Objective: This study aimed to investigate the impact of PPP2R2B mutation in Spinocerebellar Ataxia Type 12 (SCA12) by analysing PPP2R2B splice variant expression, subcellular localization, and apoptosis in patient-derived neurons

Background: SCA12 is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansion mutation in PPP2R2B gene, which encodes regulatory B subunit of PP2A phosphatase enzyme. Alternative splicing of PPP2R2B generates isoforms with distinct subcellular localization, in cytosol and mitochondria. Dysregulation of this splicing balance may alter PP2A compartmentalization and contribute to neuronal dysfunction in SCA12

Method: Peripheral blood mononuclear cells(PBMCs) from a genetically confirmed SCA12 patient and an unaffected sibling[table1] were reprogrammed into induced pluripotent stem cells(iPSCs), differentiated into neuronal precursor cells and matured into neurons[figure1],followed by analysis of gene and protein expression by qPCR and Western blotting. PPP2R2B subcellular localization was examined by immunostaining with PPP2R2B, mitochondrial marker TOM20 and neuronal marker MAP2. Apoptosis was assessed using TUNEL assay with DAPI nuclear counterstaining}

Results: Gene expression analysis revealed significant downregulation of PPP2R2B splice variant 3(p=0.0012) and 5 (p=0.0098), while variants 10 and 11 showed a decreasing trend in SCA12 neurons. In contrast, mitochondrial variant 4 was significantly upregulated (p=0.0134). Variant6[figure4] and total PPP2R2B expression showed trend of upregulation. While PPP2R2B protein levels were comparable[figure3], subcellular analysis revealed a significant increase in Pearson’s colocalization coefficient between PPP2R2B and TOM20 (p≤ 0.0001) [figure5], indicating enhanced mitochondrial association. TUNEL assay further demonstrated a significant increase in TUNEL positive apoptotic nuclei in SCA12 neurons (p=0.0016) [figure6]

Conclusion: PPP2R2B mutation in SCA12 leads to altered splice variant expression, with reduced cytosolic variants and increased expression of the mitochondrial variant. Consistent with the genetic finding, we found enhanced mitochondrial association of PPP2R2B, though total protein level remained unchanged. Altered subcellular localization of PPP2R2B may disrupt mitochondrial homeostasis causing neuronal vulnerability in SCA12

Clinical and Demographic Details

Representation of Workflow and Characterization

Gene and Protein Expression

Mitochondrial (TOM20) colocalization of PPP2R2B

TUNEL assay for detection of apoptosis

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MDS Abstracts - https://www.mdsabstracts.org/abstract/ppp2r2b-splice-variant-dysregulation-promotes-mitochondrial-association-and-neuronal-apoptosis-in-ipsc-derived-neurons-from-spinocerebellar-ataxia-type-12-patient/