 \nTau phosphorylation, whereas HIV produces a more potent and broad er activation of RSK1, resulting in 565 \nstronger downstream Tau modification. These findings provide mechanistic insight into how viral infection and 566 \nsubstance use converge on a shared signaling pathway to promote Tau pathology. 567 \nTo delineate the immediate signaling responses triggered by cocaine and HIV, we examined the acute 568 \nactivation of RSK1 in H80 neuronal cells following short term exposure to each stimulus. H80 cells were 569 \ncultured in eight independent dishes, providing two biological replicates for each of the four treatment 570 \nconditions (Control, cocaine, HIV, and cocaine + HIV). Cells were exposed acutely for 15 minutes ( Figure 571 \n3E), after which they were harvested and lysed. Protein lysates from each dish were prepared and quantified 572 \nindividually, and equivalent amounts of total protein were resolved by immunoblotting to evaluate 573 \nphosphorylation of RSK1 at the activating sites Ser380 and Thr359/Ser363, alongside total RSK1 levels, 574 \nusing actin or total protein as loading controls. Acute exposure to either cocaine or HIV elicited rapid and 575 \nrobust activation of the RSK1 signaling pathway in H80 cells. Immunoblot analysis revealed marked increases 576 \nin RSK1 phosphorylation at both Ser380 and Thr359/Ser363 relative to controls ( Figure 3F and 3G ). This 577 \nactivation was consistently observed across biological replicate lanes corresponding to each treatment group 578 \n(lanes 3–4, 5 –6, and 7 –8 compared with lanes 1 –2), demonstrating strong reproducibility of these rapid 579 \nphosphorylation events. Notably, the magnitude of RSK1 activation differed between stimuli. While cocaine 580 \ninduced a clear and reproducible increase in phosphorylation at both regulatory sites (p-RSK-1 Thr359/S363 581 \nand S380), HIV exposure elicited a substantially stronger response, producing the highest levels of RSK1 582 \nactivation among all acute treatment conditions.  583 \nTogether, these findings establish that RSK1 is a rapidly responsive kinase activated within minutes of cocaine 584 \nor HIV exposure, and that the amplitude of this response is greater under HIV stimulation than under cocaine 585 \nalone. This further reinforces the pattern observed under chronic exposure conditions, underscoring the 586 \nconsistency of cocaine and HIV ‑driven enhancement of RSK ‑1 activation across temporal paradigms , 587 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nreinforcing its central role as an early signaling mediator linking cocaine and HIV exposure to downstream 588 \nneuronal stress responses and Tau pathology.  589 \nHIV or Cocaine converge on GSK3β inhibition through Ser9 phosphorylation 590 \nGSK3β is a key regulator of neuronal signaling and a well -established mediator of Tau phosphorylation [59-591 \n63]. The enzymatic activity of GSK3β is primarily controlled through its phosphorylation at the serine-9 (Ser9) 592 \nresidue, (p-GSK3β-Ser9), which serves as an inhibitory modification that limits substrate access and thereby 593 \ninhibits GSK3β kinase activity. Given the well-established role of GSK3β in mediating Tau pathology, we next 594 \nexamined whether HIV and cocaine influence the regulation of this kinase. As shown in Figures 2 and 3 , 595 \nboth HIV and cocaine exposure markedly enhanced Tau phosphorylation, raising the possibility that these 596 \neffects are mediated, at least in part, through altered GSK3β activity. To investigate further, we specifically 597 \nassessed the phosphorylation status of GSK3β at its inhibitory Ser9 residue, thereby determining whether 598 \nHIV and cocaine relieve the inhibitory regulation of GSK3β and contribute to the observed increase in Tau 599 \nphosphorylation. Using total cellular lysates from Figure 2A, we examined the phosphorylation status of 600 \nGSK3β at the inhibitory Ser9 site by immunoblotting. Surprisingly, o ur results demonstrated a marked 601 \nincrease in Ser9 phosphorylation in response to HIV infection in Jurkat cells, while the levels of total GSK3β 602 \nremained unchanged (Figure 4A). This increase in inhibitory phosphorylation suggests that GSK3β becomes 603 \ninactivated upon HIV infection or under conditions of ongoing viral infection. 604 \nTo determine whether HIV exposure directly modulates GSK3β activity, we exposed H80 cells for 15 min to 605 \nthe supernatant derived from HIV-infected cells (Jurkat infected with HIV), while supernatant from uninfected 606 \ncell cultures (Jurkat uninfected with HIV)  was used as a control, as shown in Figure 2A . After 15 min 607 \nexposure, the cells were harvested, and protein lysates were subjected to immunoblot analysis to evaluate 608 \nthe phosphorylation status of GSK3β at Ser9 site, marking functionally inactive form of GSK3β. We observed 609 \na pronounced increase in Ser9 phosphorylation  of GSK3β in HIV-exposed cells compared to the control 610 \n(Figure 4B and 4C). Since phosphorylation at Ser9 is known to suppress GSK3β catalytic activity, this 611 \nincrease strongly suggests that acute HIV exposure enhance posttranslational modification of GSK3β at Se9, 612 \nwhich functionally inactivates GSK3β. These findings indicate that HIV exposure can rapidly influence host 613 \nkinase signaling pathways.  614 \nSubsequently, we examined how acute exposure to cocaine and HIV modulates GSK3β signaling in H80 615 \ncells. To evaluate these rapid effects, cells were cultured in eight independent dishes, providing two biological 616 \nreplicates per treatment condition, and exposed for 15 minutes to cocaine, HIV, or both. Following treatment, 617 \ncells were harvested and lysed, and protein lysates from each dish were prepared and quantified individually. 618 \nEqual amounts of total protein were subjected to immunoblot analysis to assess phosphorylation of GSK3β 619 \nat Ser9, with total protein levels serving as loading controls. Both cocaine and HIV independently elicited a 620 \nclear increase in Ser9 phosphorylation of GSK3β  (Lane 3-4, lane 5-6 and lane 7-8 compared to lane 1 -2), 621 \nindicating enhanced inhibitory modification of the kinase (Figure 4E and 4F). Notably, the consistent elevation 622 \nof Ser9 phosphorylation in HIV exposed samples further confirms the reproducibility of HIV-mediated GSK3β 623 \ninactivation, as also observed in Figures 4B and 4C . These findings demonstrate that acute exposure to 624 \neither cocaine or HIV is sufficient to rapidly inactivate GSK3β, revealing a shared regulatory mechanism by 625 \nwhich both stimuli attenuate GSK3β activity in H80 cells. Importantly, this occurs despite the observed 626 \nincrease in Tau phosphorylation, further supporting the notion that cocaine - and HIV -induced Tau 627 \ndysregulation proceeds through GSK3β-independent signaling pathways, likely involving alternative kinases 628 \nsuch as RSK1. 629 \nTo further substantiate our finding that both HIV and cocaine are independently able to inactivate GSK3β, we 630 \nperformed immunoblot analysis to assess inhibitory phosphorylation of GSK3β at Ser9. H80 cells were 631 \nchronically exposed for 48 hours to cocaine, HIV virions, or a combination of both. Under each condition, 632 \ncocaine alone, HIV alone, or combined HIV plus cocaine exposure, we observed a robust increase in Ser9 633 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nphosphorylation relative to untreated controls, whereas total GSK3β protein levels remained unchanged. 634 \nThese results indicate that the effects of HIV and cocaine on GSK3β are mediated through post-translational 635 \ninhibitory modification, rather than changes in protein abundance. Notably, combined exposure to HIV and 636 \ncocaine also produced a clear increase in p -GSK3β Ser9 compared with control cells ( Figure 4G and H ), 637 \nconfirming that both stimuli converge on functional inactivation of GSK3β. The consistency of this response 638 \nacross all treatment groups strongly supports the conclusion that HIV and cocaine each suppress GSK3β 639 \nactivity in H80 neuronal cells. This finding is particularly striking because GSK3β is widely recognized as a 640 \nmajor Tau kinase. Accordingly, if GSK3β is the main Tau kinase, inactivation of GSK3β would be expected 641 \nto reduce Tau phosphorylation. In contrast, we observed the opposite outcome: despite clear evidence of 642 \nGSK3β inactivation, Tau phosphorylation was markedly increased following exposure to HIV and/or cocaine 643 \n(Figures 2 and 3). This apparent paradox strongly suggests that Tau phosphorylation in this setting is driven 644 \nthrough an alternative pathway that is independent of GSK3β activity. Our data point towards RSK1 as a likely 645 \nupstream mediator of this effect. Indeed, both HIV and cocaine induced significant upregulation and activation 646 \nof RSK1, coinciding with enhanced Tau phosphorylation under conditions in which GSK3β remained 647 \ninactivated. These observations support a model in which RSK1-driven signaling bypasses the need for active 648 \nGSK3β and sustains pathological Tau phosphorylation, thereby promoting tauopathy -associated neuronal 649 \nstress responses. 650 \nCollectively, these findings demonstrate that HIV and cocaine independently converge on GSK3β inactivation 651 \nvia Ser9 phosphorylation yet simultaneously promote Tau hyperphosphorylation through a distinct upstream 652 \nmechanism, most likely involving RSK1. This convergence on inhibitory GSK3β signaling, together with 653 \nactivation of an alternative Tau -phosphorylating pathway, identifies a critical molecular axis by which viral 654 \nexposure and substance use disrupt neuronal signaling, ultimately contributing to Tau dysregulation and the 655 \nCNS impairments, including neuropathological processes associated with HAND.  656 \nCocaine, but not HIV  exposure, activates AKT 1 signaling through phosphorylation at Thr308 and 657 \nSer473. 658 \nPhosphorylation of GSK3β at Ser9, a critical inhibitory modification, is tightly regulated by upstream kinases, 659 \nmost notably AKT, which plays a central role in neuronal signaling cascades and survival pathways [34]. AKT-660 \nmediated phosphorylation of GSK3β at Ser9 serves as a key inhibitory checkpoint that suppresses GSK3β 661 \ncatalytic activity and prevents excessive substrate phosphorylation. Given our findings that both HIV and 662 \ncocaine independently increase Ser9 phosphorylation on GSK3β, thereby promoting its inactivation, we next 663 \nsought to determine whether these are direct effect are mediated through activation of the AKT signaling 664 \npathway. To investigate this, we examined the phosphorylation status of AKT at its regulatory sites that control 665 \nits functional activity . We hypothesized that stimulation of  AKT activity would restrict GSK3β activity by 666 \ncatalyzing its phosphorylation at Ser9 (p-GSK3β-Ser9). This approach allowed us to directly evaluate whether 667 \nHIV and cocaine converge upstream on AKT to regulate GSK3β activity and thus,  contribute to Tau 668 \nhyperphosphorylation. 669 \nTo determine the regulation of AKT signaling pathway upon  HIV and cocaine exposure, we performed 670 \nimmunofluorescence staining for phosphorylated AKT at Ser473 (p-AKT-Ser473), a well-established marker 671 \nof AKT activation and a critical modification required for full kinase activity. H80 cells were chronically exposed 672 \nfor 2 days to cocaine, HIV virions, or both, after which AKT1 phosphorylation status was evaluated (Figure 673 \n5A). Cocaine -treated cells displayed a robust , reproducible and significant increase in p -AKT-Ser473 674 \nfluorescence compared with untreated controls, indicating that cocaine strongly activates AKT signaling  675 \npathway (Figure 5B). In contrast, HIV exposure alone did not show any effect in p-AKT-Ser473 levels under 676 \nthe same conditions (Supplementary Figure S3), suggesting that HIV does not directly induce AKT activation 677 \nin this context. Notably, total AKT protein levels were unaffected across all conditions, confirming that the 678 \nobserved changes were attributable to post -translational regulation of phosphorylation rather than changes 679 \nin protein expression or stability.  680 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nTo further validate our data obtained through immunofluorescence analyses and confirm the diverse impact 681 \nof cocaine and HIV on AKT signaling, we performed immunoblot analysis using cell lysates from H80 cells 682 \nfollowing 2 days  of chronic exposure to cocaine, HIV, or a combination of both and evaluate both the 683 \nphosphorylation sites of AKT. Immunoblotting revealed that cocaine treatment induces a significant increase 684 \nin phosphorylation of AKT at both Thr308 and Ser473 compared with untreated controls  (Figure 5C). Since 685 \nphosphorylation at Thr308 and Ser473 are both essential for full activation of AKT, the simultaneous increase 686 \nin phosphorylation at these two regulatory sites strongly confirms that cocaine induces a robust activation of 687 \nthe AKT signaling pathway. In contrast, HIV exposure alone did not alter phosphorylation at either site (further 688 \nvalidating our immunofluorescence results), demonstrating that HIV exposure alone does not enhance or 689 \nactivate AKT signaling pathway. Notably, combined treatment with cocaine and HIV reproduced the increase 690 \nin phosphorylation pattern of AKT observed with cocaine alone, indicating that cocaine exerts a dominant 691 \nstimulus in activating  AKT signaling, even in the presence of viral exposure. This suggests that cocaine 692 \noverrides any potential influence of HIV on this pathway. Quantitative densitometric analysis  (Figure 5D) 693 \nprovided further support, showing a significant increase in AKT phosphorylation at both Thr308 and Ser473 694 \nin cocaine alone and HIV+ cocaine-exposed cells, while HIV exposure alone had no measurable impact 695 \nrelative to controls. Importantly, total AKT protein levels remained constant across all conditions, confirming 696 \nthat the observed changes reflect post-translational modifications rather than at gene expression. Together, 697 \nthese results establish that cocaine, but not HIV, selectively activates AKT signaling in H80 cells, with cocaine 698 \ndriving strong phosphorylation of AKT at both activation sites and dominating over HIV when both stimuli are 699 \npresent. 