KAUST Research Workshop on Innovative Technologies to Study Brain Energy Metabolism
University of Bristol, UK
AG Teschemacher, B Vaccari Cardoso, V Mosienko, BH Liu, S Kasparov
Astrocytes are thought to be the main source of extracellular L-lactate (LL) in the brain under physiological conditions. LL levels respond dynamically to neuronal network activity and metabolic triggers and may serve neurones as additional energy substrate during periods of high activity. In addition, increasing evidence indicates that LL may have a signalling role in the brain and this could have implications for regulation of sleep/wake and attention states, learning and memory, and cardiorespiratory control.
We currently lack the means for selective manipulation of LL release from astrocytes for further investigation of its actions in the brain. We therefore set out to develop an array of astrocyte-selective viral vectors that will allow us to specifically limit LL release by expressing enzymes which break down or decrease LL synthesis. Such enzymes can be 'borrowed' from bacteria, for example D-lactate dehydrogenase (DLDH; EC 126.96.36.199), lactate monooxygenase (LMO; E.C. 188.8.131.52), or L-lactate oxidase (LOx; E.C. 184.108.40.206). DLDH catalyses the NADH-dependent reversible conversion of pyruvate to DL, instead of the LL which mammalian cells produce normally. LMO is an oxidoreductase which decarboxylates LL to acetate with comparatively slow kinetics. LOx, in contrast, rapidly converts LL to pyruvate, releasing peroxide as a by-product. Each of these strategies has its merits and potential limitations which we are currently investigating.
We show, using imaging, fluorimetry, and amperometry, in HEK293 cells and primary astrocytes, that prototypes of these constructs significantly decrease intracellular LL pool sizes (see also poster by B Vaccari Cardoso). We anticipate that astrocyte-selective expression of these novel constructs via adeno- and lentiviral vectors will facilitate studies into brain energy metabolism and central metabolic signalling.