Neurodevelopmental signatures of narcotic and neuropsychiatric risk factors in 3D human-derived forebrain organoids

Abstract

It is widely accepted that narcotic use during pregnancy and specific environmental factors (e.g., maternal immune activation and chronic stress) may increase risk of neuropsychiatric illness in offspring. However, little progress has been made in defining human-specific in utero neurodevelopmental pathology due to ethical and technical challenges associated with accessing human prenatal brain tissue. Here we utilized human induced pluripotent stem cells (hiPSCs) to generate reproducible organoids that recapitulate dorsal forebrain development including early corticogenesis. We systemically exposed organoid samples to chemically defined “enviromimetic” compounds to examine the developmental effects of various narcotic and neuropsychiatric-related risk factors within tissue of human origin. In tandem experiments conducted in parallel, we modeled exposure to opiates (μ-opioid agonist endomorphin), cannabinoids (WIN 55,212-2), alcohol (ethanol), smoking (nicotine), chronic stress (human cortisol), and maternal immune activation (human Interleukin-17a; IL17a). Human-derived dorsal forebrain organoids were consequently analyzed via an array of unbiased and high-throughput analytical approaches, including state-of-the-art TMT-16plex liquid chromatography/mass-spectrometry (LC/MS) proteomics, hybrid MS metabolomics, and flow cytometry panels to determine cell-cycle dynamics and rates of cell death. This pipeline subsequently revealed both common and unique proteome, reactome, and metabolome alterations as a consequence of enviromimetic modeling of narcotic use and neuropsychiatric-related risk factors in tissue of human origin. However, of our 6 treatment groups, human-derived organoids treated with the cannabinoid agonist WIN 55,212-2 exhibited the least convergence of all groups. Single-cell analysis revealed that WIN 55,212-2 increased DNA fragmentation, an indicator of apoptosis, in human-derived dorsal forebrain organoids. We subsequently confirmed induction of DNA damage and apoptosis by WIN 55,212-2 within 3D human-derived dorsal forebrain organoids. Lastly, in a BrdU pulse-chase neocortical neurogenesis paradigm, we identified that WIN 55,212-2 was the only enviromimetic treatment to disrupt newborn neuron numbers within human-derived dorsal forebrain organoids. Cumulatively this study serves as both a resource and foundation from which human 3D biologics can be used to resolve the non-genomic effects of neuropsychiatric risk factors under controlled laboratory conditions. While synthetic cannabinoids can differ from naturally occurring compounds in their effects, our data nonetheless suggests that exposure to WIN 55,212-2 elicits neurotoxicity within human-derived developing forebrain tissue. These human-derived data therefore support the long-standing belief that maternal use of cannabinoids may require caution so to avoid any potential neurodevelopmental effects upon developing offspring in utero.

The Role of Cannabinoids in CNS Development: Focus on Proliferation and Cell Death

“…Therefore, these data show a clear CB1R-mediated apoptosis in forebrain cultures and in the neuroblastoma lineage NG 108 − 15 (Kenichi Tomiyama and Funada 2011). Dorsal forebrain organoids derived from human induced pluripotent stem cells (hiPSCs) treated with WIN-55,212-2 were selectively enriched for cells exhibiting both cleaved PARP (one of several known cellular substrates of caspases) and DNA fragmentation, which occurs when endonucleases cleave chromatin into nucleosomal units and is therefore a marker of apoptosis (Notaras et al 2021).…”

Prenatal nicotine alters development of the laterodorsal tegmentum: Possible role for attention-deficit/hyperactivity disorder and drug dependence

“…When taken together with the fact that there is currently no human data available regarding structural development within the LDT associated with PNE, experimentally examining the issue of PNE-associated changes in the LDT is warranted if we wish to understand fully the mechanisms underlying the higher risk of these maladaptive, cognitive-based behaviors in PNE individuals. While three-dimensional human-derived brain organoid models have recently been used to examine effects on neural development of environmental factors, including nicotine, they do not allow for examination of behavioral associations[ 112 ] (for review, see[ 113 ]). Accordingly, for studies examining synaptic changes that could underlie behavioral outcomes, we require animal models of PNE in which both cellular and behavioral studies can be conducted.…”

An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry

“…Common SILAC labels apply 13 C or 15 N isotopes within Arg or Lys in media to 2D cell culture systems, or in heavy labelled food of animals such as zebrafish, newts and mouse. [17][18][19][20][21].…”

“…As such, the proteome-wide study of protein dynamics in organoids presents a unique opportunity to uncovering novel mechanisms of neurodegeneration. Other studies have used quantitative mass spectrometry to profile differential protein expression in brain organoids following drug treatment [14,15]. However, there are currently no established methods to measure protein turnover in these systems.…”

“…Other studies have validated the use of heavy leucine in turnover measurements by feeding Drosophila melanogaster [5,5,5]-deuterium-3-leucine (D3-Leu) food that was incorporated into the flies over time [24]. Likewise, organoid models can be cultured with 13 C, 15 N-labeled lysine and arginine to characterize growth and protein abundance under different conditions [25]. Here, we report a robust proteomic method that measures the half-life of proteins in iPSC-derived organoid tissue using D3-Leu as a tracer.…”