700 \nCollectively, these findings establish cocaine, but not HIV, as a potent activator of the AKT signaling pathway 701 \nin H80 cells. Mechanistically, cocaine promotes sustained AKT phosphorylation at Thr308 and Ser473, which 702 \nsubsequently promotes inactivation of GSK3β by catalyzing its phosphorylation at Ser9 (p-GSK3β-Ser9). 703 \nThus, cocaine promotes GSK3β inactivation (p-GSK3β-Ser9) both directly through AKT stimulation and also 704 \nvia RSK1 activation. In contrast, HIV inactivates GSK3β exclusively through an AKT-independent 705 \nmechanisms, primarily through RSK1 signaling.  706 \nThus, cocaine and HIV converge on a shared downstream effector, GSK3 β inactivation, but diverge in their 707 \nupstream regulatory pathways. Cocaine acts through an AKT-dependent pathway that also involves RSK1 708 \nstimulation, whereas HIV acts predominantly through an AKT-independent, RSK1-driven pathway. This dual 709 \nconvergence and divergence highlight the complexity of signaling networks regulating Tau phosphorylation 710 \nand underscore how viral exposure and substance use engage distinct yet overlapping molecular pathways 711 \nto drive neurotoxicity and tauopathy.  712 \nHIV and Cocaine upregulate RSK1 to drive  Tau phosphorylation through a GSK3 β-independent 713 \nmechanism 714 \nOur findings thus far indicate that HIV and cocaine share a common upstream signaling pathway through the 715 \nupregulation of RSK1 (Figure 2 and 3). However, their downstream signaling pathways diverge in a stimulus-716 \nspecific manner. Notably, HIV does not activate the AKT signaling pathway ( Figure 5), whereas cocaine 717 \nexposure leads to robust AKT activation, as evidenced by increased phosphorylation at both key regulatory 718 \nsites, Thr308 and Ser473 ( Figure 5A-D). Despite this divergence, both stimuli ultimately converge on the 719 \ninactivation of GSK3 β, as demonstrated by increased inhibitory phosphorylation at Ser9 ( Figure 4).  This 720 \nshared downstream effect establishes GSK3 β as a critical point of signaling convergence. Importantly, this 721 \nconvergence results in a common pathological outcome, the enhanced Tau phosphorylation of Tau (Figure 2 722 \nand 3), a hallmark of neurodegenerative processes and tauopathy.  723 \nThese observations suggest a model in which distinct upstream signaling pathways (AKT -dependent for 724 \ncocaine and AKT -independent for HIV) converge on shared downstream nodes, while simultaneously 725 \nengaging alternative kinase pathways, such as RSK1, to drive Tau phosphorylation. The persistence of Tau 726 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nphosphorylation despite GSK3 β inactivation further underscores the involvement of GSK3 β-independent 727 \nmechanisms, likely mediated by RSK1.  Based on these findings, we next sought to delineate the relative 728 \ncontributions and mechanistic interplay between RSK1 and GSK3 β in mediating Tau phosphorylation 729 \nfollowing HIV and cocaine exposure. This approach aims to clarify how these signaling pathways integrate to 730 \nproduce a shared neurotoxic phenotype, thereby providing deeper insight into the mechanistic basis of HIV- 731 \nand substance use-mediated tauopathy and neurodegeneration. 732 \nTo validate our findings and further define the effects of cocaine and HIV on Tau phosphorylation, we 733 \nperformed a comprehensive immunoblot analysis using lysates from H80 cells following 48 hours of chronic 734 \nexposure to cocaine, HIV, or their combination. Cells were seeded into 12 independent culture dishes across 735 \n≥3 passages/days, yielding three biological replicates per condition (Control, cocaine, HIV, and cocaine + 736 \nHIV). After treatment, cells from each dish were harvested and lysed individually, and equal amounts of total 737 \nprotein were subjected to immunoblot analysis. Consistent with our earlier observations ( Figures 2 and 3), 738 \nimmunoblotting revealed that cocaine, HIV, and their combination each induced a significant increase in Tau 739 \nphosphorylation at Ser396 compared with untreated controls ( Figures 6A and 6B ). These results robustly 740 \nconfirm and extend our previous findings, demonstrating that both stimuli, independently and in combination, 741 \npromote sustained Tau hyperphosphorylation under chronic exposure conditions. Phosphorylation of Tau at 742 \nSer396 is widely recognized as a marker of pathological Tau, and the observed increase at this site strongly 743 \nconfirms that cocaine exposure, HIV exposure, and their combination each induce Tau hyperphosphorylation. 744 \nIn parallel, we assessed the activity of GSK3 β under these conditions. Interestingly, despite the increase in 745 \nTau phosphorylation, GSK3 β was found to be functionally inactivated, as evidenced by enhanced 746 \nphosphorylation at its inhibitory Ser9 residue (Figures 6A and 6B). These findings demonstrate that both HIV 747 \nvirion exposure and chronic cocaine treatment promote Tau phosphorylation while simultaneously restricting 748 \nGSK3β activity, indicating that Tau hyperphosphorylation occurs through a GSK3β-independent mechanism. 749 \nImportantly, although both stimuli converge on GSK3β inactivation, they do so via distinct upstream pathways. 750 \nHIV induces GSK3 β inactivation in an AKT -independent manner, whereas cocaine mediates this effect 751 \nthrough AKT activation, as reflected by increased phosphorylation at both Thr308 and Ser473. Together, these 752 \nresults highlight a critical mechanistic distinction in upstream signaling while reinforcing a shared downstream 753 \noutcome, Tau hyperphosphorylation despite GSK3 β inhibition, suggesting the involvement of alternative 754 \nkinases, such as RSK1, in driving Tau pathology.  755 \nFurthermore, to delineate the relative contributions of RSK1 and GSK3β to Tau phosphorylation in response 756 \nto exposures HIV and cocaine, we employed highly specific small molecular pharmacological inhibitors. Our 757 \nrationale was that if RSK1 is the common mediator of Tau phosphorylation induced by these stimuli, then its 758 \ninhibition should suppress this effect, whereas inhibition of GSK3β would not.  H80 cells were pretreated for 759 \n24 hours  with BI -D1870 (a selective RSK1 inhibitor) or CHIR -99021 (a highly specific GSK3 β inhibitor), 760 \nfollowed by exposure to HIV, cocaine, or both. After treatment , total protein lysates were analyzed by 761 \nimmunoblotting to assess signaling and phosphorylation dynamics (Figure 6C and 6D). Consistent with our 762 \nearlier findings (Figures 2 and 3), both HIV and cocaine exposures resulted in robust upregulation of RSK1 763 \nactivity compared to untreated controls  (Figure 6 C and 6D). Importantly, pretreatment with BI -D1870 764 \neffectively suppressed RSK1 activation, whereas CHIR-99021 had no effect on RSK1 activity, indicating that 765 \nRSK1 functions independently of, and upstream from, GSK3β in this signaling cascade (Figure 6C and 6D). 766 \nWe next examined the effect of these inhibitors on GSK3β activity. As shown previously (Figure 4), both HIV 767 \nand cocaine exposures led to inactivation of GSK3β, as evident from enhanced phosphorylation at S9 (Figure 768 \n6C and 6D). Notably, inhibition of RSK1 with BI-D1870 reduced Ser9 phosphorylation, suggesting restoration 769 \nof GSK3β activity and indicating that RSK1 contributes to GSK3β inactivation (Figure 6C and 6D). In contrast, 770 \ndirect inhibition of GSK3β with CHIR-99021 resulted in sustained inactivation, confirming the specificity and 771 \neffectiveness of the inhibitors. To evaluate the downstream effects of these perturbations, we examined Tau 772 \nphosphorylation and found that both HIV and cocaine exposure markedly increased Tau phosphorylation, 773 \nconsistent with RSK1 activation. (Figures 6C and 6D).  774 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nImportantly, RSK1 inhibition with BI-D1870 markedly suppressed Tau phosphorylation, whereas inhibition of 775 \nGSK3β with CHIR -99021 did not alter Tau phosphorylation induced by HIV  exposure or cocaine. These 776 \nfindings demonstrate that Tau phosphorylation in this context is primarily mediated through an RSK1 -777 \ndependent, GSK3β-independent mechanism. Interestingly, prolonged treatment (≥24 hours) with BI -D1870 778 \nalso resulted in a reduction in total RSK1 protein levels, while housekeeping controls (GAPDH) remained 779 \nunchanged. This suggests that sustained pharmacological inhibition may influence not only RSK1 activity but 780 \nalso its protein stability or turnover. 781 \nCollectively, these results establish a hierarchical signaling relationship in which RSK1 acts upstream of 782 \nGSK3β and plays a central role in mediating Tau phosphorylation following HIV and cocaine exposure. 783 \nFurthermore, they highlight RSK1 as a critical therapeutic target, as its inhibition effectively attenuates Tau 784 \npathology while also modulating downstream kinase signaling. We next investigated the temporal dynamics 785 \nof acute cocaine- and HIV-induced signaling in H80 cells to determine whether rapid inactivation of GSK3 β, 786 \nreflected by increased phosphorylation at the inhibitory Ser9 site, occurs in parallel with changes in Tau 787 \nphosphorylation at the pathological Ser396 residue. To assess time-dependent effects, cells were exposed to 788 \ncocaine or HIV for 1, 3, and 6 hours (Figure 6E). Protein lysates collected at each time point were analyzed 789 \nby immunoblotting for p -GSK3β Ser9, p-Tau Ser396, total Tau, and actin. Both cocaine and HIV induced a 790 \nsustained increase in GSK3β Ser9 phosphorylation at the 3- and 6-hour time points (lanes 3–4 vs. lane 1; 791 \nlanes 7–8 vs. lane 5), indicating persistent inhibition of GSK3β activity during acute exposure. Notably, this 792 \ninhibitory modification did not lead to a reduction in Tau phosphorylation. Instead, we observed a progressive 793 \nand robust increase in p -Tau Ser396 over time, demonstrating that Tau phosphorylation continues to 794 \naccumulate despite functional inactivation of GSK3 β. The simultaneous suppression of GSK3 β activity and 795 \nenhancement of Tau phosphorylation provides strong evidence for a GSK3β-independent mechanism of Tau 796 \nregulation under both cocaine and HIV exposure. These findings implicate alternative kinases, most 797 \nprominently RSK1, as key drivers of Tau phosphorylation at Ser396, even in the absence of active GSK3 β. 798 \nCollectively, these data demonstrate that acute cocaine and HIV exposures sustain Tau hyperphosphorylation 799 \nindependently of GSK3β activity, highlighting RSK1 as a dominant upstream kinase in this process. These 800 \nresults are consistent with our chronic exposure studies, further reinforcing a model in which RSK1-dependent 801 \nsignaling persistently drives Tau phosphorylation across temporal contexts. 802 \nRSK1 knockout impairs AKT signaling, activates GSK3β, and suppresses Tau phosphorylation 803 \nTo further confirm the direct role of RSK1 in regulating Tau phosphorylation, we generated RSK1 knockout 804 \n(KO) H80 cells using CRISPR-Cas9. Immunoblot analysis confirmed efficient loss/reduction of RSK1 protein, 805 \nenabling us to investigate the downstream signaling pathways.  Strikingly, RSK1 ablation led to a marked 806 \nreduction in Tau phosphorylation at Ser396 (p -Tau S396), while total Tau levels remained unchanged, 807 \nindicating that RSK1 regulates Tau primarily through post -translational mechanisms rather than 808 \ntranscriptional or translational control. Quantitative analyses across independent experiments confirmed a 809 \nsignificant decrease in p-Tau S396 in RSK1 KO cells compared with controls (Figure 7A and B), establishing 810 \nthat RSK1 is required for efficient phosphorylation of Tau at this pathological site.  811 \nInterestingly, RSK1 knockout also impacted GSK3β signaling. Specifically, loss of RSK1 resulted in a 812 \nreduction of inhibitory phosphorylation of GSK3β at Ser9, indicating reactivation of GSK3β kinase activity. 813 \nThese findings demonstrate that RSK1 acts upstream of GSK3β and contributes to its inactivation, consistent 814 \nwith our pharmacological inhibition data (Figure 6). Notably, however, reactivation of GSK3β did not restore 815 \nTau phosphorylation at Ser396, strongly documenting that RSK1 drives site -specific Tau phosphorylation 816 \nindependently of GSK3β (Figure 7). This observation highlights the complexity of Tau regulatory networks 817 \nand indicates that RSK1 is the main kinase controlling pathological Tau modification (Tau-S396), rather than 818 \nmerely modulating canonical Tau kinases such as GSK3β.  819 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nWe further evaluate the impact of RSK1 loss on AKT signaling pathway. RSK1 knockout resulted in severely 820 \nimpaired AKT signaling pathway, as evidenced by a marked reduction in phosphorylation at both Thr308 and 821 \nSer473. Notably, total AKT protein levels were also reduced, suggesting that RSK1 contributes not only to 822 \nAKT activation but also to AKT protein stability and/or abundance. These findings identify RSK1 as a positive 823 \nregulator of AKT signaling at both functional and protein stability levels (Figure 7C and D). Collectively, the 824 \ncoordinated effects of RSK1 deletion, including suppression of Tau phosphorylation, reactivation of GSK3β, 825 \nand attenuation of AKT signaling, establish RSK1 as a central upstream regulator of interconnected kinase 826 \nnetworks governing Tau pathology. Importantly, the inability of GSK3β reactivation to rescue Tau 827 \nphosphorylation further underscores the primary role of RSK1 in mediating Tau-S396 phosphorylation. 