Select antiretrovirals from multiple classes induce excitatory synapse loss in primary rodent hippocampal cultures

Abstract

The introduction of antiretroviral drugs (ARVs) has radically improved the prognoses for people living with HIV (PLWH) and is estimated to have averted almost 21 million deaths. Despite the widespread use of highly suppressive ARV treatments, approximately half of PLWH still suffer from HIV-associated neurocognitive disorders (HAND), an overall rate virtually unchanged from before their introduction. Additionally, ARVs have been identified in causing their own neuropsychiatric adverse effects, raising the question of whether they may be contributing to the persistence of HAND in virologically suppressed PLWH. As synapse loss is correlated with cognitive decline in HAND, it seems possible that ARVs themselves induce synaptic deficits that contribute both to their own neuropsychiatric effects and to the persistence of HAND. This theory is particularly bolstered by work showing that efavirenz, an ARV well known for its adverse neuropsychiatric effects, induces dendritic spine loss.I have used an automated, high-content imaging assay and cultured rat hippocampal neurons expressing PSD95-eGFP to label excitatory synapses and mCherry to fill neuronal structures to assess synaptic changes induced by a 24 hour exposure to 10 µM concentrations of 25 clinically used ARVs and one neurotoxic ARV metabolite across 6 mechanistic classes. Five ARVs elicited significant synapse loss over 24 hours: the integrase strand transfer inhibitor bictegravir, the non-nucleoside reverse transcriptase inhibitors etravirine and the 8-OH metabolite of efavirenz, the capsid inhibitor lenacapavir, and the protease inhibitors nelfinavir and saquinavir. Concentration-response assays indicated that, of the compounds assessed, only lenacapavir induced synapse loss at putative drug concentrations free in the plasma, although all four “hit” compounds evoked synapse loss at maximal total plasma concentrations, suggesting that drug interactions could raise free plasma concentrations to toxic levels. Time course studies indicated that synapse loss induced by these drugs is fully reached by 24 hours and can persist for at least three days. Therapeutically relevant antiretroviral combinations did not induce synergistic synapse loss in my study. Investigations into the mechanisms underlying synapse loss revealed that bictegravir-induced synapse loss is partially through activation of L-type voltage gated calcium channels and that bictegravir, etravirine, and lenacapavir produced synapse loss through ionotropic glutamate receptors. These results suggest that some ARVs are synaptotoxic at micromolar concentrations, and that lenacapacvir is synaptotoxic at nanomolar concentrations, indicating that if given in combination with drugs that also bind serum proteins or in disease states in which excitatory synaptic function is already potentiated, select ARVs may contribute to neuropsychiatric effects and neurocognitive deficits. The high content imaging assay used in this study provides an efficient means to evaluate new drugs and drug combinations for potential neuro- and synaptotoxicity as part of preclinical drug development.