Visualizing a Convergence of Three Mitochondrial Molecule mRNAs for Drp1/Mfn2/Ucp4 on Soma of Cerebellar Purkinje Cells by RNAscope

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.

Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and GABA-A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology

NADK2 encodes the mitochondrial isoform of NAD Kinase, which phosphorylates nicotinamide adenine dinucleotide (NAD). Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here we describe two chemically-induced mouse mutations in Nadk2, S326L and S330P, which cause a severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on brain, muscle, liver, and spinal cord at the same ages. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency, and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNAseq and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, such as translation, and those processes that are gene-specific such as glycolysis and acetylcholine binding. These findings improve our understanding of the pathophysiology of both Nadk2 and Pla2g6 mutations, as well as pathways common to neuromuscular/neurodegenerative diseases.

Low-intensity treadmill exercise protects cognitive impairment by enhancing cerebellar mitochondrial calcium retention capacity in a rat model of chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion (CCH) is caused by reduced blood flow to the brain representing gradually cognitive impairment. CCH induces mitochondrial dysfunction and neuronal cell death in the brain. Exercise is known to have a neuroprotective effect on brain damage and cognitive dysfunction. This study aimed to clarify the neuroprotective effect of low-intensity treadmill exercise (LITE) by enhancing cerebellar mitochondrial calcium retention capacity in an animal model of CCH. Wistar rats were divided into the sham group, the bilateral common carotid arteries occlusion (BCCAO) group, and the BCCAO and treadmill exercise (BCCAO+Ex) group. BCCAO+Ex group engaged the LITE on a treadmill for 30 min once a day for 8 weeks before the BCCAO surgery to investigate the protective effect of LITE on cognitive impairment. CCH induced by BCCAO resulted in mitochondrial dysfunction in the cerebellum, including impaired calcium homeostasis. CCH also decreased cerebellar Purkinje cells including of calbindin D28k and parvalbumin, resulting in cognitive impairment. The impairment of mitochondrial function, loss of cerebellar Purkinje cells, and cognitive dysfunction ameliorated by exercise. The present study showed that LITE hindered the deficit of spatial working memory and loss of Purkinje cell in the cerebellum induced by CCH. We confirmed that the protective effect of LITE on Purkinje cell by enhanced the mitochondrial calcium retention capacity. We suggest that LITE may protect against cognitive impairment, and further studies are needed to develop the intervention for patients who suffered from CCH.

A spike-sorting method based on hierarchical clustering to discriminate extracellularly recorded simple spikes and complex spikes from cerebellar Purkinje cells

Background: In the cerebellar cortex, Purkinje cells are the only output neurons and exhibit two types of discharge. Most Purkinje cell discharges are simple spikes, which are commonly appearing action potentials exhibiting a rich variety of firing patterns with a rate of up to 400 Hz. More infrequent discharges are complex spikes, which consist of a short burst of impulses accompanied by a massive increase in dendritic Ca2+ with a firing rate of around 1 Hz. The discrimination of these spikes in extracellular single-unit recordings is not always straightforward, as their waveforms vary depending on recording conditions and intrinsic fluctuations. New Method: To discriminate complex spikes from simple spikes in the extracellular single-unit data, we developed a semiautomatic spike-sorting method based on divisive hierarchical clustering. Results: Quantitative evaluation using parallel in vivo two-photon Ca2+ imaging of Purkinje cell dendrites indicated that 96.6% of the complex spikes were detected using our spike-sorting method from extracellular single-unit recordings obtained from anesthetized mice. Comparison with Existing Method(s): No reports have conducted a quantitative evaluation of spike-sorting algorithms used for the classification of extracellular spikes recorded from cerebellar Purkinje cells. Conclusions: Our method could be expected to contribute to research in information processing in the cerebellar cortex and the development of a fully automatic spike-sorting algorithm by providing ground-truth data useful for deep learning.

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

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.

Mice With Monoallelic GNAO1 Loss Exhibit Reduced Inhibitory Synaptic Input To Cerebellar Purkinje Cells

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. In this report, we used a Gnao1 mouse model G203R previously described as a gain-of-function Gnao1 mutant with movement abnormalities and enhanced seizure susceptibility. Here, we report an unexpected second mutation resulting in a loss-of-function Gαo protein and describe alterations in central synaptic transmission. Whole cell patch clamp recordings from Purkinje cells (PCs) in acute cerebellar slices from Gnao1 mutant mice showed significantly lower frequencies of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) compared to WT mice. There was no significant change in sEPSCs or mEPSCs. Whereas mIPSC frequency was reduced, mIPSC amplitudes were not affected, suggesting a presynaptic mechanism of action. A modest decrease in the number of molecular layer interneurons was insufficient to explain the magnitude of IPSC suppression. Paradoxically, Gi/o inhibitors (pertussis toxin), enhanced the mutant-suppressed mIPSC frequency and eliminated the difference between WT and Gnao1 mice. While GABAB receptor regulates mIPSCs, neither agonists nor antagonists of this receptor altered function in the mutant mouse PCs. This study is the first electrophysiological investigation of the role of Gi/o protein in cerebellar synaptic transmission using an animal model with a loss-of-function Gi/o protein.