Effects of acute low-moderate dose ionizing radiation to human brain organoids

Abstract

Human exposure to low-to-moderate dose ionizing radiation (LMD-IR) is increasing via environmental, medical, occupational sources. Acute exposure to LMD-IR can cause subclinical damage to cells, resulting in altered gene expression and cellular function within the human brain. It has been difficult to identify diagnostic and predictive biomarkers of exposure using traditional research models due to factors including lack of 3D structure in monolayer cell cultures, limited ability of animal models to accurately predict human responses, and technical limitations of studying functional human brain tissue. To address this gap, we generated brain/cerebral organoids from human induced pluripotent stem cells to study the radiosensitivity of human brain cells, including neurons, astrocytes, and oligodendrocytes. While organoids have become popular models for studying brain physiology and pathology, there is little evidence to confirm that exposing brain organoids to LMD-IR will recapitulate previous in vitro and in vivo observations. We hypothesized that exposing brain organoids to proton radiation would (1) cause a time- and dose-dependent increase in DNA damage, (2) induce cell type-specific differences in radiosensitivity, and (3) increase expression of oxidative stress and DNA damage response genes. Organoids were exposed to 0.5 or 2 Gy of 250 MeV protons and samples were collected at 30 minute, 24 hour, and 48 hour timepoints. Using immunofluorescence and RNA sequencing, we found time- and dose-dependent increases in DNA damage in irradiated organoids; no changes in cell populations for neurons, oligodendrocytes, and astrocytes by 24 hours; decreased expression of genes related to oligodendrocyte lineage, astrocyte lineage, mitochondrial function, and cell cycle progression by 48 hours; increased expression of genes related to neuron lineage, oxidative stress, and DNA damage checkpoint regulation by 48 hours. Our findings demonstrate the possibility of using organoids to characterize cell-specific radiosensitivity and early radiation-induced gene expression changes within the human brain, providing new avenues for further study of the mechanisms underlying acute neural cell responses to IR exposure at low-to-moderate doses.

A human-specific, concerted repression of microcephaly genes contributes to radiation-induced growth defects in forebrain organoids

“…Oyefeso et al, also performed RNA-seq at 48 h post-IR. The findings of these studies, were overall in line with some of our own observations, namely a dose-and time-dependent efficiency of DNA repair, and the activation of genes related to neuronal pathways while cell cycle genes were reduced (40).…”

“…Lately, efforts have been made to understand some of the mechanisms based on the principle of Adverse Outcome Pathways, which hinted at some affected pathways (84,85), although without experimental findings from human studies the picture would never be complete. A few studies have previously used the organoid model to investigate radiation effects, however, none of these studies were performed in the context of radiation-induced microcephaly and mostly focused on DNA damage repair kinetics (40,41,54). Oyefeso et al, also performed RNA-seq at 48 h post-IR.…”

“…A few previous studies have investigated short-term effects of radiation on human brain organoids. However, these studies focused on the early DDR and were not conducted in the context of microcephaly (40,41).…”

Strategies for modeling aging and age-related diseases

“…Cerebral organoid cultured under hypoxic conditions exhibited BBB dysfunction, increased oxidative stress, and elevated secretion of inflammatory cytokines such as IL-1β, TNF-α and IL-6 41 . Moreover, Organoids irradiated with 0.5 or 2 Gy of 250 MeV protons for 30 mins, 24 h, and 48 h exhibited time and dose-dependent increase in DNA damage respectively 42 . Pharmacological inhibition of telomerase activity by 10 μM telomerase inhibitor BIBR1532 increases the percentage of short telomeres by 4.8% in PINK1 and 2.12% in PARKIN mutant-derived midbrain dopaminergic (mDA) neuron at differentiation day 65.…”

“…Furthermore, aging phenotype can be induced by exposure to ROS inducing agents, ionizing radiation and by shortening telomeres of human IPSC-derived lineages 39 – 42 . Cerebral organoid cultured under hypoxic conditions exhibited BBB dysfunction, increased oxidative stress, and elevated secretion of inflammatory cytokines such as IL-1β, TNF-α and IL-6 41 .…”