Poster AA17 - Development of an Engineered Model of Neurovascular Coupling: Validating GABAergic Interneuron Nitric Oxide Production

Introduction: An estimated sixty-five million people globally suffer from Alzheimer’s disease (AD), and Parkinson's disease. Much of the cognitive decline associated with these and other neurodegenerative diseases is linked to dysfunctional neurovascular coupling (NVC). NVC refers to the complex biochemical signaling that links neuronal activity to modulation of nearby resistance vessel diameters and thus local vascular resistance. Multiple studies have identified nitric oxide (NO) as playing a key role in multiple NVC pathways. Indeed, one of the most established signaling pathways involves glutamate binding to the N-methyl-D-aspartate receptor 1 (NMDA-R1) of GABAergic interneurons containing nitric oxide synthase (nNOS), a protein which can then convert L-Arginine to N Hydroxy-L-Arginine to form L-Citrulline and NO. Once released, NO has strong local vasodilation effects through activating soluble guanylate cyclase in smooth muscle cells. This interaction increases local blood flow as neuronal activity increases. While many microphysiological models of the neurovascular unit have been previously demonstrated, to date none have focused on this crucial process. As a first step towards a microphysiological model of NVC, we aim to replicate the pathway described above. Because of the wide heterogeneity of GABAergic interneuron subpopulations, we first need to validate the presence of nNOS and NMDA-R1 in the human iPSC-derived GABAergic interneurons we are employing.

Materials and

Methods: We investigated the feasibility of NO production from human iPSC-derived GABAergic interneurons by immunostaining for nNOS and NMDA-R1 as well as GAD67 (GABAergic interneuron-specific marker) and TUBB3 (pan-neuronal marker). After GABAergic interneurons (Elixirgen Scientific, GA-mRNA-CW50065) had reached maturity at day seven, they were stained in two groups: 1) nNOS and NMDA-R1 with primary antibodies (BD Biosciences, AB_397700 and Invitrogen, AB_2112003) added at a concentration of 1:50 and 1:100, respectively. Secondary antibodies (Invitrogen, AB_2534069, and Abcam, AB_150080) were added at a concentration of 1:100 and 1:200, respectively. 2) TUBB3 and GAD67 with primary antibodies (Invitrogen, AB_10987356, AB_1070721) added at a concentration of 1:100 and 1:50, respectively. Secondary antibodies (Invitrogen, AB_2534102, AB_2762830) added at a concentration of 1:400 and 1:200, respectively. Both groups were stained with DAPI as a nuclear stain at a 6:1000 concentration. After staining neurons were imaged using a confocal microscope (Zeiss, LSM 710).

Results, Conclusions, and Discussions: TUBB3 and GAD67 staining confirmed the GABAergic interneuron identity of the cells (Figure 1). Additionally, we found strong immunofluorescence for both nNOS and NMDA-R1 (Figure 2), suggesting that these cells have the necessary components to synthesize NO in response to glutamate transmission. Control experiments without primary or secondary antibodies presented minimal signal (data not shown).

This study demonstrated the presence of nNOS and NMDA-R1 in human iPSC-derived GABAergic interneurons. These results are a meaningful first step towards the development of a microphysiological model of the glutamate-NMDAR-nNOS-NO pathway of NVC. With this knowledge, we will now focus on demonstrating that these cells produce detectable levels of NO upon stimulation with glutamate.

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