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BACKGROUND: Ataxia Telangiectasia (AT) is a genetic disorder characterized by compromised DNA repair, cerebellar degeneration, and immune dysfunction. Understanding the molecular mechanisms driving AT pathology is crucial for developing targeted therapies. METHODS: In this study, we conducted a comprehensive analysis to elucidate the molecular mechanisms underlying AT pathology. Using publicly available RNA-seq datasets comparing control and AT samples, we employed in silico transcriptomics to identify potential genes and pathways. We performed differential gene expression analysis with DESeq2 to reveal dysregulated genes associated with AT. Additionally, we constructed a Protein-Protein Interaction (PPI) network to explore the interactions between proteins implicated in AT. RESULTS: The network analysis identified hub genes, including TYROBP and PCP2, crucial in immune regulation and cerebellar function, respectively. Furthermore, pathway enrichment analysis unveiled dysregulated pathways linked to AT pathology, providing insights into disease progression. CONCLUSION: Our integrated approach offers a holistic understanding of the complex molecular landscape of AT and identifies potential targets for therapeutic intervention. By combining transcriptomic analysis with network-based methods, we provide valuable insights into the underlying mechanisms of AT pathogenesis.

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Utilizing cell type-specific knockout mice has been an excellent tool for decades not only to explore the role of a gene in a specific cell, but also to unravel the underlying mechanism in diseases. To investigate the mechanistic association between dysfunction of the peroxisomal protein multifunctional protein 2 (MFP2) and retinopathy, we generated and phenotyped multiple transgenic mouse models with global or cell type-specific MFP2 deletion. These studies pointed to a potential role of MFP2 specifically in rod bipolar cells. To explore this, we aimed to create rod bipolar cell specific knockout mice of Mfp2 by crossing Mfp2L/L mice with L7Cre-2 mice (also known as PCP2Cre), generating L7-Mfp2-/- mice. L7Cre-2 mice express Cre recombinase under the control of the L7 promoter, which is believed to be exclusively expressed in rod bipolar cells and cerebellar Purkinje cells. Unexpectedly, only sporadic Cre activity was observed in the rod bipolar cells of L7-Mfp2-/- mice, despite efficient Cre recombination in cerebellar Purkinje cells. Moreover, a variable fraction of photoreceptors was targeted, which does not correspond with the supposed specificity of L7Cre-2 mice. These observations indicate that L7Cre-2 mice can be exploited to manipulate Purkinje cells in the cerebellum, whereas they cannot be used to generate rod bipolar cell specific knockout mice. For this aim, we suggest utilizing an independently generated mouse line named BAC-L7-IRES-Cre.

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Spinocerebellar ataxia (SCA) is a hereditary neurodegenerative disease. We have generated SCA17 transgenic mice bearing human TBP with 109 CAG repeats under the Purkinje cell-specific L7/pcp2 promoter. These mice recapitulate the patients' phenotypes and are suitable for the study of the SCA17 pathomechanism. Magnetic resonance imaging (MRI) and immunostainings were performed to identify the neuroimaging spectrum during disease progression. The results indicate that despite an overall normal appearance at birth, postnatal brain damage takes place rapidly in SCA17. Cerebellar atrophy, fourth-ventricle enlargement, and reduced cerebellar N-acetylaspartate levels were detected at the presymptomatic stage, when the mice were juvenile. The aberrations, which included reductions in body weight; cerebral size; striatal size; and the mean, radial, and axial diffusivities of the cerebellum, became more salient as the disease progressed to the old, late-symptomatic stage. Phosphorylated H2A histone family, member X (γH2AX) immunostaining revealed that the cerebellum underwent severe cell senescence in the old stage while the striatum appeared relatively unaffected by aging. Morphometric analysis indicated that the cerebellar atrophy occurred in all subregions with aging. The data establish that the SCA17 mouse brain appears normal at birth but becomes aberrant at the presymptomatic/juvenile stage. More widespread deficits add to the pathological spectrum at the old stage. The study provides information for the expression and expansion of L7/pcp2 promoter and implies the disease progression of SCA17 patients.

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Cell-type-specific promoters in combination with viral vectors and gene-editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood-brain barrier confirmed the Purkinje cell specificity of the L7-6 in the CNS. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.

