Fig 2: RSK1 overexpression modulates AKT and GSK3β signaling in H80 cells. / RSK1 as a central upstream modulator of AKT and GSK3β signaling(A–B) Immunoblot analysis confirmed robust overexpression of RSK1 following transient transfection with a CMV-driven RSK1 construct, as evidenced by a marked increase in total RSK1 protein and elevated levels of RSK isoforms (RSK1/2/3). Overexpression also resulted in a significant increase in phosphorylation of RSK1 at Ser380, an autophosphorylation site associated with partial catalytic activation, while phosphorylation at Thr348 decreased relative to empty vector transfected control. (C–D) RSK1 overexpression induced a robust increase in AKT phosphorylation at both Thr308 and Ser473, two canonical regulatory sites required for full AKT activation, without altering total AKT protein levels. These findings indicate that RSK1 positively regulates AKT activity predominantly through post-translational mechanisms. (E–F) Overexpression of RSK1 also enhanced phosphorylation of GSK3β at Ser9, a canonical inhibitory site that suppresses GSK3β kinase activity, while total GSK3β levels remained unchanged. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total protein, with values expressed relative to the empty vector transfected control. Immunoblots are representative of at least three independent biological replicates. Data are presented as mean ± S.D. Statistical significance was assessed using an unpaired, two tailed Student’s t test. Significance is indicated as P < 0.01 (**) and ns denotes not significant.

Fig 3: Cocaine activates and upregulates RSK1 and promotes Tau phosphorylation in H80 cells.(A) Schematic representation of the protocol for the IF and Immunoblot assay detailing treatment with the chronic cocaine and HIV exposure (B) Immunofluorescence analysis of H80 cells chronically exposed to cocaine (twice daily for 2 days) revealed a marked increase in phosphorylated Tau at Ser396 (p-Tau-Ser396) compared with untreated controls, while Tau levels remained unchanged, indicating that cocaine enhances Tau phosphorylation without altering overall Tau protein. Yellow arrows indicate representative phosphorylation-positive sites in the immunofluorescence images. The scale bar represents 10 μm. (C–D) Immunoblot analysis of whole-cell lysates from H80 cells treated with cocaine, HIV, or both revealed that cocaine modestly increased total RSK1 expression and its phosphorylation at Ser380, a marker of catalytic activation, while minimally affecting Thr348 phosphorylation. HIV exposure produced a robust increase in total RSK1 and phosphorylation at multiple sites (Ser380, Thr348, Thr359, and Ser363), far exceeding the effects of cocaine alone. Both cocaine and HIV significantly elevated p-Tau-Ser396 relative to untreated controls, with HIV exerting a stronger effect. Co-exposure to cocaine and HIV resulted in a higher, though not strictly additive, increase in Tau phosphorylation, suggesting convergence on overlapping signaling pathways. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total protein, with values expressed relative to the control (ctrl/No HIV). (E) Schematic representation of the protocol for immunoblots with acute cocaine and HIV exposure. (F-G) H80 cells of different passages were cultured in eight independent dishes (two biological replicates per condition), and whole-cell lysates were collected 15 min after exposure to cocaine, HIV, or the combined treatment. Protein lysates were prepared separately from each dish and quantified. Equal amounts of protein were loaded and analyzed by immunoblotting for p-RSK1 S380, p-RSK1 Thr359 and S363, and RSK1, with actin as loading controls. Immunoblotting confirmed activation of RSK1, increased phosphorylation of RSK1 at S380, Thr359 and S363, in HIV-exposed H80 cells. Densitometric analysis of immunoblots was performed by normalizing band intensities to RSK1, with values expressed relative to the control (Ctrl/No HIV). Immunoblots are representative of at least three independent biological replicates. Data are presented as mean ± S.D. Statistical significance was assessed using one way ANOVA with Dunnett’s multiple comparisons test. Significance is indicated as P < 0.05 (*) and P < 0.01 (**). Together, these findings implicate RSK1 activation as a key mediator of cocaine- and HIV-driven Tau phosphorylation, highlighting a shared molecular axis underlying neurodegenerative processes in the context of substance use and HIV exposure.

Fig 4: CRISPR-Cas9-mediated RSK1 knockout reveals its essential role in Tau phosphorylation and AKT-GSK3β signaling. / RSK1 as a critical upstream regulator of Tau phosphorylation and AKT signaling, functioning hierarchically above GSK3β and contributing to multiple signaling pathways relevant to neurodegeneration.(A–B) Immunoblot analysis confirmed successful RSK1 knockout (RSK1 KO) in H80 cells and demonstrated reduced inhibitory phosphorylation of GSK3β at Ser9 (p-GSK3β-Ser9), indicating reactivation of GSK3β kinase activity upon loss of RSK1. RSK1 KO also led to a significant reduction in Tau phosphorylation at Ser396 (p-Tau-S396) compared with control cells, while total Tau levels remained unchanged. Despite GSK3β reactivation, Tau phosphorylation did not recover, suggesting that RSK1 mediates site-specific Tau phosphorylation independently of GSK3β. Quantitative analysis from multiple independent experiments confirmed a significant decrease in p-Tau-S396 in RSK1 KO cells, establishing RSK1 as essential for efficient Tau phosphorylation. (C-D) Analysis of AKT signaling revealed that RSK1 knockout severely impaired AKT activation, as evidenced by a marked reduction in phosphorylation at Thr308 and Ser473, and also decreased total AKT protein levels. These findings indicate that RSK1 positively regulates both AKT activation and AKT protein stability. Densitometric analysis of immunoblots was performed by normalizing band intensities to β actin or total protein, with values expressed relative to the control. Immunoblots are representative of at least three independent biological replicates. Data are presented as mean ± S.D. Statistical significance was assessed using one way ANOVA with Dunnett’s multiple comparisons test. Significance is indicated as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

Fig 5: Model summarizing HIV- and cocaine-induced Tau phosphorylation in neuronal cells.Proposed schematic illustrating distinct yet convergent signaling mechanisms by which HIV exposure and cocaine promote Tau phosphorylation in H80 neuronal cells. HIV exposure induces a robust and sustained activation and upregulation of RSK1, which drives Tau phosphorylation through a pathway that is independent of AKT signaling while concurrently promoting inhibitory phosphorylation of GSK3β at Ser9. In contrast, cocaine exposure engages a partially overlapping but mechanistically distinct pathway, characterized by modest RSK1 induction and strong activation of AKT, as evidenced by phosphorylation at Thr308 and Ser473. Activated AKT subsequently catalyzes inhibitory phosphorylation of GSK3β at Ser9, leading to its functional inactivation. Despite GSK3β inactivation under both conditions, Tau phosphorylation persists, indicating the existence of a GSK3β-independent mechanism regulated by RSK1. Collectively, these findings identify RSK1 as a central signaling hub that integrates viral and substance-induced signaling to drive Tau dysregulation, highlighting its critical role in neurodegenerative processes relevant to HIV-associated neurocognitive disorders.