Abstract
HIV and cocaine are known to disrupt neuronal signaling and contribute to neurocognitive dysfunction, yet the underlying molecular mechanisms are not clear. In this study, we delineate the underlying molecular mechanism by which HIV and/or cocaine enhance Tau phosphorylation (p-Tau S396), a marker of Tau-mediated neuropathies. Furthermore, we elucidate how these two independent neuropathogenic factors, cocaine and HIV, exploit distinct yet convergent signaling pathways to drive this pathological event. We demonstrate that HIV robustly activates and upregulates RSK1, which functions upstream of AKT and promotes Tau phosphorylation through an AKT-independent mechanism while simultaneously inactivating GSK3β via serine-9 phosphorylation (p-GSK3β S9). However, cocaine not only activates RSK1 but also strongly stimulates AKT1, resulting in sustained GSK3β inhibition and persistent Tau phosphorylation. Notably, Tau phosphorylation persists even under conditions of GSK3β inactivation in both HIV and cocaine exposure, revealing a previously unrecognized GSK3β-independent mechanism of Tau modification. Collectively, these findings identify RSK1 as the primary mediator of Tau phosphorylation upon HIV and/or cocaine exposure, and uncover a novel RSK1-driven, GSK3β-independent pathway contributing to Tauopathy. Through a combination of immunofluorescence, immunoblotting, genetic knockout, and overexpression approaches, we establish RSK1 as a central signaling hub linking the AKT-GSK3β pathway to Tau phosphorylation. We demonstrate that RSK1 operates as a critical upstream regulator of AKT and GSK3β signaling, playing dual roles, both activating AKT and suppressing GSK3β, thereby uncovering a novel layer of pathways that regulates Tau phosphorylation. The reproducibility of these main signaling pathways across SH-SY5Y neurons, mixed cell 3D spheroids, and human brain organoids underscores the robustness and biological relevance of this mechanism. Collectively, these findings reveal mechanistic convergence of HIV and cocaine on RSK1-dependent signaling and provide critical insight into how diverse neuropathic / neuropathological factors remodel neuronal signaling to drive Tau-associated dysfunction. These findings provide novel mechanistic insight into the molecular underpinnings of neuro-HIV and substance abuse associated Tauopathy. By identifying RSK1 as a master regulator and demonstrating that Tau phosphorylation can bypass GSK3β inhibition, our study advances understanding of signaling complexity and highlights new opportunities for therapeutic intervention. Targeting RSK1 may represent a promising strategy to mitigate Tau pathology, induced due to insoluble aggregates of phosphorylated Tau, a common factor promoting cognitive decline not only in individuals with Alzheimer's disease but also in those exposed to cocaine or/and infected with HIV. SIGNIFICANCES: This study demonstrates that exposure to HIV and/or cocaine induces Tau phosphorylation at serine 396 (S396), a well-established marker of Tau pathology, and delineates how these two independent neuropathogenic factors engage distinct yet convergent signaling pathways to drive this pathogenic event. We show that HIV exposure drives robust RSK1 activation, positioning it upstream of AKT to promote Tau phosphorylation via an AKT-independent mechanism, while concurrently suppressing GSK3β activity through serine-9 phosphorylation. In contrast, cocaine, while only moderately activating RSK1, primarily enhances AKT signaling, leading to sustained GSK3β inhibition and increased Tau phosphorylation. Notably, Tau phosphorylation persists even under conditions of GSK3β inactivation in both settings, revealing a previously unrecognized, RSK1-centered, GSK3β-independent pathway of Tau modification. Overall, our findings demonstrate that Tau phosphorylation in the context of HIV infection and cocaine exposure is a complex, multi-layered regulatory process involving multiple signaling nodes. Importantly, we identify RSK1 as a central integrative hub linking viral and substance-induced signaling to downstream Tau pathology. This work advances our understanding of the molecular mechanisms underlying neuroHIV and substance abuse-associated neurodegeneration. Furthermore, it highlights RSK1 as a novel and promising therapeutic target for mitigating Tauopathy in both cocaine-using and non-using people with HIV (PWH). HIGHLIGHTED POINTS: RSK1 acts as a central regulator of Tau phosphorylation, capable of driving this process through a GSK3β-independent mechanism.HIV promotes Tau phosphorylation primarily via robust upregulation and activation of RSK1, operating largely independent of AKT1, while concurrently inducing GSK3β inactivation.Drugs of abuse, such as cocaine induces Tau phosphorylation through dual activation of AKT1 and RSK1, alongside sustained inactivation of GSK3β.Tau phosphorylation persists despite GSK3β inhibition, revealing a complex AKT1-RSK1 signaling axis and underscoring the dominant role of GSK3β-independent mechanisms in Tau pathology following HIV and cocaine exposure.HIV and cocaine engage distinct yet convergent signaling pathways that disrupt neuronal homeostasis and drive tauopathy, providing mechanistic insight into neuroHIV and substance abuse-associated neurodegeneration.RSK1 functions as a key upstream modulator of AKT and GSK3β pathways, positively regulating AKT signaling while negatively regulating GSK3β activity.RSK1 emerges as a potential therapeutic target, offering new opportunities for intervention in HIV-associated neurocognitive disorders (HAND) and drug-induced neurodegeneration.Established and characterized H80 cells as a novel neuronal cell model and demonstrated their suitability for studying neuron-specific signaling pathways, including Tau phosphorylation.The conserved and widespread nature of the signaling cascade driving Tau phosphorylation in response to HIV and/or cocaine exposure was validated across multiple model systems, including both 2D neuronal cell cultures and 3D systems such as human brain organoids and spheroids. STRENGTH OF THE STUDY: This original study provides novel mechanistic insight into how HIV and cocaine, two independent neuropathological factors, converge and diverge on intracellular signaling pathways to regulate Tau phosphorylation. By integrating immunofluorescence, immunoblotting, genetic knockout, and overexpression approaches, we identified RSK1 as a master regulator of Tau phosphorylation. Importantly, we discovered that HIV robustly upregulates and activates RSK1 to promote Tau phosphorylation through an AKT-independent route while simultaneously inactivating GSK3β. On the other hand, cocaine exerts a moderate effect on RSK1 but strongly stimulates AKT to induce GSK3β inactivation and drive Tau phosphorylation. A key strength of this work is the discovery that Tau phosphorylation persists despite GSK3β inactivation, revealing a complex, GSK3β-independent mechanism, involving RSK1 in Tau pathology. Moreover, our study, for the first time, identify RSK1 as an upstream regulator of AKT-GSK3β signaling cascade, enhancing AKT signaling while simultaneously inhibiting GSK3β activity, thereby underscoring the critical role of RSK1 in Tau phosphorylation and associated illnesses, such as HAND and Alzheimer's disease. Together, these findings not only advance our understanding of the molecular underpinnings of neuroHIV and substance abuse associated tauopathy but also highlight RSK1 as a promising therapeutic target for not only HIV and cocaine induced neurotoxicity but also other neurodegenerative diseases, such as Alzheimer's disease. Another key strength of this study is the establishment and characterization of H80 cells as a novel neuronal model, demonstrating their suitability for investigating neuron-specific signaling pathways, including Tau phosphorylation. The combination of comparative signaling analysis, genetic perturbations, and integrative mechanistic modeling makes this study both conceptually and technically novel, besides broadly relevant to the fields of neurovirology, addiction neuroscience, neurodegeneration, and cognitive impairments.