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Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively-a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV-L7-ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.

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We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture.

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Objective: To construct the extracellular region of PCP-2(PCP-2EC) and the immunoglobin IgG Fc fusi on protein expression vector,and then express and purify the soluble PCP-2EC/Fc fusion protein for the study of its function in neuronal adhesion. Methods: PCP-2 extracellular region was amplified and cloned into an expression vector pIGplus containing human IgG Fc; PCP-2EC/Fc fusion protein was expressed by COS-7 and 293 cells transfected by the constructed plasmid and purified by protein A. The purified fusion protein was used as substrate to study its function in neuronal adhesion. Results: PCP2 extracellular region was cloned into IgG Fc expression vector successfully; PCP 2EC/Fc fusion protein was expressed and purified in mammal cells; and the purified fusion protein promoted neuronal adhesion. Conclusion:PCP 2EC/Fc fusion protein expression system is successfully constructed and the purified fusion protein can promote neuronal adhesion. These results lay a foundation for the research on the PCP-2 function in neuronal adhesion and the further functional study in the nervous system and other fields.

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Objective: To construct the extracellular region of PCP-2(PCP-2EC) and the immunoglobin IgG Fc fusi on protein expression vector,and then express and purify the soluble PCP-2EC/Fc fusion protein for the study of its function in neuronal adhesion. Methods: PCP-2 extracellular region was amplified and cloned into an expression vector pIGplus containing human IgG Fc; PCP-2EC/Fc fusion protein was expressed by COS-7 and 293 cells transfected by the constructed plasmid and purified by protein A. The purified fusion protein was used as substrate to study its function in neuronal adhesion. Results: PCP2 extracellular region was cloned into IgG Fc expression vector successfully; PCP 2EC/Fc fusion protein was expressed and purified in mammal cells; and the purified fusion protein promoted neuronal adhesion. Conclusion:PCP 2EC/Fc fusion protein expression system is successfully constructed and the purified fusion protein can promote neuronal adhesion. These results lay a foundation for the research on the PCP-2 function in neuronal adhesion and the further functional study in the nervous system and other fields.

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Objective: To construct the extracellular region of PCP-2(PCP-2EC) and the immunoglobin IgG Fc fusi on protein expression vector,and then express and purify the soluble PCP-2EC/Fc fusion protein for the study of its function in neuronal adhesion. Methods: PCP-2 extracellular region was amplified and cloned into an expression vector pIGplus containing human IgG Fc; PCP-2EC/Fc fusion protein was expressed by COS-7 and 293 cells transfected by the constructed plasmid and purified by protein A. The purified fusion protein was used as substrate to study its function in neuronal adhesion. Results: PCP2 extracellular region was cloned into IgG Fc expression vector successfully; PCP 2EC/Fc fusion protein was expressed and purified in mammal cells; and the purified fusion protein promoted neuronal adhesion. Conclusion:PCP 2EC/Fc fusion protein expression system is successfully constructed and the purified fusion protein can promote neuronal adhesion. These results lay a foundation for the research on the PCP-2 function in neuronal adhesion and the further functional study in the nervous system and other fields.

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Objective: To construct the extracellular region of PCP-2(PCP-2EC) and the immunoglobin IgG Fc fusi on protein expression vector,and then express and purify the soluble PCP-2EC/Fc fusion protein for the study of its function in neuronal adhesion.Methods: PCP-2 extracellular region was amplified and cloned into an expression vector pIGplus containing human IgG Fc; PCP-2EC/Fc fusion protein was expressed by COS-7 and 293 cells transfected by the constructed plasmid and purified by protein A.The purified fusion protein was used as substrate to study its function in neuronal adhesion.Results:PCP-2 extracellular region was cloned into IgG Fc expression vector successfully; PCP-2EC/Fc fusion protein was expressed and purified in mammal cells; and the purified fusion protein promoted neuronal adhesion.Conclusion:PCP-2EC/Fc fusion protein expression system is successfully constructed and the purified fusion protein can promote neuronal adhesion.These results lay a foundation for the research on the PCP-2 function in neuronal adhesion and the further functional study in the nervous system and other fields.