Evaluating Neurodevelopmental Consequences of Perinatal Exposure to Antiretroviral Drugs: Current Challenges and New Approaches

Abstract

As antiretroviral therapy (ART) becomes increasingly affordable and accessible to women of childbearing age across the globe, the number of children who are exposed to Human Immunodeficiency Viruses (HIV) but remain uninfected is on the rise, almost all of whom were also exposed to ART perinatally. Although ART has successfully aided in the decline of mother-to-child-transmission of HIV, the long-term effects of in utero exposure to ART on fetal and postnatal neurodevelopment remain unclear. Evaluating the safety and efficacy of therapeutic drugs for pregnant women is a challenge due to the historic limitations on their inclusion in clinical trials and the dynamic physiological states during pregnancy that can alter the pharmacokinetics of drug metabolism and fetal drug exposure. Thus, much of our data on the potential consequences of ART drugs on the developing nervous system comes from preclinical animal models and clinical observational studies. In this review, we will discuss the current state of knowledge and existing approaches to investigate whether ART affects fetal brain development, and describe novel human stem cell-based strategies that may provide additional information to better predict the impact of specific drugs on the human central nervous system. Graphical Abstract Approaches to evaluate the impact of drugs on the developing brain. Dysregulation of the developing nervous system can lead to long-lasting changes. Integration of data from animal models, clinical observations, and cell culture studies is needed to predict the safety of therapeutic antiretroviral drugs during pregnancy. New approaches include human induced pluripotent stem cell (iPSC)-based 2D and 3D models of neuronal networks and brain regions, as well as single cell profiling in response to drug exposure.

Keywords: Neurodevelopment; antiretroviral drugs; iPSCs; organoids.

Fig. 1. Impact of antiretroviral therapy (ART) drugs on the HIV life cycle

HIV infects and replicates in a seven-stage life cycle. (1) Once HIV is in the host bloodstream, the HIV virion binds to the host’s T-cell membrane via the CD4 receptor and the CCR5 co-receptor. Entry inhibitors, comprised of post-attachment inhibitors and CCR5 antagonists, block this stage of the life cycle by preventing HIV virion binding to the CD4 receptor and CCR5 co-receptor, respectively. (2) After binding, the HIV virion fuses with the T-cell membrane to release its RNA into the host cell. Fusion inhibitors block this stage. (3) In the cell cytoplasm, the HIV RNA is reverse transcribed into DNA. Nucleoside reverse transcriptase inhibitors (NRTI) or non-nucleoside reverse transcriptase inhibitors (NNRTI) block this stage by competitively and non-competitively binding reverse transcriptase, respectively. (4) In the nucleus, HIV DNA can integrate into the host’s DNA using a viral enzyme, HIV integrase. HIV integrase and host DNA integration can be blocked with integrase inhibitors. (5) Following integration, viral DNA undergoes replication (6) and the generation of additional HIV RNA that encodes for HIV proteins, which are assembled by translation in the cytoplasm. (7) After protein assembly, the new HIV virions form and bud from the host cell. The HIV protease enzyme cleaves immature viral proteins, allowing for maturation and infection of other cells, which can be blocked by protease inhibitors (PI).

Fig. 2. Overview of human brain developmental processes

Human brain development undergoes many critical stages of maturation from conception through late adolescence and early adulthood. Neurodevelopment begins approximately two to three weeks after conception with the formation of the neural tube, or neurulation, in which the lateral ends of the neural plate fold and fuse. Following neurulation is the process of neurogenesis in which morphogen gradients determine the identity of neural progenitors that give rise to neurons. This stage continues through gestation until birth, with some evidence of neurogenesis occurring after birth. Between four to 16 gestational weeks, microglia migrate into the developing brain to establish the pool of resident immune cells. Following neurogenesis, synaptogenesis begins at approximately 12 weeks in utero, and pruning of excess synapses and dendritic processes continue after birth. At 18 weeks, apoptosis eliminates overproduction of cells to refine cell populations and to ensure proper development and synaptic connectivity in the mature brain. At 22 weeks, gliogenesis results in the production of region- and subtype-specific glia, which continues through adulthood. At approximately 32 gestational weeks, glial cells aid in myelination of the neurons, and this process continues after birth into adulthood.

Fig. 3. In vitro models to study the effects of antiretroviral therapy on neurodevelopment

Induced pluripotent stem cells (iPSCs) offer new opportunities to study the effects of environmental factors and drugs on neurodevelopment. Targeted differentiated protocols can be used to generate neural progenitor cells (NPCs) and further patterning can lead to specific populations of neurons or glial cells. 2D cultures allow for highly enriched populations to study the effects of drugs in specific cell types. 3D culture approaches allow for the generation of organoids, which can recapitulate the heterogeneity of multiple cell populations and self-organization that is characteristic of fetal brain development. Forebrain cortical organoids exhibit proliferative ventricular zones and laminar stratification of neuronal layers reminiscent of the cerebral cortex. Microglia are generated from a distinct lineage and can be co-cultured with either 2D monolayer populations or 3D organoids to model virus-host interactions.