Visualizing a 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 dynamics regulates in the Purkinje cells. To explore it, we first set up the Gad2-cre:ZsGreen-tdTomatofl/fl mice where Purkinje cells expressed tdTomato in the cerebellum. Secondly, double stainings verified tdTomato cells were Calbinin (CB)-positive Purkinje cells, but colocalized neither with astrocyte marker GFAP nor with microglia marker Iba1. Thirdly, application of RNAscope in situ hybridization with the identification of mRNAs of mitofusin 2 (Mfn2), calcium transporter (Mcu and Nclx) and uncoupling proteins (Ucp2 and Ucp4) were used onto Purkinje cells for specific spatial analysis. Our findings demonstrated that Mfn2 mRNAs expression was evident in Purkinje cells. And few expressions of Ucp4 mRNAs were presented in dendritic shafts of Purkinje cells. It should be noted that Mcu, Nclx, and Ucp2 mRNAs expression were only scattered on both soma and dendrites in Purkinje cells. The double RNAscope profiling of mitochondrial molecules showed Mfn1 mRNAs are presented only in the soma of the Purkinje cells. Double RNAscope showed none of Drp1 mRNAs were co-localized with Mcu mRNAs, as well as almost none of Ucp2 mRNAs were co-localized with Mfn2 mRNAs. All of these results showed the mitochondrial Drp1/Mfn2/Ucp4 convergence on the Purkinje cells. Finally, present research focuses on developing new and more specific molecules tuning the activity of the Purkinje cells activate or inactivate and opening therapeutic windows for Purkinje cells-related diseases. The molecular identification of potential drug targets, mechanism of action, and structural basis of their activity will crucially enable preclinical development.