828 \nTaken together, these results position RSK1 as a critical signaling hub integrating AKT and GSK3β pathways 829 \nto regulate Tau phosphorylation and identify it as a promising therapeutic target for Tauopathies, including 830 \nHIV-associated neurocognitive disorders (HAND) and cocaine-associated neurodegeneration. 831 \nRSK1 functions as an upstream regulator of AKT- GSK3β signaling cascade. 832 \nAs detailed above, we identified RSK1 as a key upstream regulator of both AKT and GSK3β signaling in H80 833 \ncells. Loss (knock out) or pharmacological inhibition of RSK1 impaired AKT activation and simultaneously 834 \nreactivated GSK3β, as evidenced by reduced phosphorylation at its inhibitory Ser9 site. To further 835 \nsubstantiate this regulatory hierarchy, we next tested whether RSK1 overexpression produces the reciprocal 836 \neffects. Based on our prior findings, we hypothesized that elevated RSK1 levels would enhance AKT 837 \nactivation while promoting GSK3β inactivation via increased Ser9 phosphorylation. 838 \nTo evaluate the signaling consequences of RSK1 upregulation, H80 cells were transiently transfected with a 839 \nCMV-driven RSK1 expression construct, and lysates were collected 48 hours post -transfection for 840 \nimmunoblot analysis. Overexpression was confirmed by a marked increase in total RSK1 protein, along with 841 \nelevated levels of RSK1/2/3 isoforms, validating robust induction of the RSK signaling axis ( Figure 8A ). 842 \nNotably, phosphorylation of RSK1 at Ser380, a key autophosphorylation site associated with catalytic 843 \nactivation, was significantly increased. However, phosphorylation at Thr348, a critical activation loop residue 844 \nin the N -terminal kinase domain that is typically phosphorylated downstream of ERK signaling, remained 845 \nunchanged or even decreased relative to vector -transfected controls. The selective increase in pSer380 846 \nwithout a corresponding increase in pThr348 suggests that RSK1 overexpression alone does not result in full 847 \nenzymatic activation and may reflect a partial or ERK -independent activation state. This phosphorylation 848 \npattern implies that RSK1 may be primed for signaling but is not fully engaged in substrate phosphorylation, 849 \npotentially limiting canonical downstream activity.  850 \nConsistent with our model, RSK1 overexpression resulted in robust activation of AKT, as demonstrated by 851 \nincreased phosphorylation at both Thr308 and Ser473, the two critical regulatory sites required for full AKT 852 \nactivity (Figure 8B and D). Importantly, total AKT protein levels remained unchanged, indicating that RSK1 853 \nenhances AKT signaling primarily through post -translational activation rather than changes in protein 854 \nabundance. 855 \nIn parallel, RSK1 overexpression led to a pronounced increase in inhibitory phosphorylation of GSK3β at Ser9 856 \n(Figure 8C and D), confirming functional inactivation of this kinase. Total GSK3β levels remained constant, 857 \nfurther supporting that this effect reflects post-translational regulation. These findings reinforce the conclusion 858 \nthat RSK1 negatively regulates GSK3β activity through phosphorylation-dependent inhibition, likely in part via 859 \nAKT activation.  860 \nCollectively, these results establish RSK1 as a central upstream modulator of the AKT-GSK3β signaling axis 861 \nin H80 cells. By simultaneously activating AKT and suppressing GSK3β, RSK1 integrates MAPK/RSK and 862 \nPI3K/AKT signaling pathways and creates a cellular environment conducive to pathological Tau 863 \nphosphorylation. This mechanistic link suggests that dysregulation of RSK1 could shift neuronal kinase 864 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nnetworks toward pathological Tau modification, with broader implications for survival, metabolism, and 865 \nneurodegenerative processes. This coordinated regulation highlights RSK1 as a critical signaling hub that 866 \ngoverns kinase network balance and promotes Tau dysregulation. Altogether, our data position RSK1 as a 867 \nkey mechanistic driver and potential therapeutic target in conditions characterized by aberrant Tau 868 \nphosphorylation, including HIV-mediated neurotoxicity (HAND) and cocaine-induced neurodegeneration. 869 \nCocaine- and HIV-induced signaling is conserved across 2D neuronal cultures, 3D spheroids, and 870 \nbrain organoid model systems  871 \nTo determine whether the signaling pathways induced by cocaine and HIV in H80 cells are conserved across 872 \nadditional neuronal systems, we extended our investigation to SH -SY5Y neuroblastoma cells ( Figure 9B), 873 \n3D neuronal spheroids ( Figure 9A and 9C ), and human iPSC-derived brain organoids (Figure 9D). This 874 \napproach allowed us to evaluate the robustness and reproducibility of the identified signaling axis across 875 \nmodels of increasing biological complexity.  876 \nFirst, to assess reproducibility in an independent neuronal cell line, SH -SY5Y cells were exposed to HIV for 877 \n48 hours, followed by immunoblot analysis of key signaling markers. Chronic HIV exposure resulted in a 878 \npronounced upregulation of RSK1, accompanied by increased inhibitory phosphorylation of GSK3β at Ser9, 879 \nwhile total GSK3β levels remained unchanged (Figure 9B; lanes 3–4 vs. 1–2). Notably, this was paralleled 880 \nby a significant increase in Tau phosphorylation at Ser396, demonstrating that HIV induces a coordinated 881 \nsignaling response involving RSK1 activation, GSK3β inactivation, and Tau dysregulation. These findings 882 \nconfirm that the signaling axis identified in H80 cells is reproducible in additional neuronal cell types. 883 \nTo further examine whether these mechanisms are preserved in a more physiologically relevant 3D context, 884 \nwe utilized a multicellular neuronal spheroid model. Spheroids were generated by co-culturing equal numbers 885 \nof H80 cells, microglia, and SH-SY5Y cells (15,000 cells total per spheroid), and subjected to control, cocaine, 886 \nHIV, or combined treatments. Following 48 -hour exposure, pooled spheroids from each condition were 887 \nanalyzed by immunoblotting. Consistent with 2D models, both cocaine and HIV treatments induced robust 888 \nRSK1 upregulation, increased GSK3β Ser9 phosphorylation, and enhanced Tau phosphorylation at Ser396 889 \n(Figure 9C ). Importantly, the presence of microglia enabled productive HIV infection within the spheroid 890 \nsystem, further increasing physiological relevance. These coordinated molecular changes demonstrate that 891 \nthe RSK1-GSK3β-Tau signaling axis is preserved within a multicellular 3D neuronal microenvironment. 892 \nWe next evaluated whether these findings extend to higher -order neural systems using human cerebral 893 \norganoids (hCOs) derived from hiPSCs. Following exposure to cocaine, HIV, or both, organoids were 894 \nprocessed for immunoblot analysis. Consistent with results from both 2D cultures and spheroids, treated 895 \norganoids exhibited marked upregulation of RSK1, increased inhibitory phosphorylation of GSK3β at Ser9, 896 \nand sustained Tau phosphorylation at Ser396 (Figure 9D). Notably, Tau phosphorylation remained elevated 897 \ndespite GSK3β inactivation, reinforcing the presence of a GSK3β -independent mechanism, likely mediated 898 \nby RSK1. These results confirm that the identified signaling pathway is conserved even in complex, human-899 \nrelevant 3D brain models. 900 \nTogether, these data demonstrate that the core signaling cascade , RSK1 activation/upregulation, GSK3β 901 \ninactivation, and pathological Tau phosphorylation , identified in H80 cells is highly reproducible across 902 \nneuronal systems of increasing complexity, including 3D cultures, multicellular spheroids, and brain 903 \norganoids. This consistency underscores the biological robustness and generalizability of this pathway and 904 \nhighlights its relevance across diverse human-derived neural contexts, strengthening its potential significance 905 \nin HIV- and cocaine-associated neurodegeneration.  906 \nOverall, our investigation identifies RSK1 as a central signaling hub that integrates and coordinates multiple 907 \nkinase pathways governing Tau phosphorylation. Both HIV and cocaine robustly activate RSK1, which 908 \neventually promotes the inactivation of GSK3β, establishing a convergent downstream signaling axis despite 909 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\ndistinct upstream regulatory mechanisms. Importantly, Tau phosphorylation persists even under conditions 910 \nof GSK3β inhibition, demonstrating that RSK1 drives pathological Tau modification through a GSK3β -911 \nindependent mechanism.  These findings establish RSK1 as an essential upstream regulator of 912 \ninterconnected kinase networks that control site-specific Tau phosphorylation.  913 \nNotably, these signaling dynamics are consistently reproduced across multiple neuronal systems , including 914 \n2D cultures, 3D spheroids, and human brain organoids , underscoring the robustness, reproducibility, and 915 \nbiological relevance of this pathway across diverse neural contexts.  Collectively, these results support a 916 \nunified model in which RSK1 serves as the primary mediator linking HIV and cocaine exposure to Tau 917 \ndysregulation and neuronal stress responses. Beyond HAND and substance use -related neurotoxicity, this 918 \nmechanism has broader implications for Tau -driven neurodegenerative diseases, including Alzheimer’s 919 \ndisease and related cognitive disorders. Thus, RSK1 emerges as a key mechanistic driver and a promising 920 \ntherapeutic target for conditions characterized by aberrant Tau phosphorylation and neurodegeneration. 921 \nDistinct yet convergent signaling mechanisms by which HIV exposure and cocaine drive Tau 922 \nphosphorylation/ pathology 923 \nTo summarize our findings, we propose the following model for HIV - and cocaine -induced Tau 924 \nphosphorylation (Figure 10). In this study, we demonstrate that exposure to HIV and cocaine leads to robust 925 \nTau phosphorylation, and we delineate the distinct yet convergent molecular mechanisms underlying this 926 \nprocess. Although both stimuli ultimately induce Tau phosphorylation, the upstream signaling pathways they 927 \nengage are mechanistically distinct. In the context of HIV exposure, we observed a pronounced and sustained 928 \nupregulation and activation of RSK1. Activated RSK1 promotes Tau phosphorylation while simultaneously 929 \ninhibiting GSK3β activity through an AKT -independent mechanism. Consistent with this pathway, HIV 930 \nexposure resulted in a marked increase in Tau phosphorylation, identifying RSK1 as a dominant mediator of 931 \nHIV-driven Tau dysregulation. On the other hand, cocaine exposure engages a partially overlapping but 932 \ndistinct signaling cascade. While cocaine induces modest activation of RSK1, it strongly stimulates AKT, as 933 \nevidenced by robust phosphorylation at Thr308 and Ser473, both required for full catalytic activation. 934 \nActivated AKT subsequently phosphorylates GSK3β at Ser9 (p -GSK3β-Ser9), leading to its functional 935 \ninactivation. Despite differences in upstream signaling intensity, cocaine also promotes Tau phosphorylation, 936 \nhighlighting a mechanism that does not rely solely on the robust RSK1 activation observed in the case of HIV. 937 \nNotably, Tau phosphorylation persists even under conditions of GSK3β inactivation in both HIV- and cocaine-938 \nexposed systems. This finding reveals the existence of a GSK3β-independent mechanism of Tau modification 939 \nand establishes RSK1 as a key regulator of Tau phosphorylation under these conditions. The persistence of 940 \nTau phosphorylation despite suppression of canonical GSK3β activity suggests the involvement of parallel or 941 \ncompensatory signaling pathways that warrant further investigation. 942 \nImportantly, we identify RSK1 as a critical upstream regulator of both AKT and GSK3β signaling, exerting 943 \npositive control over AKT activation while negatively regulating GSK3β activity. This dual regulatory capacity 944 \npositions RSK1 as a central signaling hub integrating viral and drug-induced pathways that converge on Tau 945 \npathology. Collectively, our findings provide mechanistic insight into how HIV and cocaine exposure, through 946 \ndistinct yet convergent pathways, drive Tau dysregulation and contribute to neurotoxicity. From a therapeutic 947 \nperspective, these results highlight RSK1 as a promising target for intervention, offering a unifying framework 948 \nfor mitigating tauopathy in neuroHIV, HAND, and cocaine-associated neurodegeneration. 949 \nDiscussion 950 \nNeuronal cell lines such as SH‑SY5Y are widely used experimental models, yet they have limitations that can 951 \ncompromise experimental robustness  [56]. Neurons cells are highly sensitive to culture conditions, exhibit 952 \nvariable growth and differentiation rates, and frequently display batch ‑to‑batch and passage ‑dependent 953 \nheterogeneity [64]. Such instability poses significant challenges for studies requiring long ‑term culture or 954 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nconsistent phenotypic behavior, particularly investigations focused on neurodegeneration, Tau ‑related 955 \npathology, or kinase‑driven signaling mechanisms. These constraints highlight the need for neuronal models 956 \nthat retain essential neuron ‑like properties while offering greater stability, reproducibility, and ease of 957 \nmaintenance. More robust and tractable cell systems not only improve experimental consistency but also 958 \nenable higher ‑throughput analyses and more reliable interpretation of signaling mechanisms relevant to 959 \nneuropathies, tauopathies, and neurotoxic exposures. 960 \nIn this study, we characterized H80 cells as a stable and experimentally tractable neuronal model and 961 \nemployed them to investigate the molecular mechanisms by which HIV and cocaine promote Tau 962 \nphosphorylation and neurotoxicity. H80 cells were selected based on their robust proliferation, low cytotoxicity, 963 \nand stable culture performance. Through immunofluorescence, qPCR, and Western blot analyses, we 964 \nconfirmed that H80 cells express key neuronal markers, including NeuN, MAP2, and Tau, consistent with a 965 \nmature neuronal phenotype (Figure 1A-C; Supplementary Figure S1). Notably, the expression of MAP2, an 966 \naxon-associated protein critical for neuronal architecture and implicated in neurodegenerative processes, 967 \nfurther supports the neuronal identity of H80 cells. Collectively, the presence of these well -established 968 \nneuronal markers demonstrates that H80 cells possess essential neuron-like features and extends their utility 969 \nbeyond glioma research.  