Novel molecular tools to selectively inhibit astrocyte-to-neurone L-lactate signalling

Abstract

Astrocyte-derived L-lactate (LL) is thought to metabolically support neurones during periods of high activity. LL also has signalling roles in the brain and its release can affect sleep/wake cycle, learning and memory, and cardiorespiratory control. We have previously shown that astrocyte-derived LL induces noradrenaline release in the locus coeruleus in vitro, and that this action was abolished by the enantiomer D-lactate. Moreover, LL application in the brainstem or pons in vivo increased sympathetic nerve activity, or caused cortical desynchronization, respectively. A new route for LL release in the brain mediated by connexin hemichannels has been described, raising the possibility that these conduits actively participate in LL effects in the brain.
During this project, I have developed a range of molecular tools to selectively limit astrocyte-derived LL release and actions in the brain, in order to better understand its roles. These vectors induce expression of bacteria-derived enzymes that either reduce the intracellular LL pool by metabolising LL (LL monooxygenase – LMO; LL oxidase – LOX), or interfere with LL -noradrenaline signalling by production of D-lactate (D-lactate dehydrogenase – DLDH). Moreover, a tool to drive astrocyte-selective knock-down of connexin-43 was created in order to investigate hemichannel-mediated LL release from astrocytes.
Functional validation of the novel molecular tools in vitro showed that the enzymes of bacterial origin are active when expressed in mammalian cells. Constitutive and forced LL release were reduced in primary dissociated cultured astrocytes as well as in organotypic brainstem slices. Moreover, expression of the DLDH-based molecular tool induced production and release of D-lactate by astrocytes. Evaluation of the impact of the new constructs on noradrenaline release suggested that expression of LOX inhibits LL -induced noradrenaline release from locus coeruleus neurons.
Therefore, these novel tools show promise for future studies into the roles of astrocytic LL in the brain in vivo.

Date of Award	28 Nov 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Anja G Teschemacher (Supervisor) & Sergey Kasparov (Supervisor)