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Title: Functional aspects of a mutation in the α2δ-2 calcium channel subunit of the ducky mouse, a model for absence epilepsy and cerebellar ataxia
Author: Brodbeck, Jens
ISNI:       0000 0001 3481 0982
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2002
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Positional cloning identified the genomic rearrangement disrupting the Cacna2d2 gene to underlie the ducky mutation, a model for human absence epilepsy and cerebellar ataxia. The mutation results in the loss of full-length Cacna2d2 transcript, encoding the α2δ-2 auxiliary calcium channel subunit, and the presence of two mutant transcripts. In situ hybridization found α2δ-2 highly expressed in +/+ cerebellar Purkinje cells, where in du/du mice mutant transcript 1 could be detected. This transcript consists of the first three exons of Cacna2d2, lacking most of the α2-2 and all of the δ-2 subunit and is therefore not normally functional. Phenotypically this leads in homozygotes to the severe neurological defects of absence seizures, cerebellar ataxia and demyelination, as well as a failure to breed or survive beyond 35 days. The aim of this study was to monitor functional consequences of this mutation on structure, protein expression as well as calcium homeostasis in neurones of the cerebellum. Cresyl violet-stained brain sections of P24 mice revealed normal foliation and laminar structure, although each layer was found to be reduced in size compared to age-matched wild-type animals. Lucifer yellow/neurobiotin injection, as well as Golgi impregnation, showed severe morphological alterations in cerebellar Purkinje neurones, implying a functional disruption of these cells. Two antibodies directed against the N-terminus of α2δ-2 could detect the truncated protein in Purkinje neurones of du/du mice. The entirely intracellular localization of this protein as well as its size has been confirmed in COS-7 cell expression. The expression levels of the calcium-binding protein Calbindin D-28K and the astroglial marker protein GFAP quantified by western blot analysis were unchanged. Caspase 3 expression level, a protein involved in the apoptosis pathway, appeared to be elevated in Purkinje neurones of du/du cerebellar sections. Western blot analysis of caspase 3 and 12 however did not show a changed expression level in du/du compared to +/+ littermates. Changes in free [Ca2+]i in Fura-2-loaded cultured cerebellar granule cells and acutely isolated Purkinje neurones were measured by digital imaging during depolarizing K+ stimulation. No difference in the resting [Ca2+]i was observed. Peak cytoplasmic [Ca2+]i transients were significantly reduced (24%) in Purkinje cells from du/du mice compared to +/+ mice. Whether the loss of full-length α2δ-2 and the presence of a truncated form of the protein, with subsequent reduction in calcium currents, is responsible for cerebellar degeneration and ataxia remains to be determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genetics