Introduction: L-lactate (lactate) is an essential energy substrate and gliotransmitter released by astrocytes with roles in neurotransmission, memory consolidation, and stress response. Current in vivo tools for modulating brain lactate levels fail to widely target astrocytes, as these are driven by the promoter for glial fibrillary acidic protein (GFAP) expressed in only 10-20% of astrocytes with high regional heterogeneity. To better understand the function of astrocytic lactate, we need tools that specifically target a wider population of astrocytes.
A recent promising strategy to reduce lactate in astrocytes is the expression of bacterial-derived Lactate Oxidase (LacOx) enzyme, which catalyses the conversion of lactate to pyruvate. Primary astrocytes transfected with AVVs carrying GFAP-driven LacOx reduces lactate release by 50%. In vivo GFAP-driven expression of LacOx in hippocampal astrocytes also results in decreased anxiety-like behaviour in a novel environment. Here, we aim to leverage LacOx to optimise a viral tool for lactate reduction in a wider astrocyte population with high specificity.
Methods: Animal procedures were performed according to the Animal Scientific Procedures Act 1986, Home Office, United Kingdom and the Animal Welfare and Ethics Review Board, University of Bristol (PPL PP5495972). An AAV LacOx construct with reversible loxP sites and an IRES-GFP (AAV-LacOx-GFP) was stereotaxically injected in the dorsal hippocampus (dHip) and prelimbic cortex (PrlC) of Aldh1l1-Cre/ERT2 BAC transgenic mice, where Tamoxifen-inducible Cre expression was controlled by Aldh1l1. Aldh1l1-Cre/ERT2 BAC mouse line was chosen for pan-astrocytic targeting, and the inducible Cre system was selected to provide temporal control of expression. A control virus lacking LacOx (AAV-YFP) was also studied. Expression of the AAVs was studied at different titres (~10^12 to 10^9 vg/ml), and in both transgenic positive (with and without Tamoxifen induction) and negative animals. We also studied the expression of LacOx by a GFAP-driven lentiviral vector (LVV-LacOx-tdTomato), and a LacOx-lacking negative control construct (LVV-tdTomato) in the PrlC.
Results and Statistics: Transgenic positive mice injected with AAV-YFP virus showed astrocyte-specific expression in both brain areas [n(animals)=2, n(slices analysed)=5 for PrlC; n(animals)=2, n(slices analysed)=8 for dHip] whereas AAV-LacOx-GFP showed exclusively neuronal expression [n(animals)=2, n(slices analysed)=6 for PrlC; n(animals)=2, n(slices analysed)=7 for dHip]. In the CA1 dHip, animals injected with AAV-LacOx-GFP showed a significantly higher proportion of fluorescent neurons than animals injected with AAV-flox-YFP control virus [n(ROIs analysed)=19 and 20, respectively; p<0.0001, Mann-Whitney test]. Neuronal expression of LacOx-IRES-GFP was also observed in the dHip of transgenic negative animals [n(animals)=2, n(slices analysed)=3] and transgenic positive animals not treated with Tamoxifen [n(animals)=1, n(slices analysed)=3]. Reduction in the titres of AAV-LacOx-GFP did not improve astrocyte-specificity. Similarly to the AAVs, animals injected with LVV-LacOx-tdTomato showed a significantly higher proportion of fluorescent neurons than animals injected with LVV-tdTomato virus in the PrlC [n(animals)=2 each, n(ROIs analysed)= 8 and 11, respectively; p<0.0001, Mann-Whitney test].
Conclusion: LacOx-containing viral vectors have a propensity for off-target and Cre-independent expression. Further studies are required, including a time-course study of viral expression and the effect of LacOx enzyme on astrocyte health, to ascertain why LacOx evades astrocytic expression.