An iPSC-derived neurotoxicity screening platform for HIV Associated Neurocognitive Disorder (HAND) demonstrates compound toxicity on neural progenitor cells and mature neurons, astrocytes, and microglia

Abstract

Individuals living with HIV, including pregnant women, receive combination antiretroviral therapy (cART) indefinitely to maintain their health and to prevent perinatal HIV transmission. Clinical studies and preclinical research suggest that cART may affect fetal brain development, and impair adult hippocampal neurogenesis, thus contributing to HIV-Associated Neurocognitive Disorder (HAND) and related neuroafflictions (NeuroHIV). Therefore, it is important to develop predictive assays for assessing the safety of cART. Here, we used cells derived from human induced pluripotent stem cells (hiPSCs) to develop and validate platforms for anti-retroviral (ARV) toxicity testing. The platforms include neural progenitor cells (NPCs) and cultures of mature neurons alone, and neurons in culture with astrocytes and microglia. After treatment with HIV anti-retroviral drugs (ARV), we assessed outcomes such as cell viability, proliferation, neurite length and synapse number, and calcium imaging. We developed assays for NPCs and mature neuron cultures in 384-well plates. In all assays, nuclei were stained with a nuclear dye for single cell identification and viability measurements. Cell proliferation was measured using Click-iT EdU kits and fluorescent calcium dyes were used for imaging calcium transients. Fixed endpoint labeling was performed with antibodies detecting neurites, pre- and post-synaptic markers for neurite measurements and synapse count. High speed imaging and fixed sample imaging was performed on Vala Science's Kinetic Image Cytometer (KIC IC200) and single cell analysis was performed on Vala's CyteSeer Image Analysis software. We observed a decrease in NPC viability and cell proliferation after treatment with the ARVs Elvitegravir (EVG) and Dolutegravir (DTG). These two ARVs also reduced viability, neurite length, and synapse count in mature neuron monocultures. In tri-cultures, we observed toxicity with EVG, with reduced neuronal firing and diminished spike amplitudes. In conclusion, we demonstrated that Elvitegravir and Dolutegravir reduce viability of NPC's and neurons, as well as affect neuron function. The toxic effects of the treatments may have implications for HAND and NeuroHIV. We are also expanding this platform to address HIV infection/replication of microglia within the cultures, so that both safety and efficacy of future ARV compounds can be assessed.