Adrenomedullin promotes interneuron migration in a dual human model for hypoxic interneuronopathy of prematurity

Abstract

Extremely preterm born individuals at < 28 postconceptional weeks (PCW) are at high risk for encephalopathy of prematurity and life-long neuropsychiatric conditions. Clinical studies and animal models of preterm brain injury suggest that encephalopathy of prematurity is strongly associated with exposure to hypoxia and/or inflammation in the perinatal period. Histologic examination of postmortem brain tissue from children born preterm demonstrates decreased numbers of cortical GABA-ergic interneurons in the cerebral cortex. However, the cellular and molecular mechanisms underlying the decreased numbers of GABA-ergic interneurons in the cerebral cortex of extremely preterm individuals remain unclear. Here, we developed a dual, complementary human cellular model to study hypoxia-induced interneuronopathies using human forebrain assembloids (hFA) derived from human induced pluripotent stem cells (hiPSCs) and ex vivo human prenatal cerebral cortex at mid-gestation. The hFA are generated through the integration of region-specific neural organoids containing either dorsal forebrain (excitatory) glutamatergic neurons or ventral forebrain (inhibitory) GABA-ergic interneurons. We discover a substantial reduction in migration of cortical interneurons during exposure to hypoxic stress in both hFA and ex vivo human prenatal cerebral cortex. Next, we identify that this migration defect is restored by supplementation of hypoxic cell culture media with exogenous adrenomedullin (ADM), a peptide hormone member of the calcitonin gene related peptide (CGRP) family. Lastly, we demonstrate that the rescue is mediated through increased activity of the PKA molecular pathway and increased pCREB-dependent expression of GABA receptors. Overall, these findings provide important insights into the cellular mechanisms possibly contributing to cortical interneuron depletion in preterm infants, and pinpoint novel therapeutic molecular pathways with translational potential for hypoxic encephalopathy of prematurity.

Hypoxia disrupts circadian rhythms in astrocytes and causes synapse engulfment defects

“…To investigate hypoxia-induced transcriptional changes in hCO-derived astrocytes, we first validated the induction of hypoxia in intact hCOs exposed to ∼30 mmHg (5% CO2, 37 °C) for 48 hr, by using HIF-1α protein stabilization (two-tailed paired t -test, P=0.001) (Figure 2a and b). These data align with our previous reports using hCOs at early stages of differentiation 17,20 .…”

“…Using an optical microsensor of oxygen tension (OXB50, PyroScience) attached to a fiber-optic multi-analyte meter (FireStingO 2 , PyroScience), we measured the partial pressure of oxygen (P O 2 ) in the control and hypoxic medium, and validated the expected decrease of P O 2 (Extended Figure 1e). We confirmed that these conditions are sufficient to induce stabilization of HIF-1α (two-tailed paired t -test, P=0.002) (Figure 1b and c), as expected based on our previously published data 17,20 .…”

“…Neural organoids have become an essential platform for scientific investigation of biological mechanisms underlying neurodevelopmental diseases. In previous work, we demonstrated the power of this platform to model developmental hypoxic cortical gray matter 17,20 , identify cellular phenotypes, define molecular mechanisms of insult and pinpoint pharmacological rescue interventions.…”

“…Cell culture medium placed in the control incubator was run in parallel. After 48hr, an optical microsensor of oxygen tension (OXB50, PyroScience) attached to a fiber-optic multi-analyte meter (FireStingO 2 , PyroScience) was immersed into the culture medium to detect oxygen tension, as previously described 17,20 .…”

“…Astrocytes were FACS sorted as described above and plated in 8-well Lab-Tek II®CC2 Slide (Thermo Scientific, 1054941). Within 2-3 days after plating, astrocytes were exposed to control or hypoxia for 48 hr as described previously, then fixed with 4% PFA for 15 mins followed by processing for RNAscope assay as described 20 . Briefly, slides were dehydrated using 50%, 70%, 100%, 100% ethanol for 5 min each.…”