Visualizing an convergence of three mitochondrial molecule mRNAs for DRP1/MFN2/UCP4 on soma of cerebellar Purkinje cells by RNAscope

Abstract

We know little about how mitochondrial dynamic are regulated in the Purkinje cells. To explore it, we first set up a transgenic mice in which Purkinje cells expressed tdTomato in the cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice. Secondly Double stainings verified tdTomato cells were Calbinin (CB)-positive Purkinje cells, but not colocalized either with astrocyte marker GFAP or with microglia marker Iba1. Thirdly, application of RNAscope in situ hybridization with the identification of mRNAs of mitochondrial fusion (Mfn2), calcium transporter (Mcu and Nclx) and uncoupling proteins (Ucp2 and Ucp4) were used onto Purkinje cells for specific spatial analysis. The RNAscope assay used a semi‑quantitative H scoring guideline to evaluate the staining results. The number of bins ranges from 0–4 according to the ACD scoring system. Moreover, ACD scoring system was used to calculate the overall H scores of Dendritic Weighted Formula (DWF) and Soma Weighted Formula (SWF). Our data for the first time demonstrated that Mfn2 mRNAs expression was evident in Prukinje cells with the H scores of DWF and SWF as 60 and 139, respectively. And few Ucp4 mRNAs expression was present in dendritic shafts of Prukinje cells with the H scores of DWF and SWF as 14 and 103, respectively. It should be noted that Mcu mRNAs, Nclx mRNAs, as well as Ucp2 mRNAs expression were only scattered on both soma and dendrites in Prukinje cells with the low H scores of DWF and SWF. Double RNAscope profiling of mitochondrial molecules showed The data showed Mfn1 mRNAs are present only in the soma of the Purkinje cells, instead of processes. Double RNAscope showed almost none of dots of Drp1 mRNAs was co-localized with dots of Mcu mRNAs, as well as almost none of dots of Ucp2 mRNAs was co-localized with dots of Mfn2 mRNAs. All of these results show the mitochondrial Drp1/Mfn2/UCP4 convergence on the Purkinje cells. Finally, a major focus of present research will be to develop new and more specific molecules that tune the activity of the Purkinje cells activate or inactivate and to address diseases for which such druglike molecules may open therapeutic windows for Purkinje cells-related manipulation in the clinic. The molecular identification of drug targets, mechanism of action, and structural basis of their activity will crucially enable preclinical development.