The Novel GABAergic Cerebral Vascular Signalling System and its Involvement in Proliferation of Endothelial Cells and Pericytes

Abstract

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter present in the human brain. GABA is responsible for diverse functions including neuronal homeostasis, cognition, learning, memory, motor control, neuronal development, and excitatory-inhibitory balance. Although the characterisation of GABA in neuronal tissues is well established, little has been elucidated regarding GABAs contribution to non-neuronal cell function. Recent investigation of the GABAergic signalling system in human cerebral microvascular endothelial cells (hCMVECs), primary human brain pericytes (hPCs) and the human postmortem vasculature have identified the expression of various GABA receptor (GABAR) subunits. Additionally, in vitro and in vivo studies of brain endothelial cells from embryonic mice conveyed a role for GABA and the GABA type A receptor(GABAAR) in angiogenesis. These findings together suggest that GABA and its cognate receptors may drive a novel cellular process involved in cerebral vascular proliferation. In this thesis, it was determined that the GABAAR β3, ε, and γ3 subunits were abundantly expressed in both hCMVECs and hPCs. All receptor subunits were co-expressed in the same cells, but no colocalisation of the β3 subunit with either the ε or γ3 subunit was observed. Proliferation was then assessed in hCMVECs exposed to GABA over 7 and 15h. Interestingly, GABA induced a significant reduction in cell count and pH3 immunoreactivity at the 15h endpoint, strongly indicative of an inhibitory effect on cell proliferation. Further investigation to determine the mechanism behind this effect was conducted via knockdown of the GABAAR β3, ε, and γ3 subunits using siRNA. Successful knockdown of β3 subunit was identified, but hCMVEC proliferation was unaffected. The same experiment was conducted in hPCs which showed significant reductions in final cell number after siRNA treatment. Finally, considering the link between Alzheimer’s disease (AD) and cerebral vascular dysfunction, hCMVEC proliferation was assessed under amyloid beta (Aβ) 1-42 exposure. Aβ1-42 also significantly attenuated proliferation compared to control without affecting cell survival. In summary GABA can influence the proliferative potential of human cerebral vascular cells. It could be postulated that both microvascular endothelial cells and human brain pericytes express a unique GABAR conformation through which GABA can drive its inhibitory effects on proliferation. Further characterisation of the cerebral vascular GABA signalling system and the mechanism at hand may provide novel avenues for future therapies. Exploiting GABAR pharmacology to rescue angiogenic deficits in the healthy and diseased brain may provide delayed onset and improved outcomes in diseases such as AD.