High-content analysis and Kinetic Image Cytometry identify toxic and epigenotoxic effects of HIV antiretrovirals on human iPSC-neurons and neural precursor cells

Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that antiretrovirals (ARVs), themselves, may contribute to HAND. We are examining the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG)) on viability, structure, and function of glutamatergic neurons and neural precursor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC-neurons and hiPSC-NPCs). In studies utilizing the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased viability of primary human NPCs and changed the distribution of histone modifications that regulate chromatin packing. These effects may reduce neurogenesis in the adult brain, a process whose inhibition may lead to loss of cognition. Developing methods to assay epigenotoxic effects in hiPSC-NPCs will enable further research into this process and enable testing of hypotheses regarding how dementia-associated gene variants may influence neurogenesis. Regarding hiPSC-neurons, we are utilizing automated digital microscopy, image analysis (high content analysis, HCA), and Kinetic Image Cytometry, and we have found that DTG, EVG, and TDF decrease hiPSC-neuron viability, neurites, and synapses, and that that DTG and EVG decrease the frequency and magnitude of intracellular calcium transients. We are also developing co-culture techniques with neurons, astrocytes, and microglia to advance the model for ARV toxicity to resemble the CNS more closely, and methods to simultaneously assay effects of test compounds on voltage and calcium activity of the cultures. Our research will establish human preclinical assays that can screen potential ARVs for CNS toxicity and develop safer cART regimens and HAND therapeutics.