Importantly, given our focus on HIV -induced neurotoxicity, we assessed whether 970 \nH80 cells express key HIV receptors and co-receptors. Our results show that H80 cells do not express CD4 971 \nor CCR5, the canonical receptor and major co -receptor for HIV entry, respectively. However, H80 does 972 \nexpress one of the HIV co -receptors, CXCR4, in approximately 20% of cells ( Figure 1D). This expression 973 \nprofile is consistent with previous reports indicating that neurons lack CD4 but express chemokine receptors 974 \nsuch as CXCR4 and CCR5.  Prior studies, including those by Kaul and colleagues, have demonstrated that 975 \nalthough due to the absence of HIV receptor, neurons are not productively infected by HIV, they remain highly 976 \nsusceptible to HIV-induced injury mediated through chemokine receptors and downstream signaling cascade, 977 \nwith CXCR4 serving as a key mediator of neurotoxic signaling in neurons [17, 57]. The absence of CCR5 and 978 \nthe selective expression of CXCR4 in H80 cells therefore provide a unique and focused system to investigate 979 \nCXCR4-dependent mechanisms of HIV-associated neuronal stress. This receptor profile enables us to dissect 980 \nhow HIV exposure perturbs neuronal signaling in the absence of CCR5 -mediated protective pathways, 981 \nthereby facilitating a clearer understanding of HIV-induced neurotoxicity. 982 \nOur study identifies a previously unrecognized mechanistic link between viral exposure and neuronal stress 983 \npathways. Using an integrated approach combining transcriptional analysis, immunofluorescence, and 984 \nbiochemical analyses, we demonstrate that exposure to HIV virions robustly activates inflammatory signaling 985 \ncascades and induces neurotoxic responses in H80 neuronal cells. Specifically, HIV exposure significantly 986 \nupregulates the transcripts of pro-inflammatory cytokines IL-1β and TNF-α (Figure 2B), revealing a previously 987 \nunderappreciated neuron-intrinsic inflammatory response. These findings indicate that direct interaction with 988 \nviral particles is sufficient to trigger canonical neuroinflammatory programs in neuronal cells. Given that IL-1β 989 \nand TNF-α are well-established mediators of neuronal injury in both HIV-associated neurocognitive disorders 990 \n(HAND) and Alzheimer’s disease, our results highlight a shared inflammatory axis between virally induced 991 \nand classical neurodegenerative processes, characterized by proinflammatory cytokines and pathologic Tau 992 \nphosphorylation.  Although prior studies have primarily attributed HIV-induced cytokine production to microglia 993 \n[65, 66] , emerging evidence suggests that neurons can also produce cytokines that modulate synaptic 994 \nfunction and central nervous system homeostasis, a fact further strengthened using our novel neuronal model 995 \nsystem. [67, 68]. The robust cytokine induction observed here further supports effective exposure of H80 cells 996 \nto HIV virions and underscores the capacity of neurons to directly engage in inflammatory signaling. 997 \nImportantly, our biochemical analyses reveal that HIV-exposed H80 cells exhibit concurrent increases in RSK1 998 \nprotein levels and Tau phosphorylation at Ser396 (p -Tau S396), with quantitative immunoblotting 999 \ndemonstrating a strong correlation between these events (Figures 2C-E). In contrast, total Tau protein levels 1000 \nwere only modestly altered, indicating that HIV primarily drives post -translational modification of Tau rather 1001 \nthan increasing its overall expression and abundance. These findings support a model in which HIV-induced 1002 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nRSK1 activation directly promotes pathogenic Tau phosphorylation. Given that phosphorylation at Ser396 is 1003 \nclosely associated with Tau protein aggregation and neuronal toxicity, our data establishes RSK1 as a critical 1004 \neffector linking HIV exposure to early tauopathic signaling. The simultaneous induction of inflammatory 1005 \ncytokines and RSK1 further suggests coordinated activation of inflammatory and stress -responsive kinase 1006 \npathways, creating a signaling environment that may initially be adaptive but ultimately becomes maladaptive. 1007 \nAs a downstream effector of MAPK signaling, RSK1 plays key roles in transcriptional regulation and cellular 1008 \nstress responses; however, its sustained activation appears to drive pathological Tau modification.  1009 \nCollectively, these findings identify RSK1 as a central molecular node connecting HIV-induced inflammatory 1010 \nsignaling to Tau pathology. By delineating this pathway, our study provides new mechanistic insight into how 1011 \nHIV exposure can initiate and accelerate neurodegenerative processes within the central nervous system.  1012 \nIn addition to the effects induced by HIV, our data demonstrate that cocaine also independently activates the 1013 \nRSK1 signaling cascade to drive Tau phosphorylation, revealing a shared, previously unappreciated kinase 1014 \ndependency underlying both viral - and drug-induced neurotoxicity underscoring the convergence of drug s 1015 \nand HIV‑mediated stress responses on a shared kinase pathway. Chronic cocaine exposure triggered robust 1016 \nphosphorylation of RSK1 at key regulatory residues (Thr348, Thr359/Ser363, and Ser380), accompanied by 1017 \na pronounced increase in Tau phosphorylation at Ser396, without any significant change in total Tau protein 1018 \nlevels (Figures 3B–D). These findings establish that cocaine drives Tau pathology primarily through post -1019 \ntranslational mechanisms rather than altering Tau expression via transcriptional or translational regulation.  1020 \nStrikingly, RSK1 activation was both rapid and highly sensitive; even a brief 15 -minute exposure to cocaine 1021 \nwas sufficient to induce multi -site phosphorylation ( Figures 3E –G). A comparable activation profile was 1022 \nobserved following acute HIV exposure, positioning RSK1 as an immediate and convergent sensor of diverse 1023 \nneurotoxic stimuli. Functional interrogation of RSK1 in mediating Tau phosphorylation unequivocally 1024 \nestablishes RSK1 as essential for Tau phosphorylation. Both pharmacological inhibition and genetic ablation 1025 \nof RSK1 completely abolished Tau -Ser396 phosphorylation induced by either HIV or cocaine ( Figure 6), 1026 \ndemonstrating that RSK1 is not merely correlative but a required driver of this process. Notably, these findings 1027 \nadd a new layer to the prevailing paradigm that GSK3β is the dominant Tau kinase, instead identifying RSK1 1028 \nas the principal effector of Tau phosphorylation under conditions of HIV and cocaine exposure. Despite distinct 1029 \nupstream dynamics, HIV and cocaine converge on a common downstream outcome, pathological Tau 1030 \nphosphorylation, through a shared RSK1 -centered signaling axis. HIV elicited more robust RSK1 activation 1031 \nthan cocaine; however, both stimuli produced comparable levels of Tau phosphorylation, indicating that RSK1 1032 \nactivity, rather than upstream signal intensity, dictates the pathological output. Importantly, combined HIV and 1033 \ncocaine exposure failed to produce additive or synergistic effects, instead reaching a plateau consistent with 1034 \nsaturation of a shared signaling pathway. Collectively, these findings redefine the molecular framework of Tau 1035 \ndysregulation by establishing RSK1 as a central and dominant integrator of viral and drug -induced neuronal 1036 \nstress. By orchestrating Tau phosphorylation, RSK1 provides a common node  through which diverse 1037 \nupstream perturbations converge on a common pathological outcome. The rapid activation of RSK1 following 1038 \nacute exposure further suggests that it may function as an early sensor of neuronal stress, initiating 1039 \ndownstream signaling cascades that culminate in Tau pathology. The rapid activation kinetics further suggest 1040 \nthat RSK1 functions as an early molecular sentinel that initiates downstream tauopathic cascades. The 1041 \nconvergence of HIV and cocaine on this shared node provides a mechanistic explanation for the heightened 1042 \nvulnerability to neurodegeneration observed in individuals exposed to either insult, particularly in the context 1043 \nof neuroHIV, where comorbid stimulant use is widespread and associated with accelerated cognitive decline. 1044 \nThe identification of RSK1 as a unifying mechanistic driver provides a compelling framework for understanding 1045 \nhow these interactions may arise and highlights a promising therapeutic target for mitigating Tau pathology 1046 \nacross diverse neurotoxic contexts. 1047 \nAn unexpected and conceptually important observation emerged from our investigation, an independent 1048 \nexposure to either HIV or cocaine consistently resulted in inactivation of GSK3 β ( an increase in 1049 \nphosphorylation at S9), a kinase implicated in driving Tau phosphorylation and neurodegenerative pathology. 1050 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nThis suppression of GSK3 β activity was evidenced by a robust increase in its inhibitory phosphorylation at 1051 \nSer9, yet Tau remained persistently hyperphosphorylated under these same conditions (Figure 4 and Figure 1052 \n6). This strongly suggests that, Tau phosphorylation proceeds through GSK3 β‑independent mechanisms, 1053 \nthereby elevating, RSK-1. Our findings highlight RSK1 as the dominant kinase responsible for maintaining 1054 \nTau phosphorylation when GSK3β is rendered inactive. Our data reveal a remarkably consistent pattern, both 1055 \nHIV and cocaine suppress GSK3β activity while Tau phosphorylation remains elevated (Figure 4 and Figure 1056 \n6). This divergence between upstream signaling and Tau modification further supports a model in which RSK1 1057 \nactivation becomes the primary driver of Tau ‑Ser396 phosphorylation under HIV and cocaine exposure. 1058 \nImportantly, this signaling paradigm proved highly reproducible across multiple experimental conditions. We 1059 \nobserved the same pattern of GSK3 β inactivation coupled with persistent Tau phosphorylation during acute 1060 \n(Figure 4C, 4D, 4E, 4F and 6E ) as well as chronic cocaine and HIV exposure (Figure 4G, 4H, 6A, 6B, 6C 1061 \nand 6D). HIV and cocaine suppress GSK3β and act through RSK1 to drive Tau phosphorylation, positioning 1062 \nRSK1 as a central mediator of their shared pathogenic effects. Altogether, our findings demonstrate that both 1063 \ncocaine and HIV decrease GSK3 β activity and instead engage RSK1 to drive Tau phosphorylation. These 1064 \nresults identify RSK1 as a central signaling node through which cocaine and HIV converge to promote shared 1065 \npathogenic mechanisms. 1066 \nFurther mechanistic dissection revealed a clear divergence in how cocaine and HIV regulate upstream kinase 1067 \nsignaling. Cocaine, but not HIV, robustly activated the AKT pathway, as evidenced by increased 1068 \nphosphorylation at Thr308 and Ser473, two critical residues required for full catalytic activation ( Figure 5). 1069 \nThis activation was accompanied by a corresponding increase in inhibitory phosphorylation of GSK3β at Ser9, 1070 \nconfirming that cocaine suppresses GSK3 β through a canonical AKT-dependent pathway. In contrast, HIV 1071 \nexposure failed to induce measurable AKT activation, indicating that HIV -mediated inhibition of GSK3 β 1072 \nproceeds via an AKT -independent mechanism. Instead, HIV selectively upregulates and activates RSK1, 1073 \nwhich in turn drives GSK3 β inactivation. Thus, while both stimuli converge on GSK3 β inactivation and Tau 1074 \nhyperphosphorylation, they do so through distinct upstream routes, cocaine engaging both AKT and RSK1, 1075 \nand HIV relying predominantly on RSK1. These findings support a bifurcated signaling model in which cocaine 1076 \nand HIV converge on a shared downstream outcome, GSK3 β inhibition and Tau hyperphosphorylation, yet 1077 \nreach this endpoint through mechanistically distinct routes. Cocaine engages both AKT and RSK1, thereby 1078 \nbroadly amplifying kinase signaling networks, whereas HIV bypasses AKT entirely and relies predominantly 1079 \non RSK1 activation. This distinction provides important biological insight: cocaine simultaneously activates 1080 \nsurvival-associated (AKT) and stress -responsive (RSK1) pathways, while HIV exerts a more targeted yet 1081 \npotent effect through selective RSK1 induction.  1082 \nPharmacological and genetic perturbation studies further establish RSK1 as the central regulator of this 1083 \nsignaling architecture. Inhibition of RSK1 with BI -D1870 markedly reduced Tau -Ser396 phosphorylation 1084 \ndespite restoration of GSK3β activity, demonstrating that RSK1, not GSK3β, is the primary kinase sustaining 1085 \nTau phosphorylation under both cocaine and HIV exposure (Figure 6). Conversely, inhibition of GSK3β with 1086 \nCHIR-99021 had no effect on RSK1 activation, confirming that RSK1 operates upstream of GSK3 β in this 1087 \nhierarchy. These findings were further validated by CRISPR -Cas9-mediated knockout of RSK1, which 1088 \nabolished Tau phosphorylation, reactivated GSK3 β, and reduced both AKT phosphorylation and total AKT 1089 \nlevels (Figure 7). Notably, the reduction in AKT abundance following RSK1 depletion suggests that RSK1 1090 \ncontributes to AKT stabilization and activation, placing it at the apex of a coordinated kinase network.  