Dataset Information

Characterization of starvation-induced autophagy in cerebellar Purkinje cells of pHluorin-mKate2-human LC3B transgenic mice.

ABSTRACT: We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To validate the usability of PK-LC3 mice, we quantified PK-LC3 puncta in Purkinje cells of mice kept in normal feeding conditions and of mice starved for 24?hours. Our results showed a significant increase in autophagosome number and in individual puncta areal size following starvation. To confirm these results, we used morphometry at the electron microscopic level to analyze the volume densities of autophagosomes and lysosomes/autolysosomes in Purkinje cells of PK-LC3 mice. The results revealed that the volume densities of autophagic structures increase significantly after starvation. Together, our data show that PK-LC3 mice are suitable for monitoring autophagy flux in Purkinje cells of the cerebellum, and potentially other areas in the central nervous system.

SUBMITTER: Oliva Trejo JA

PROVIDER: S-EPMC7295967 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Characterization of starvation-induced autophagy in cerebellar Purkinje cells of pHluorin-mKate2-human LC3B transgenic mice.

Oliva Trejo Juan Alejandro JA Tanida Isei I Suzuki Chigure C Kakuta Soichiro S Tada Norihiro N Uchiyama Yasuo Y

Scientific reports 20200615 1

We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To va ...[more]

PMID: 32541814

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Project description:Mutations in the SCN8A gene encoding the voltage-gated sodium channel α-subunit Nav1. 6 have been reported in individuals with epilepsy, intellectual disability and features of autism spectrum disorder. SCN8A is widely expressed in the central nervous system, including the cerebellum. Cerebellar dysfunction has been implicated in autism spectrum disorder. We investigated conditional Scn8a knockout mice under C57BL/6J strain background that specifically lack Scn8a expression in cerebellar Purkinje cells (Scn8a flox/flox , L7Cre + mice). Cerebellar morphology was analyzed by immunohistochemistry and MR imaging. Mice were subjected to a battery of behavioral tests including the accelerating rotarod, open field, elevated plus maze, light-dark transition box, three chambers, male-female interaction, social olfaction, and water T-maze tests. Patch clamp recordings were used to evaluate evoked action potentials in Purkinje cells. Behavioral phenotyping demonstrated that Scn8a flox/flox , L7Cre + mice have impaired social interaction, motor learning and reversal learning as well as increased repetitive behavior and anxiety-like behaviors. By 5 months of age, Scn8a flox/flox , L7Cre + mice began to exhibit cerebellar Purkinje cell loss and reduced molecular thickness. At 9 months of age, Scn8a flox/flox , L7Cre + mice exhibited decreased cerebellar size and a reduced number of cerebellar Purkinje cells more profoundly, with evidence of additional neurodegeneration in the molecular layer and deep cerebellar nuclei. Purkinje cells in Scn8a flox/flox , L7Cre + mice exhibited reduced repetitive firing. Taken together, our experiments indicated that loss of Scn8a expression in cerebellar Purkinje cells leads to cerebellar degeneration and several ASD-related behaviors. Our study demonstrated the specific contribution of loss of Scn8a in cerebellar Purkinje cells to behavioral deficits characteristic of ASD. However, it should be noted that our observed effects reported here are specific to the C57BL/6 genome type.

Dataset Information

Characterization of starvation-induced autophagy in cerebellar Purkinje cells of pHluorin-mKate2-human LC3B transgenic mice.

Publications

Characterization of starvation-induced autophagy in cerebellar Purkinje cells of pHluorin-mKate2-human LC3B transgenic mice.

Similar Datasets

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