Proteomic characterisation of HIV-Tat associated degeneration at various stages of neuronal development

Murugan, Brandon

Proteomic characterisation of HIV-Tat associated degeneration at various stages of neuronal development

Thesis / Dissertation

2026

Publisher

University of Cape Town

Department

Department of Integrative Biomedical Sciences (IBMS)

Faculty

Faculty of Health Sciences

Abstract

Combined antiretroviral therapy has significantly reduced the proportion of infected children born to mothers living with the Human Immunodeficiency Virus (HIV). However, the negative consequences of HIV exposure in utero, particularly neurodevelopmental defects remain a regular occurrence, despite maternal viral suppression. The protein HIV-Trans-Activator of Transcription (Tat) has been previously implicated in neurological dysfunction in fully developed neurons but less is understood about the effect of HIV-Tat exposure during earlier stages of embryonic neuronal development. This thesis aims to characterise the effects of HIV-Tat exposure in vitro, using hindbrain neural precursor cells derived from 6-week-old foetuses to model neurogenesis. I assessed the molecular effects of extracellular HIV-Tat at various stages of neural development using proteomic mass spectrometry, specifically, the immediate exposure of proliferating embryonic neural precursor cells, immature neurons exposed throughout their differentiation, and exposure of postmitotic neurons. Data-dependent (Top10) shotgun mass spectrometry in a Q Exactive mass spectrometer was used for all analysis. First, the immediate signalling consequences of exposure to HIV-Tat were assessed using phospho-proteomic mass spectrometry. SILAC (stable isotope labelling by amino acids in cell culture)-labelled stem cells were treated with HIV-Tat and phosphorylation measured at 15-, 30-, 45-, and 60-minutes post-exposure. Kinase substrate enrichment analysis highlighted the activation of several key cyclin-dependant kinases and iii associated proteins. Thus, exposure of neural precursors to HIV-Tat promoted cell cycle progression and increased proliferation, which has the potential to impact brain structural development. Next, the effect of continuous HIV-Tat exposure throughout the differentiation process was measured by examining protein abundance changes at differentiation day 12. Cells were treated with HIV-Tat once per day for 12 days, followed by mass spectrometry. As expected, continuous exposure to HIV-Tat negatively perturbed proteins involved in neural growth, structure and transport, and synapse functionality. These data show that exposure of embryonic neural precursors to HIV-Tat negatively affects downstream differentiation. Finally, the impact of HIV-Tat exposure on postmitotic neurons was investigated. Differentiated neurons were treated for 48 hours and functionality assessed using electrophysiology patch clamp analysis to measure voltage and current values of sodium and potassium. Treated neurons were also processed and subjected to mass spectrometry. Exposure of postmitotic neurons to HIV-Tat resulted in a significant decrease in the total number of action potentials (spikes), and decreased sodium and potassium currents. These findings suggest that HIV-Tat significantly decreases intrinsic membrane excitability by means of decreased ion channel expression. Furthermore, proteomic analysis revealed that tubulin microtubule proteins were significantly under-expressed post HIV-Tat exposure. Since these proteins are essential for transport of ion channels to the neuronal membrane, this mechanism may drive the observed decrease in intrinsic excitability. Overall, these data show that HIV-Tat perturbs vital cellular processes at various stages of embryonic neurodevelopment. These important findings highlight the detrimental effects of HIV-Tat on later brain development due to exposure in utero. Deeper investigations into the impact of Tat and other HIV-virulence proteins at earlier stages of neurodevelopment are needed, to ultimately design strategies to mitigate the impact of maternal HIV infection on cognitive development.