1091 \nCollectively, these results define a unified signaling paradigm in which RSK1 functions as a central integrator 1092 \nlinking HIV and cocaine exposure to pathological Tau hyperphosphorylation. This RSK1 -driven mechanism 1093 \noperates independently of GSK3β and, in the case of cocaine, is further reinforced by AKT activation, thereby 1094 \nintegrating distinct upstream perturbations into a common pathological outcome. The convergence of viral 1095 \nand drug-induced signaling on RSK1 provides a mechanistic explanation for the heightened vulnerability to 1096 \nTau-associated neurodegeneration observed in neuroHIV, particularly in the context of stimulant drug use. 1097 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nImportantly, the demonstration that pharmacological inhibition of RSK1 reverses Tau hyperphosphorylation 1098 \nand restores kinase balance underscores its translational potential. These findings position RSK1 as a 1099 \ntractable and previously underappreciated therapeutic target for mitigating Tauopathy in HIV -associated 1100 \nneurocognitive disorders (HAND), as well as in broader neurodegenerative conditions such as Alzheimer’s 1101 \ndisease. Future studies in preclinical models, including humanized mouse models, will be essential to 1102 \ndetermine whether targeting RSK1 can attenuate neuroinflammation, prevent Tau pathology, and ultimately 1103 \nslow or halt neurodegenerative progression. 1104 \nPrior studies have shown that the Tat protein of HIV  can activate GSK3 β and contributes to Tat-mediated 1105 \nneurotoxicity [19, 69, 70] . However, in this study we examined neuronal responses to intact, replication -1106 \ncompetent HIV virions (HIV-1 strain 93/TH/051; dual-tropic, R5/X4), thereby capturing the integrated effects 1107 \nof the full viral particle without infection and replication. Notably, both acute and chronic exposure to these 1108 \ndual-tropic virions consistently resulted in functional inactivation of GSK3 β, as evidenced by increased 1109 \nphosphorylation at the inhibitory Ser9 site ( Figures 4 and 6). These observations likely reflect the complex 1110 \ncomposition of intact virions, which contain multiple structural and accessory proteins capable of exerting both 1111 \nactivating and inhibitory influences on intracellular kinase networks, ultimately shifting the balance toward 1112 \nGSK3β suppression. Strikingly, cocaine exposure produced a similar biochemical signature, robust Ser9 1113 \nphosphorylation and inactivation of GSK3 β. This convergence suggests that HIV and cocaine, despite 1114 \nengaging distinct upstream signaling pathways, AKT-dependent in the case of cocaine and AKT-independent 1115 \nfor HIV, ultimately suppress GSK3 β through a shared downstream mechanism. Our data identify RSK1 as 1116 \nthe central integrator of this process, coordinating GSK3 β inhibition and sustaining Tau phosphorylation. 1117 \nThese findings indicate that virion-mediated effects are not merely additive but instead converge on a common 1118 \nRSK1-driven intracellular signaling axis that governs neuronal stress responses and Tau pathology. 1119 \nFuture studies will focus on defining the specific viral determinants within the intact virion that initiate RSK1 1120 \nactivation, as well as identifying the neuronal receptors involved, including the potential role of CXCR4 -1121 \nmediated signaling. Elucidating the precise regulatory sites on RSK1 that mediate downstream suppression 1122 \nof GSK3 β, particularly those governing Ser9 -directed inhibitory phosphorylation, while simultaneously 1123 \nsustaining Tau phosphorylation at Ser396 will be critical for resolving the hierarchical organization of this 1124 \nsignaling network. In parallel, comparative analyses of cocaine- and HIV-mediated pathways will be essential 1125 \nto determine whether these distinct stimuli converge on shared upstream sensors or utilize overlapping 1126 \nsignaling modules. Such investigations will help establish whether a common molecular node integrates viral 1127 \nand environmental stressors to modulate neuronal vulnerability. 1128 \nImportantly, our findings identify RSK1 as a major effector of HIV -induced Tau phosphorylation at Ser396, 1129 \nestablishing a direct mechanistic link between viral exposure and tauopathic processes ( Figure 2 ). HIV 1130 \nexposure activates inflammatory signaling cascades, leading to upregulation of RSK1 and subsequent 1131 \nphosphorylation of Tau at Ser396, a modification strongly associated with neurofibrillary tangle formation in 1132 \nAlzheimer’s disease and HAND. The concurrent induction of pro-inflammatory cytokines, including IL-1β and 1133 \nTNF-α, further implicates inflammatory stress as a key upstream driver of this pathway.  Collectively, these 1134 \nresults support a model in which HIV -induced inflammatory and stress -responsive signaling converge on 1135 \nRSK1 to drive Tau pathology. This RSK1 -centered mechanism provides a unifying framework linking viral 1136 \nexposure, neuroinflammation, and neurodegeneration, and offers important insight into how HIV infection may 1137 \ninitiate or accelerate Tau-mediated neuronal dysfunction within the CNS. 1138 \nIn summary, our findings support a unified signaling network in which RSK1 functions as a central molecular 1139 \nnode linking both HIV and cocaine exposure to Tau hyperphosphorylation. We show that Tau phosphorylation 1140 \ncan be sustained through a GSK3 β independent mechanism under conditions of HIV and cocaine induced 1141 \nstress, extending current models of Tau regulation. In the context of cocaine exposure, our data indicates the 1142 \npresence of a dual axis signaling architecture in which RSK1 cooperates with AKT, integrating stress 1143 \nresponsive and survival signaling pathways into a convergent downstream outcome. Collectively, these 1144 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nresults indicate that RSK1 is not merely associated with Tau dysregulation but plays a functional role in 1145 \nmediating Tau phosphorylation in the setting of HIV and cocaine exposure. 1146 \nThrough complementary genetic and pharmacological approaches, we demonstrate that RSK1 acts as a 1147 \ndominant and convergent driver of Tau pathology, redefining the hierarchical organization of kinase signaling 1148 \nnetworks that govern Tau modification. These findings fill a critical gap in our understanding of how diverse 1149 \nupstream insults, viral infection, and substance abuse , converge on shared molecular pathways to drive 1150 \nneurodegeneration. The significance of this work lies in its ability to bridge mechanistic and clinical 1151 \nobservations. HIV infection and stimulant use are independently associated with accelerated cognitive 1152 \ndecline, yet the molecular basis of their interaction has remained poorly understood. By identifying RSK1 as 1153 \na unifying signaling hub, our study provides a mechanistic framework that explains how these factors 1154 \nsynergistically promote Tauopathy in HIV -associated neurocognitive disorders (HAND) and related 1155 \nneurodegenerative conditions, including Alzheimer’s disease (AD). This conceptual advance establishes a 1156 \nnew foundation for investigating the intersection of neuroHIV and substance abuse –associated 1157 \nneuropathology. 1158 \nImportantly, our findings have immediate translational implications. We demonstrate that pharmacological 1159 \ninhibition of RSK1 reverses Tau hyperphosphorylation and restores kinase homeostasis, identifying RSK1 as 1160 \na tractable and high -value therapeutic target. Targeting RSK1 offers the potential to intercept pathogenic 1161 \nsignaling cascades upstream of irreversible neuronal damage, representing a fundamentally new strategy for 1162 \nmitigating Tau-driven neurodegeneration. Future studies will focus on evaluating the therapeutic efficacy of 1163 \nRSK1 inhibition in physiologically relevant preclinical models, including humanized mouse systems, to 1164 \ndetermine whether targeting this pathway can attenuate neuroinflammation, suppress Tau pathology, and 1165 \npreserve neuronal function. Successful validation of this approach has the potential to transform therapeutic 1166 \nstrategies for HAND and other Tau-associated disorders by targeting a shared and central molecular driver 1167 \nof disease. 1168 \nLimitation 1169 \nThe main limitation of the study is that while NeuN, MAP2, and Tau serve as well -established neuronal 1170 \nmarkers, future studies should incorporate additional proteins associated with synaptic activity and neuronal 1171 \nfunction, such as synaptophysin, neurofilament, and neuron -specific enolase (NSE), to further validate 1172 \nwhether H80 cells exhibit fully functional neuronal behavior. H80 cells also require further characterization to 1173 \ndetermine whether they correspond to distinct neuronal lineages, including dopaminergic, glutamatergic, 1174 \nGABAergic, or cholinergic neurons. Moreover, it remains unclear whether neuronal protein expression in H80 1175 \ncells arises from intrinsic differentiation potential, genetic reprogramming, or adaptive responses to the tumor 1176 \nmicroenvironment. Elucidating these mechanisms will be essential for defining the broader biological 1177 \nsignificance of our observations. However, despite limitations, our study provides compelling evidence that 1178 \nH80 cells possess neuronal lineage features, as demonstrated by the expression of NeuN, MAP2, and Tau. 1179 \nThese findings expand the characterization of H80 cells and underscore their potential as a hybrid model 1180 \nsystem at the intersection of glioma biology and neurodegenerative research.  Nevertheless, some of the 1181 \nsalient findings have been confirmed in well -established neuronal models, such as SH -SY5Y neuronal cell 1182 \nline, and 3D models, such as spheroid and organoids containing either neuronal cell line (SHSY5Y) or iPSCs-1183 \nderived neurons, respectively, which substantially enhance the rigor and robustness of the findings.   1184 \nConclusion 1185 \nThese findings collectively support a unified model in which RSK1 functions as a central signaling hub 1186 \nintegrating diverse upstream perturbations into a common downstream outcome, pathological Tau 1187 \nphosphorylation. The convergence of HIV- and cocaine-induced signaling on RSK1 provides a mechanistic 1188 \nframework for understanding how viral infection and substance abuse jointly exacerbate neurodegenerative 1189 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nprocesses, particularly in the context of neuroHIV.  Importantly, this RSK1 -driven mechanism appears to 1190 \noperate independently of GSK3 β, indicating the presence of an additional regulatory layer beyond the 1191 \nestablished role of GSK3β in Tau phosphorylation. In the context of cocaine exposure, this pathway is further 1192 \nreinforced through coordinated activation of AKT, supporting a dual -axis signaling network that integrates 1193 \nsurvival and stress -responsive pathways into a shared pathological outcome. This study provides direct 1194 \nevidence that RSK1 is not merely associated with but is functionally required for Tau phosphorylation in the 1195 \nsetting of viral and substance -induced neuronal stress. By identifying RSK1 as a dominant and convergent 1196 \ndriver of Tau pathology, our work redefines the regulatory hierarchy of Tau -directed kinase signaling and 1197 \nuncovers a critical, previously underappreciated mechanism underlying neurodegeneration.  From a 1198 \ntranslational perspective, the identification of RSK1 as a dominant and druggable driver of Tau pathology has 1199 \nimportant implications. The ability of RSK1 inhibition to reverse Tau hyperphosphorylation and restore kinase 1200 \nbalance suggests that targeting this pathway may offer a viable therapeutic strategy for mitigating Tauopathy 1201 \nin HIV -associated neurocognitive disorders, as well as in broader neurodegenerative conditions. More 1202 \nbroadly, these results provide mechanistic clarity to longstanding clinical observations linking HIV infection 1203 \nand stimulant use with accelerated cognitive decline, and position RSK1 as a promising point of intervention 1204 \nfor preventing or slowing neurodegeneration. Future studies will focus on evaluating whether pharmacologic 1205 \ninhibition of RSK1 in physiologically relevant preclinical models, including humanized mouse systems, can 1206 \nattenuate neuroinflammation, suppress Tau pathology, and preserve neuronal function. Such investigations 1207 \nwill be critical for establishing the therapeutic viability of RSK1-targeted interventions and may ultimately pave 1208 \nthe way for novel treatment strategies aimed at preventing or slowing neurodegeneration in HAND and related 1209 \nTau-associated disorders. 1210 \nAcknowledgement 1211 \nWe thank the AIDS Research and Reagent Program, Division of AIDS, National Institute of Allergy, and 1212 \nInfectious Diseases, US National Institutes of Health. We thank Dr. Jonathan Karn and his laboratory for 1213 \nproviding the C20 human microglial cell line. We acknowledge the NIH HIV Reagent Program (Division of 1214 \nAIDS, NIAID, NIH) and Dr. Douglas Richman for providing MT 4 cells (ARP 120). This study utilized services 1215 \noffered by core facilities of Thomas Jefferson University (FACS and Imaging) and the Comprehensive 1216 \nNeuroHIV Center (CNHC) at Temple University Lewis Katz School of Medicine. Moreover, we would like to 1217 \nthank the Center for Translational Medicine, Thomas Jefferson University, including all staff members for their 1218 \ntechnical support and assistance in conducting the experiments for this study. 1219 \nFunding  1220 \nResearch reported in this publication was supported by the National Institutes of Health under Award Number 1221 \nR01DA041746 and 1R21MH126998 -01A1 to M.T.; Institutional TJU grant (908107) to M.T. The content is 1222 \nsolely the responsibility of the authors and does not necessarily represent the official views of the National 1223 \nInstitutes of Health. 1224 \nAuthors’ Contribution  1225 \nConceptualization, A.L.S. and M.T.; methodology, A.L.S. and I.K.S.; software, A.L.S., I.K.S., and M.T.; 1226 \nvalidation, A.L.S., I.K.S., and M.T.; formal analysis, A.L.S., I.K.S., U.P.N., and M.T.; investigation, A.L.S., I.K.S., 1227 \nU.P .N., and M.T.; data curation, A.L.S., I.K.S., U.P.N., and M.T.; writing—original draft preparation and review, 1228 \nA.L.S., I.K.S., U.P.N., and M.T.; project supervision and funding acquisition, M.T.; all authors have read and 1229 \napproved the final version of the manuscript. 1230 \nDeclaration of interests 1231 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nThe authors declare no competing interests 1232 \nEthics statement 1233 \nNo ethical approval required 1234 \nGenerative AI statement 1235 \nThe authors declare that no generative AI was used in the creation of this manuscript 1236 \nResource availability 1237 \nLead contact  1238 \nRequests for further information and resources should be directed to and will be fulfilled by the lead contact, 1239 \nMudit Tyagi (Mudit.tyagi@jefferson.edu).  1240 \nMaterials availability  1241 \nThis study did not generate new unique reagents.  1242 \nData and code availability  1243 \n• This paper does not report original code.  1244 \n• Any additional information required to reanalyze the data reported in this paper is available from the lead 1245 \ncontact upon request. 1246 \nKey resources table 1247 \nREAGENT or RESOURCE SOURCE IDENTIFIER \nAntibodies   \np-RSK-1 (pS380.20A) Santa Cruz Biotechnology sc-136476 \np-RSK-1 (Thr 348) Santa Cruz Biotechnology sc-101770 \nPhospho-p90RSK (Thr359/Ser363) Antibody Cell signaling technology #9344 \nRSK1 Antibody Cell signaling technology #9333 \nRSK1/RSK2/RSK3 (D7A2H) Rabbit mAb Cell signaling technology #14813 \nPhospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb Cell signaling technology #5558 \nGSK-3β (D5C5Z) XP® Rabbit mAb Cell signaling technology #12456 \nPhospho-Akt (Thr308) (244F9) Rabbit mAb Cell signaling technology #4056 \nPhospho-Akt (Ser473) (D9E) XP® Rabbit mAb Cell signaling technology #4060 \nAkt1 (C73H10) Rabbit mAb Cell signaling technology #2938 \nPhospho-Tau (Ser396) (PHF13) Mouse mAb Cell signaling technology #9632 \nTau (D1M9X) XP® Rabbit mAb Cell signaling technology #46687 \nAnti-HIV1 p55 + p24 + p17 antibody Abcam ab63917 \nMAP2 Polyclonal antibody proteintech 17490-1-AP \nAlexa Fluor 647 Anti-RBFOX3 (NeuN) BioLegend 608453 \nAlexa Fluor 647 anti-Tau phosphor (Ser396) BioLegend 829005 \nAnti-Tau BioLegend 806701 \nActin antibody Santa Cruz Biotechnology sc-47778 \nGAPDH antibody Santa Cruz Biotechnology sc-25778 \nAlexa Fluor 488 goat anti-rabbit Invitrogen A11008 \nAlexa Fluor 568 goat anti-mouse Invitrogen A11004 \nIRDye 680RD Li-cor (Lincoln, NE, USA) Cat# 926-68071; RRID: \nAB_10956166 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nIRDye 680LT Li-cor (Lincoln, NE, USA) Cat# 926-68022; RRID: \nAB_10715072 \nIRDye 800CW Li-cor (Lincoln, NE, USA) Cat# 926-32211; RRID: AB_621843 \nAPC anti-human CD4 BioLegend Cat# 317416 \nPE anti-human CD184 (CXCR4) BioLegend Cat# 306505 \nPE anti-human CD4 BioLegend Cat# 357403 \nAPC/Cyanine& anti0human CD195 (CCR5) BioLegend Cat# 359110 \nChemicals   \nCocaine NIH  \nLipofectamine 2000 Reagent Invitrogen 11668027 \nTrizma Base Sigma-Aldrich T1503 \nGlycine Fisher Chemical G46-1 \nSodium chloride Fisher Chemical S271-1 \nSodium dodecyl sulfate Bio-Rad 161-0302 \nAcrylamide Fisher Chemical O1065-500 \nBis-acrylamide Hoefer GR142-100 \nEDTA Sigma-Aldrich E6758-500G \nPotassium chloride Sigma-Aldrich P9541-1KG \nBSA (Fraction V) RPI Research Products A30075-100.0 \nAmmonium persulfate Fisher Chemical S25178 \nTEMED Fisher Chemical BP150-20 \nPierce BCA Reagent A Thermo Scientific 23228 \nPierce BCA Reagent B Thermo Scientific 23224 \n2-Mercaptoethanol Sigma-Aldrich M3148-250ML \n1-Butanol Fisher Chemical A399-500 \nRPMI-1640 Medium (1X) Cytiva HyClone Laboratories SH30027.02 \nPenicillin-Streptomycin Gibco 15140-122 \nFetal Bovine Serum Gibco 10082147 \nNonidet P-40 Substitute Sigma-Aldrich 74385-1L \nTriton X-100 Sigma-Aldrich T8787 \nDL-Dithiothreitol Sigma-Aldrich D0632-1G \nHEPES Buffer Corning 25-060-Cl \nPMSF Thermo Fisher Scientific 36978 \nPageRuler Prestained Ladder Thermo Fisher Scientific 26617 \nNitrocellulose blotting membrane Amersham 10600006 \nRNeasy Plus Mini Kit Qiagen 74134 \nHigh-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific 4374967 \nHoechst 33342,  Invitrogen H1399 \nProLong glass Antifade Mountant Invitrogen P36980 \nAnti-Adherence Rinsing solution Stemcell Technologies 07010 \nInhibitors   \nBI-D1870 Selleckhem S2843 \nCHIR 99021 Tocris 4423 \nCell lines   \nH80 cell line A gift  \nJurkat cell line ATCC TIB-152 \nMT-4 cell line NIH AIDS reagent ARP-120 \nU937 cell line ATCC CRL-1593.2 \nHEK293T cell line ATCC CRL-3216 \nSH-SY5Y cell line ATCC CRL-2266 \nMicroglial cell line (C20) A gift  \nVirus   \nHIV replication-competent virus (HIV-1 strain \n93/TH/051; R5- and X4-tropic virus isolated from a \nseropositive individual in Thailand) \nNIH AIDS reagent ARP-2165 \nSoftware   \nPrism 9 GraphPad Version 9.1.2 \nOdyssey Infrared Imaging LI-COR Version 3.0.30 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nImageJ NIH Version 1.53e \nBiorender   \nOthers   \n1.5 mL microcentrifuge tubes Caplugs Evergreen 214-3721-010 \nBiofloat 3D cell culture plates Sarstedt 83.3925.400 \nEppendorf Centrifuge 5810R Eppendorf 5811000015 \nSorvall™ Legend™ Micro 21R Microcentrifuge Thermo Scientific™ 75002447 \nVWR Analog Heat Block VWR 12621-104 \nCHROMATE-4300-N Awareness Technology 4300 \nMini Trans-Blot Transfer Cell Bio-Rad 1703930 \nEVOS M7000 Imaging System  Thermo Fisher Scientific AMF7000 \n 1248 \nReferences  1249 \n1. De Cock KM, Jaffe HW, Curran JW: Reflections on 40 Years of AIDS . Emerg Infect Dis 2021, 1250 \n27(6):1553-1560. 1251 \n2. Swinton MK, Sundermann EE, Pedersen L, Nguyen JD, Grelotti DJ, Taffe MA, Iudicello JE, Fields JA: 1252 \nAlterations in Brain Cannabinoid Receptor Levels Are Associated with HIV -Associated 1253 \nNeurocognitive Disorders in the ART Era: Implications for Therapeutic Strategies Targeting the 1254 \nEndocannabinoid System. Viruses 2021, 13(9). 1255 \n3. Irollo E, Luchetta J, Ho C, Nash B, Meucci O: Mechanisms of neuronal dysfunction in HIV -1256 \nassociated neurocognitive disorders. Cell Mol Life Sci 2021, 78(9):4283-4303. 1257 \n4. Heaton RK, Clifford DB, Franklin DR, Jr., Woods SP, Ake C, Vaida F, Ellis RJ, Letendre SL, Marcotte 1258 \nTD, Atkinson JH  et al : HIV-associated neurocognitive disorders persist in the era of potent 1259 \nantiretroviral therapy: CHARTER Study. Neurology 2010, 75(23):2087-2096. 1260 \n5. Marino J, Maubert ME, Mele AR, Spector C, Wigdahl B, Nonnemacher MR: Functional impact of 1261 \nHIV-1 Tat on cells of the CNS and its role in HAND. Cell Mol Life Sci 2020, 77(24):5079-5099. 1262 \n6. Sil S, Hu G, Liao K, Niu F, Callen S, Periyasamy P, Fox HS, Buch S: HIV-1 Tat-mediated astrocytic 1263 \namyloidosis involves the HIF-1alpha/lncRNA BACE1-AS axis. PLoS Biol 2020, 18(5):e3000660. 1264 \n7. Avila J, Lucas JJ, Perez M, Hernandez F: Role of tau protein in both physiological and 1265 \npathological conditions. Physiol Rev 2004, 84(2):361-384. 1266 \n8. Morris M, Maeda S, Vossel K, Mucke L: The many faces of tau. Neuron 2011, 70(3):410-426. 1267 \n9. Iqbal K, Liu F, Gong CX, Grundke-Iqbal I: Tau in Alzheimer disease and related tauopathies. Curr 1268 \nAlzheimer Res 2010, 7(8):656-664. 1269 \n10. Wang Y, Mandelkow E: Tau in physiology and pathology. Nat Rev Neurosci 2016, 17(1):5-21. 1270 \n11. Harris RB, Martin RJ: Metabolic response to a specific lipid -depleting factor in parabiotic rats. 1271 \nAm J Physiol 1986, 250(2 Pt 2):R276-286. 1272 \n12. Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR: Tau protein isoforms, phosphorylation 1273 \nand role in neurodegenerative disorders. Brain Res Brain Res Rev 2000, 33(1):95-130. 1274 \n13. Arriagada PV, Growdon JH, Hedley -Whyte ET, Hyman BT: Neurofibrillary tangles but not senile 1275 \nplaques parallel duration and severity of Alzheimer's disease . Neurology 1992, 42(3 Pt 1):631-1276 \n639. 1277 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\n14. Braak H, Braak E: Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 1278 \n1991, 82(4):239-259. 1279 \n15. Chen Y , Yu Y: Tau and neuroinflammation in Alzheimer's disease: interplay mechanisms and 1280 \nclinical translation. J Neuroinflammation 2023, 20(1):165. 1281 \n16. Zhang J, Zhang Y , Wang J, Xia Y, Zhang J, Chen L: Recent advances in Alzheimer's disease: 1282 \nMechanisms, clinical trials and new drug development strategies. Signal Transduct Target Ther 1283 \n2024, 9(1):211. 1284 \n17. Kaul M, Garden GA, Lipton SA: Pathways to neuronal injury and apoptosis in HIV -associated 1285 \ndementia. Nature 2001, 410(6831):988-994. 1286 \n18. Gonzalez-Scarano F, Martin-Garcia J: The neuropathogenesis of AIDS . Nat Rev Immunol 2005, 1287 \n5(1):69-81. 1288 \n19. Maggirwar SB, Tong N, Ramirez S, Gelbard HA, Dewhurst S: HIV-1 Tat-mediated activation of 1289 \nglycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity. J Neurochem 1999, 1290 \n73(2):578-586. 1291 \n20. Kaul M, Lipton SA: Mechanisms of neuronal injury and death in HIV-1 associated dementia. Curr 1292 \nHIV Res 2006, 4(3):307-318. 1293 \n21. Buch S, Yao H, Guo M, Mori T, Su TP , Wang J: Cocaine and HIV -1 interplay: molecular 1294 \nmechanisms of action and addiction. J Neuroimmune Pharmacol 2011, 6(4):503-515. 1295 \n22. Sonti S, Tyagi K, Pande A, Daniel R, Sharma AL, Tyagi M: Crossroads of Drug Abuse and HIV 1296 \nInfection: Neurotoxicity and CNS Reservoir. Vaccines (Basel) 2022, 10(2). 1297 \n23. Clare K, Park K, Pan Y , Lejuez CW, Volkow ND, Du C: Neurovascular effects of cocaine: relevance 1298 \nto addiction. Front Pharmacol 2024, 15:1357422. 1299 \n24. Tyagi M, Weber J, Bukrinsky M, Simon GL: The effects of cocaine on HIV transcription. J Neurovirol 1300 \n2016, 22(3):261-274. 1301 \n25. Sahu G, Farley K, El-Hage N, Aiamkitsumrit B, Fassnacht R, Kashanchi F, Ochem A, Simon GL, Karn 1302 \nJ, Hauser KF et al: Cocaine promotes both initiation and elongation phase of HIV-1 transcription 1303 \nby activating NF-kappaB and MSK1 and inducing selective epigenetic modifications at HIV -1 1304 \nLTR. Virology 2015, 483:185-202. 1305 \n26. Sharma AL, Shafer D, Netting D, Tyagi M: Cocaine sensitizes the CD4(+) T cells for HIV infection 1306 \nby co-stimulating NFAT and AP-1. iScience 2022, 25(12):105651. 1307 \n27. Tyagi M, Bukrinsky M, Simon GL: Mechanisms of HIV Transcriptional Regulation by Drugs of 1308 \nAbuse. Curr HIV Res 2016, 14(5):442-454. 1309 \n28. Sharma AL, Tyagi P , Khumallambam M, Tyagi M: Cocaine-Induced DNA-Dependent Protein Kinase 1310 \nRelieves RNAP II Pausing by Promoting TRIM28 Phosphorylation and RNAP II 1311 \nHyperphosphorylation to Enhance HIV Transcription. Cells 2024, 13(23). 1312 \n29. Zicari S, Sharma AL, Sahu G, Dubrovsky L, Sun L, Yue H, Jada T, Ochem A, Simon G, Bukrinsky M 1313 \net al: DNA dependent protein kinase (DNA-PK) enhances HIV transcription by promoting RNA 1314 \npolymerase II activity and recruitment of transcription machinery at HIV LTR. Oncotarget 2020, 1315 \n11(7):699-726. 1316 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\n30. Naim A, Farooqui AM, Badruddeen, Khan MI, Akhtar J, Ahmad A, Islam A: The Role of Kinases in 1317 \nNeurodegenerative Diseases: From Pathogenesis to Treatment . Eur J Neurosci 2025, 1318 \n61(11):e70156. 1319 \n31. Jiang G, Xie G, Li X, Xiong J: Cytoskeletal Proteins and Alzheimer's Disease Pathogenesis: 1320 \nFocusing on the Interplay with Tau Pathology. Biomolecules 2025, 15(6). 1321 \n32. Rankin CA, Sun Q, Gamblin TC: Pre-assembled tau filaments phosphorylated by GSK -3b form 1322 \nlarge tangle-like structures. Neurobiol Dis 2008, 31(3):368-377. 1323 \n33. Rankin CA, Sun Q, Gamblin TC: Tau phosphorylation by GSK -3beta promotes tangle -like 1324 \nfilament morphology. Mol Neurodegener 2007, 2:12. 1325 \n34. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase 1326 \nkinase-3 by insulin mediated by protein kinase B. Nature 1995, 378(6559):785-789. 1327 \n35. Sen T, Saha P, Jiang T, Sen N: Sulfhydration of AKT triggers Tau-phosphorylation by activating 1328 \nglycogen synthase kinase 3beta in Alzheimer's disease . Proc Natl Acad Sci U S A 2020, 1329 \n117(8):4418-4427. 1330 \n36. Sayas CL, Avila J: GSK-3 and Tau: A Key Duet in Alzheimer's Disease. Cells 2021, 10(4). 1331 \n37. Domise M, Didier S, Marinangeli C, Zhao H, Chandakkar P , Buee L, Viollet B, Davies P , Marambaud 1332 \nP, Vingtdeux V: AMP-activated protein kinase modulates tau phosphorylation and tau pathology 1333 \nin vivo. Sci Rep 2016, 6:26758. 1334 \n38. Yoshida H, Goedert M: Phosphorylation of microtubule-associated protein tau by AMPK-related 1335 \nkinases. J Neurochem 2012, 120(1):165-176. 1336 \n39. Yoshimura Y, Ichinose T, Yamauchi T: Phosphorylation of tau protein to sites found in Alzheimer's 1337 \ndisease brain is catalyzed by Ca2+/calmodulin -dependent protein kinase II as demonstrated 1338 \ntandem mass spectrometry. Neurosci Lett 2003, 353(3):185-188. 1339 \n40. Kirouac L, Rajic AJ, Cribbs DH, Padmanabhan J: Activation of Ras-ERK Signaling and GSK-3 by 1340 \nAmyloid Precursor Protein and Amyloid Beta Facilitates Neurodegeneration in Alzheimer's 1341 \nDisease. eNeuro 2017, 4(2). 1342 \n41. Rawat P , Sehar U, Bisht J, Selman A, Culberson J, Reddy PH: Phosphorylated Tau in Alzheimer's 1343 \nDisease and Other Tauopathies. Int J Mol Sci 2022, 23(21). 1344 \n42. Shimamura A, Ballif BA, Richards SA, Blenis J: Rsk1 mediates a MEK -MAP kinase cell survival 1345 \nsignal. Curr Biol 2000, 10(3):127-135. 1346 \n43. Doehn U, Hauge C, Frank SR, Jensen CJ, Duda K, Nielsen JV, Cohen MS, Johansen JV, Winther BR, 1347 \nLund LR et al: RSK is a principal effector of the RAS -ERK pathway for eliciting a coordinate 1348 \npromotile/invasive gene program and phenotype in epithelial cells. Mol Cell 2009, 35(4):511-522. 1349 \n44. Vassal M, Cruz AC, Rebelo S, Martins F: Neurons in a Dish: A Review of In Vitro Cell Models for 1350 \nStudying Neurogenesis. J Neurochem 2026, 170(1):e70344. 1351 \n45. Jarabo P, de Pablo C, Herranz H, Martin FA, Casas -Tinto S: Insulin signaling mediates 1352 \nneurodegeneration in glioma. Life Sci Alliance 2021, 4(3). 1353 \n46. Portela M, Venkataramani V, Fahey-Lozano N, Seco E, Losada -Perez M, Winkler F, Casas-Tinto S: 1354 \nGlioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that 1355 \nenhances glioblastoma progression and neurodegeneration. PLoS Biol 2019, 17(12):e3000545. 1356 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\n47. Slanzi A, Iannoto G, Rossi B, Zenaro E, Constantin G: In vitro Models of Neurodegenerative 1357 \nDiseases. Front Cell Dev Biol 2020, 8:328. 1358 \n48. Rai SN, Dilnashin H, Birla H, Singh SS, Zahra W, Rathore AS, Singh BK, Singh SP: The Role of 1359 \nPI3K/Akt and ERK in Neurodegenerative Disorders. Neurotox Res 2019, 35(3):775-795. 1360 \n49. Pan J, Yao Q, Wang Y, Chang S, Li C, Wu Y, Shen J, Yang R: The role of PI3K signaling pathway 1361 \nin Alzheimer's disease. Front Aging Neurosci 2024, 16:1459025. 1362 \n50. Chu E, Mychasiuk R, Hibbs ML, Semple BD: Dysregulated phosphoinositide 3-kinase signaling 1363 \nin microglia: shaping chronic neuroinflammation. J Neuroinflammation 2021, 18(1):276. 1364 \n51. Tyagi M, Pearson RJ, Karn J: Establishment of HIV latency in primary CD4+ cells is due to 1365 \nepigenetic transcriptional silencing and P-TEFb restriction. J Virol 2010, 84(13):6425-6437. 1366 \n52. Sanjana NE, Shalem O, Zhang F: Improved vectors and genome -wide libraries for CRISPR 1367 \nscreening. Nat Methods 2014, 11(8):783-784. 1368 \n53. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, 1369 \nOrchard R et al: Optimized sgRNA design to maximize activity and minimize off -target effects 1370 \nof CRISPR-Cas9. Nat Biotechnol 2016, 34(2):184-191. 1371 \n54. Richards SA, Dreisbach VC, Murphy LO, Blenis J: Characterization of regulatory events 1372 \nassociated with membrane targeting of p90 ribosomal S6 kinase 1 . Mol Cell Biol 2001, 1373 \n21(21):7470-7480. 1374 \n55. Donadoni M, Cakir S, Bellizzi A, Swingler M, Sariyer IK: Modeling HIV-1 infection and NeuroHIV in 1375 \nhiPSCs-derived cerebral organoid cultures. J Neurovirol 2024, 30(4):362-379. 1376 \n56. Prisacar M, Esser S, Hausherr M, Karacora B, Vyushkova Y , Eisenacher M, Grugel R, Marcus K, 1377 \nEggers B: Systematic Analysis of SH -SY5Y Differentiation Protocols and Neuronal Subtype 1378 \nAbundance. Cell Mol Neurobiol 2025, 45(1):104. 1379 \n57. Kaul M, Ma Q, Medders KE, Desai MK, Lipton SA: HIV-1 coreceptors CCR5 and CXCR4 both 1380 \nmediate neuronal cell death but CCR5 paradoxically can also contribute to protection . Cell 1381 \nDeath Differ 2007, 14(2):296-305. 1382 \n58. Buckley S, Byrnes S, Cochrane C, Roche M, Estes JD, Selemidis S, Angelovich TA, Churchill MJ: The 1383 \nrole of oxidative stress in HIV-associated neurocognitive disorders. Brain Behav Immun Health 1384 \n2021, 13:100235. 1385 \n59. Hanger DP, Hughes K, Woodgett JR, Brion JP, Anderton BH: Glycogen synthase kinase-3 induces 1386 \nAlzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes 1387 \nand neuronal localisation of the kinase. Neurosci Lett 1992, 147(1):58-62. 1388 \n60. Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, Hanger D, Mulot S, 1389 \nMarquardt B, Stabel S  et al : Alzheimer's disease -like phosphorylation of the microtubule -1390 \nassociated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr Biol 1391 \n1994, 4(12):1077-1086. 1392 \n61. Cho JH, Johnson GV: Glycogen synthase kinase 3beta phosphorylates tau at both primed and 1393 \nunprimed sites. Differential impact on microtubule binding. J Biol Chem 2003, 278(1):187-193. 1394 \n62. Hooper C, Killick R, Lovestone S: The GSK3 hypothesis of Alzheimer's disease . J Neurochem 1395 \n2008, 104(6):1433-1439. 1396 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\n63. Hernandez F, Lucas JJ, Avila J: GSK3 and tau: two convergence points in Alzheimer's disease. J 1397 \nAlzheimers Dis 2013, 33 Suppl 1:S141-144. 1398 \n64. Forster JI, Koglsberger S, Trefois C, Boyd O, Baumuratov AS, Buck L, Balling R, Antony PM: 1399 \nCharacterization of Differentiated SH -SY5Y as Neuronal Screening Model Reveals Increased 1400 \nOxidative Vulnerability. J Biomol Screen 2016, 21(5):496-509. 1401 \n65. Mamik MK, Hui E, Branton WG, McKenzie BA, Chisholm J, Cohen EA, Power C: HIV-1 Viral Protein 1402 \nR Activates NLRP3 Inflammasome in Microglia: implications for HIV -1 Associated 1403 \nNeuroinflammation. J Neuroimmune Pharmacol 2017, 12(2):233-248. 1404 \n66. Zink WE, Zheng J, Persidsky Y , Poluektova L, Gendelman HE: The neuropathogenesis of HIV -1 1405 \ninfection. FEMS Immunol Med Microbiol 1999, 26(3-4):233-241. 1406 \n67. Zipp F, Bittner S, Schafer DP: Cytokines as emerging regulators of central nervous system 1407 \nsynapses. Immunity 2023, 56(5):914-925. 1408 \n68. Hashimoto O, Hepler TD, Tynan A, Torres A, Li JH, Brines M, Tracey KJ, Chavan SS: Central neurons 1409 \nencode interleukin-1beta signals and mediate stress -induced inflammation. J Exp Med 2026, 1410 \n223(4). 1411 \n69. Sui Z, Sniderhan LF, Fan S, Kazmierczak K, Reisinger E, Kovacs AD, Potash MJ, Dewhurst S, Gelbard 1412 \nHA, Maggirwar SB: Human immunodeficiency virus-encoded Tat activates glycogen synthase 1413 \nkinase-3beta to antagonize nuclear factor-kappaB survival pathway in neurons. Eur J Neurosci 1414 \n2006, 23(10):2623-2634. 1415 \n70. Kehn-Hall K, Guendel I, Carpio L, Skaltsounis L, Meijer L, Al -Harthi L, Steiner JP , Nath A, Kutsch O, 1416 \nKashanchi F: Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta 1417 \ninhibitors. Virology 2011, 415(1):56-68. 1418 \n 1419 \nFigure Legends 1420 \nFigure 1: Neuronal characteristics and HIV co -receptor profile of H80 cells.  (A) Immunofluorescence 1421 \nstaining of H80 cells for NeuN using specific primary antibodies, with Hoechst (blue) counterstaining and 1422 \nunstained as controls. NeuN was detected, confirming the neuronal -like phenotype of H80 cells. (B) 1423 \nComparative immunofluorescence staining of H80 cells, microglial cells, SH-SY5Y cells (positive control), and 1424 \nHEK293T cells (negative control) demonstrated that MAP2 expression was restricted to H80 and SH -SY5Y 1425 \ncells. The inclusion of HEK293T as a negative control and SH -SY5Y as a positive control confirmed assay 1426 \nspecificity. Detection of MAP2 in H80 cells indicates that these cells exhibit features of differentiated neurons. 1427 \n(C) Immunoblotting verified MAP2 expression in H80 cells but not in HEK293T or microglial cells, further 1428 \nsupporting the neuronal identity of H80 cells. Collectively, these findings demonstrate that H80 cells express 1429 \ncanonical neuronal markers, including NeuN and MAP2, positioning them as a relevant model for investigating 1430 \nneuron-related molecular mechanisms, implicated in neurodegenerative disease and HIV/drug -induced 1431 \nneurotoxicity. (D) Flow cytometry analysis of HIV receptor expression on HEK293T, U937, and H80 cells. 1432 \nCells were blocked with 2% BSA plus Fc block and co-stained for either CD4 and CXCR4 (APC anti-human 1433 \nCD4, BioLegend cat. no. 317416; PE anti-human CD184 \\[CXCR4], BioLegend cat. no. 306505) or CD4 and 1434 \nCCR5 (PE anti -human CD4, BioLegend cat. no. 357403; APC/Cyanine7 anti -human CD195 \\[CCR5], 1435 \nBioLegend cat. no. 359110). HEK293T cells (negative control) lacked CD4, CXCR4, and CCR5 expression, 1436 \nwhereas U937 cells (positive control) expressed all three markers. H80 cells did not express CD4 or CCR5 1437 \nbut showed detectable CXCR4 expression. These findings suggest that H80 cells lack main HIV receptor 1438 \nCD4 and chemokine receptor CCR5 for HIV entry, despite expressing CXCR4. 1439 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 2: HIV exposure induces RSK1 upregulation and promotes Tau phosphorylation in H80 cells. 1440 \n(A) Immunoblot analysis of total cell lysates from Jurkat T cells infected with replication -competent HIV or 1441 \nuninfected (Ctrl), confirming HIV infection by detection of the HIV p24 capsid protein. Culture supernatants 1442 \nfrom uninfected Jurkat cells (control) and from HIV -infected cells (containing replication competent virion 1443 \nparticles) were collected and used to expose H80 cells via spinoculation (2 h at 1,000 rpm). Cells were 1444 \nsubsequently reseeded, and on the following day subjected to a second round of spinoculation with HIV 1445 \nvirions under the same conditions, followed by seeding onto 100 -mm plates. (B) Twenty-four hours post -1446 \nexposure, total RNA was extracted from H80 cells and analyzed by reverse transcription –quantitative PCR 1447 \n(RT–qPCR) for the expression of IL-1β, TNF-α, and RSK1, relative normalized to GAPDH. HIV-exposed cells 1448 \nshowed transcriptional upregulation of IL -1β, TNF-α, and RSK1 relative to No HIV controls. (C) After 48 h 1449 \npost-exposure, cells were subjected to immunofluorescence staining using antibodies against phosphorylated 1450 \nTau at Ser396 and Tau. HIV exposure enhanced increased Tau phosphorylation compared to No HIV exposed 1451 \ncontrols. Yellow arrows indicate representative phosphorylation -positive sites in the immunofluorescence 1452 \nimages. The scale bar represents 10 µm. (D–E) To validate these findings, H80 cells were cultured in four 1453 \nindependent dishes (two biological replicates per condition), and whole cell lysates were collected 48 h after 1454 \nHIV exposure. Protein lysates were prepared separately from each dish and quantified. Equal amounts of 1455 \nprotein were loaded and analyzed by immunoblotting for RSK1, RSK1/2/3, phosphorylated Tau (p-Tau-S396), 1456 \nand Tau, with actin or total protein as loading controls. Immunoblotting confirmed upregulation of RSK1, 1457 \nincreased phosphorylation of Tau at Ser396, and a moderate elevation in Tau protein levels in HIV-exposed 1458 \nH80 cells. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or 1459 \ntotal protein, with values expressed relative to the control (Ctrl/No HIV). (F) To assess whether H80 cells were 1460 \nsusceptible to HIV infection, lysates from HIV‑exposed and No HIV exposed H80 cells were examined for HIV 1461 \np24 by immunoblotting, alongside Jurkat T cells included as positive (infected) and negative (uninfected) 1462 \ncontrols. Immunoblots are representative of at least three independent biological replicates. Data are 1463 \npresented as mean ± S.D. Statistical significance was assessed using an unpaired, two tailed Student’s t test. 1464 \nSignificance is indicated as P < 0.05 (*) and P < 0.01 (**). 1465 \nFigure 3: Cocaine activates and upregulates RSK1 and promotes Tau phosphorylation in H80 cells.  1466 \n(A) Schematic representation of the protocol for the IF and Immunoblot assay detailing treatment with the 1467 \nchronic cocaine and HIV exposure (B) Immunofluorescence analysis of H80 cells chronically exposed to 1468 \ncocaine (twice daily for 2 days) revealed a marked increase in phosphorylated Tau at Ser396 (p-Tau-Ser396) 1469 \ncompared with untreated controls, while Tau levels remained unchanged, indicating that cocaine enhances 1470 \nTau phosphorylation without altering overall Tau protein. Yellow arrows indicate representative 1471 \nphosphorylation-positive sites in the immunofluorescence images. The scale bar represents 10 µm. ( C–D) 1472 \nImmunoblot analysis of whole -cell lysates from H80 cells treated with cocaine, HIV, or both revealed that 1473 \ncocaine modestly increased total RSK1 expression and its phosphorylation at Ser380, a marker of catalytic 1474 \nactivation, while minimally affecting Thr348 phosphorylation. HIV exposure produced a robust increase in 1475 \ntotal RSK1 and phosphorylation at multiple sites (Ser380, Thr348, Thr359, and Ser363), far exceeding the 1476 \neffects of cocaine alone. Both cocaine and HIV significantly elevated p -Tau-Ser396 relative to untreated 1477 \ncontrols, with HIV exerting a stronger effect. Co-exposure to cocaine and HIV resulted in a higher, though not 1478 \nstrictly additive, increase in Tau phosphorylation, suggesting convergence on overlapping signaling pathways. 1479 \nDensitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total 1480 \nprotein, with values expressed relative to the control (ctrl/No HIV). (E) Schematic representation of the 1481 \nprotocol for immunoblots with acute cocaine and HIV exposure. (F-G) H80 cells of different passages were 1482 \ncultured in eight independent dishes (two biological replicates per condition), and whole ‑cell lysates were 1483 \ncollected 15 min after exposure to cocaine, HIV, or the combined treatment. Protein lysates were prepared 1484 \nseparately from each dish and quantified. Equal amounts of protein were loaded and analyzed by 1485 \nimmunoblotting for p -RSK1 S380, p -RSK1 Thr359 and S363, and RSK1, with actin as loading controls. 1486 \nImmunoblotting confirmed activation of RSK1, increased phosphorylation of RSK1 at S380, Thr359 and S363, 1487 \nin HIV -exposed H80 cells. Densitometric analysis of immunoblots was performed by normalizing band 1488 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nintensities to RSK1, with values expressed relative to the control (Ctrl/No HIV). Immunoblots are 1489 \nrepresentative of at least three independent biological replicates. Data are presented as mean ± S.D. 1490 \nStatistical significance was assessed using one way ANOVA with Dunnett’s multiple comparisons test. 1491 \nSignificance is indicated as P < 0.05 (*) and P < 0.01 (**). Together, these findings implicate RSK1 activation 1492 \nas a key mediator of cocaine - and HIV-driven Tau phosphorylation, highlighting a shared molecular axis 1493 \nunderlying neurodegenerative processes in the context of substance use and HIV exposure. 1494 \nFigure 4: HIV and cocaine converge on GSK3β inactivation via Ser9 phosphorylation. (A-B) Immunoblot 1495 \nanalysis of Jurkat T cells infected with HIV or uninfected (control) revealed a marked increase in GSK3 β 1496 \nphosphorylation at the inhibitory Ser9 site (p-GSK3β-Ser9) upon HIV infection, while GSK3β levels remained 1497 \nunchanged, indicating functional inactivation of GSK3 β during viral infection in immune cells. (C-D) Acute 1498 \nexposure of H80 cells (15 min) to supernatants from HIV-infected Jurkat cells induced a pronounced increase 1499 \nin Ser9 phosphorylation compared to control (uninfected) supernatant, demonstrating that HIV exposure 1500 \nrapidly modulates host kinase signaling even in non -permissive cells lacking CD4. (E-F) H80 cells were 1501 \ncultured in eight independent dishes (two biological replicates) under each identical condition and whole-cell 1502 \nlysates were collected 15 min after cocaine, HIV exposure and cocaine plus HIV exposure. Protein lysates 1503 \nwere prepared separately from each dish and quantified. Equal amounts of protein were loaded and analyzed 1504 \nby immunoblotting for p-GSK3β S9, and GSK3β. Immunoblotting confirmed inactivation of GSK3β, increased 1505 \nphosphorylation of GSK3β at S9, in cocaine, HIV exposed and cocaine along with HIV -exposed H80 cells 1506 \ncompared to Ctrl/No HIV. Densitometric analysis of immunoblots was performed by normalizing band 1507 \nintensities to GSK3 β, with values expressed relative to the control (Ctrl/No HIV). Immunoblots are 1508 \nrepresentative of at least three independent biological replicates. (G-H) Immunoblot analysis of H80 cells 1509 \nexposed for 48 h to cocaine (chronic exposure), HIV virions, or both (Schematic representation in Figure 3A) 1510 \nrevealed robust Ser9 phosphorylation under all conditions, while GSK3β levels remained constant, confirming 1511 \npost-translational regulation rather than changes in protein abundance. Combined exposure to HIV and 1512 \ncocaine produced an inhibitory effect of GSK3 β similar to individual treatments. Densitometric analysis of 1513 \nimmunoblots was performed by normalizing band intensities to GSK3β, with values expressed relative to the 1514 \ncontrol (Ctrl/No HIV).  Immunoblots are representative of at least three independent biological replicates. Data 1515 \nare presented as mean ± S.D. Statistical significance was assessed using an unpaired, two tailed Student’s t 1516 \ntest (for B and D) or one way ANOVA with Dunnett’s multiple comparisons test (for F and H). Significance is 1517 \nindicated as P < 0.05 (*) and P < 0.01 (**). 1518 \nFigure 5: Cocaine activates AKT signaling in H80 cells, whereas HIV exposure does not activate AKT. 1519 \n(A) Schematic representation of the protocol for the IF and Immunoblot assay detailing treatment with the 1520 \nchronic cocaine and HIV exposure (B) Immunofluorescence analysis of H80 cells chronically exposed to 1521 \ncocaine (twice daily for 2 days) revealed a robust increase in phosphorylated AKT at Ser473 (p-AKT-Ser473) 1522 \ncompared with untreated controls, indicating strong activation of the AKT signaling pathway. Hoechst staining 1523 \nwas used for nuclear visualization. AKT levels remained unchanged, confirming that the observed effect 1524 \nreflects post -translational regulation rather than changes in protein abundance. Yellow arrows indicate 1525 \nrepresentative phosphorylation-positive sites in the immunofluorescence images. The scale bar represents 1526 \n10 µm. HIV exposure alone did not alter p -AKT-Ser473 levels under the same conditions (data in 1527 \nsupplementary). (C) Immunoblot analysis of whole -cell lysates from H80 cells exposed to cocaine, HIV, or 1528 \nboth for 48 h demonstrated that cocaine significantly increased phosphorylation of AKT at both Thr308 and 1529 \nSer473, modifications essential for full kinase activation. HIV exposure alone did not affect AKT 1530 \nphosphorylation, while combined treatment mirrored the effect of cocaine alone, indicating that cocaine exerts 1531 \na dominant influence on AKT activation. (D) Densitometric quantification confirmed a significant increase in 1532 \nAKT phosphorylation at Thr308 and Ser473 in cocaine-treated and HIV+cocaine-treated cells, whereas HIV 1533 \nalone had no measurable impact. AKT protein levels remained constant across all conditions. Densitometric 1534 \nanalysis of immunoblots was performed by normalizing band intensities to AKT, with values expressed relative 1535 \nto the control (Ctrl/No HIV). Immunoblots are representative of at least three independent biological replicates. 1536 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nData are presented as mean ± S.D. Statistical significance was assessed using one way ANOVA with 1537 \nDunnett’s multiple comparisons test. Significance is indicated as P < 0.01 (**) and ns denotes not significant. 1538 \nFigure 6: RSK1 functions upstream of GSK3 β to mediate Tau phosphorylation induced by HIV 1539 \nexposure and cocaine.  (A & B ) H80 cells were cultured in twelve independent dishes (three biological 1540 \nreplicates per condition) under each identical condition and whole -cell lysates were collected 48 h after 1541 \ncocaine, HIV exposure and cocaine plus HIV exposure. Protein lysates were prepared separately from each 1542 \ndish and quantified. Equal amounts of protein were loaded and analyzed by immunoblotting. Immunoblot 1543 \nanalysis revealed a significant increase in Tau phosphorylation at Ser396 (p -Tau-S396), a marker of 1544 \npathological Tau, and RSK-1 expression under all conditions (cocaine, HIV exposure and both) compared 1545 \nwith untreated controls. Concurrent analysis demonstrated enhanced phosphorylation of GSK3β at Ser9 (p-1546 \nGSK3β-Ser9), indicating functional inactivation of GSK3 β under all conditions, while GSK3 β and Tau levels 1547 \nremained unchanged. Densitometric analysis of immunoblots was performed by normalizing band intensities 1548 \nto β actin or total protein, with values expressed relative to the control (Ctrl/No HIV). (C & D) To delineate the 1549 \nrelative contributions of RSK1 and GSK3 β, H80 cells were pretreated for 24 h with selective inhibitors BI -1550 \nD1870 (RSK1 inhibitor) or CHIR -99021 (GSK3 β inhibitor) prior to HIV and/or cocaine exposure. 1551 \nImmunoblotting and densitometry revealed that BI -D1870 effectively suppressed RSK1 activation and 1552 \nsignificantly reduced Tau phosphorylation, whereas CHIR-99021 failed to alter Tau phosphorylation induced 1553 \nby HIV or cocaine, confirming that Tau modification is primarily mediated through RSK1-dependent signaling. 1554 \nInhibition of RSK1 also reversed GSK3 β inactivation, as evidenced by reduced Ser9 phosphorylation, 1555 \nsuggesting a hierarchical relationship in which RSK1 lies upstream of GSK3 β. Densitometric analysis of 1556 \nimmunoblots was performed by normalizing band intensities to GAPDH, with values expressed relative to the 1557 \ncontrol (Ctrl/No HIV). (E) An acute time point study was conducted for 1 h, 3 h and 6 h with cocaine and 1558 \ncocaine along with HIV, Immunoblot results show enhanced phosphorylation of GSK3 β at Ser9 (p-GSK3β-1559 \nSer9), indicating functional inactivation of GSK3 β at 6h while simultaneously seen the enhanced tau 1560 \nphosphorylation at S396. Immunoblots are representative of at least three independent biological replicates. 1561 \nData are presented as mean ± S.D. Statistical significance was assessed using one way ANOVA with 1562 \nDunnett’s multiple comparisons test. Significance is indicated as P < 0.05 (*) and P < 0.01 (**). 1563 \nFigure 7: CRISPR -Cas9-mediated RSK1 knockout reveals its essential role in Tau phosphorylation 1564 \nand AKT-GSK3β signaling. / RSK1 as a critical upstream regulator of Tau phosphorylation and AKT 1565 \nsignaling, functioning hierarchically above GSK3 β and contributing to multiple signaling pathways 1566 \nrelevant to neurodegeneration. (A–B) Immunoblot analysis confirmed successful RSK1 knockout (RSK1 1567 \nKO) in H80 cells and demonstrated reduced inhibitory phosphorylation of GSK3 β at Ser9 (p-GSK3β-Ser9), 1568 \nindicating reactivation of GSK3 β kinase activity upon loss of RSK1. RSK1 KO also led to a significant 1569 \nreduction in Tau phosphorylation at Ser396 (p-Tau-S396) compared with control cells, while total Tau levels 1570 \nremained unchanged. Despite GSK3 β reactivation, Tau phosphorylation did not recover, suggesting that 1571 \nRSK1 mediates site -specific Tau phosphorylation independently of GSK3 β. Quantitative analysis from 1572 \nmultiple independent experiments confirmed a significant decrease in p -Tau-S396 in RSK1 KO cells, 1573 \nestablishing RSK1 as essential for efficient Tau phosphorylation. (C-D) Analysis of AKT signaling revealed 1574 \nthat RSK1 knockout severely impaired AKT activation, as evidenced by a marked reduction in phosphorylation 1575 \nat Thr308 and Ser473, and also decreased total AKT protein levels. These findings indicate that RSK1 1576 \npositively regulates both AKT activation and AKT protein stability. Densitometric analysis of immunoblots was 1577 \nperformed by normalizing band intensities to β actin or total protein, with values expressed relative to the 1578 \ncontrol. Immunoblots are representative of at least three independent biological replicates. Data are 1579 \npresented as mean ± S.D. Statistical significance was assessed using one way ANOVA with Dunnett’s multiple 1580 \ncomparisons test. Significance is indicated as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). 1581 \nFigure 8: RSK1 overexpression modulates AKT and GSK3β signaling in H80 cells. / RSK1 as a central 1582 \nupstream modulator of AKT and GSK3 β signaling (A–B) Immunoblot analysis confirmed robust 1583 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\noverexpression of RSK1 following transient transfection with a CMV-driven RSK1 construct, as evidenced by 1584 \na marked increase in total RSK1 protein and elevated levels of RSK isoforms (RSK1/2/3). Overexpression 1585 \nalso resulted in a significant increase in phosphorylation of RSK1 at Ser380, an autophosphorylation site 1586 \nassociated with partial catalytic activation, while phosphorylation at Thr348 decreased relative to empty vector 1587 \ntransfected control. (C–D) RSK1 overexpression induced a robust increase in AKT phosphorylation at both 1588 \nThr308 and Ser473, two canonical regulatory sites required for full AKT activation, without altering total AKT 1589 \nprotein levels. These findings indicate that RSK1 positively regulates AKT activity predominantly through post-1590 \ntranslational mechanisms. (E–F) Overexpression of RSK1 also enhanced phosphorylation of GSK3β at Ser9, 1591 \na canonical inhibitory site that suppresses GSK3 β kinase activity, while total GSK3 β levels remained 1592 \nunchanged. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin 1593 \nor total protein, with values expressed relative to the empty vector transfected control. Immunoblots are 1594 \nrepresentative of at least three independent biological replicates. Data are presented as mean ± S.D. 1595 \nStatistical significance was assessed using an unpaired, two tailed Student’s t test. Significance is indicated 1596 \nas P < 0.01 (**) and ns denotes not significant. 1597 \nFigure 9: HIV exposure and cocaine induce RSK1 ‑dependent Tau phosphorylation in neuronal 1598 \nmonolayers, 3D spheroid, and brain organoid models.  (A) Schematic representation of the immunoblot 1599 \nexperimental workflow illustrating HIV and cocaine exposure in a three‑dimensional spheroid culture system. 1600 \n(B) SH‑SY5Y neuronal cells were exposed to HIV for 48 h, followed by cell lysis and immunoblot analysis. 1601 \nHIV exposure resulted in upregulation of RSK1, increased inhibitory phosphorylation of GSK3β at Ser9, and 1602 \nenhanced phosphorylation of Tau at Ser396. (C) These immunoblot findings were recapitulated in a 3D 1603 \nmulticellular spheroid model composed of H80 neurons, microglia, and SH ‑SY5Y cells, confirming the 1604 \nreproducibility of HIV ‑induced signaling responses in a heterogeneous cellular context. (D) Immunoblot 1605 \nanalysis in a three ‑dimensional organoid model further validated HIV ‑ and cocaine‑induced activation and 1606 \nregulation of RSK1, phosphorylation GSK3 β S9, and Tau phosphorylation S396, demonstrating the 1607 \nrobustness of this signaling axis across increasingly complex neuronal systems. 1608 \nFigure 10:  Model summarizing HIV ‑ and cocaine ‑induced Tau phosphorylation in neuronal cells.  1609 \nProposed schematic illustrating distinct yet convergent signaling mechanisms by which HIV exposure and 1610 \ncocaine promote Tau phosphorylation in H80 neuronal cells. HIV exposure induces a robust and sustained 1611 \nactivation and upregulation of RSK1, which drives Tau phosphorylation through a pathway that is independent 1612 \nof AKT signaling while concurrently promoting inhibitory phosphorylation of GSK3 β at Ser9. In contrast, 1613 \ncocaine exposure engages a partially overlapping but mechanistically distinct pathway, characterized by 1614 \nmodest RSK1 induction and strong activation of AKT, as evidenced by phosphorylation at Thr308 and Ser473. 1615 \nActivated AKT subsequently catalyzes inhibitory phosphorylation of GSK3β at Ser9, leading to its functional 1616 \ninactivation. Despite GSK3β inactivation under both conditions, Tau phosphorylation persists, indicating the 1617 \nexistence of a GSK3β‑independent mechanism regulated by RSK1. Collectively, these findings identify RSK1 1618 \nas a central signaling hub that integrates viral and substance ‑induced signaling to drive Tau dysregulation, 1619 \nhighlighting its critical role in neurodegenerative processes relevant to HIV ‑associated neurocognitive 1620 \ndisorders. 1621 \n 1622 \n 1623 \n 1624 \n 1625 \n 1626 \n 1627 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure1 1628 \n 1629 \n 1630 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 2 1631 \n 1632 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 3 1633 \n 1634 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 4 1635 \n 1636 \n 1637 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 5 1638 \n 1639 \n 1640 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 6 1641 \n 1642 \n 1643 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 7 1644 \n 1645 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 8 1646 \n 1647 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 9 1648 \n 1649 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint \n\nFigure 10 1650 \n 1651 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.14.718541doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}