Differential neuronal and nuclear targeting properties of calcium channel 4 splice variants and their involvement in gene regulation / by Solmaz Etemad-Ahari
VerfasserEtemad-Ahari, Solmaz
Betreuer / BetreuerinnenFlucher, Bernhard
Umfang134 S. : Ill., graph. Darst.
HochschulschriftInnsbruck, Med. Univ., Diss., 2015
Datum der AbgabeApril 2015
Schlagwörter (DE)Voltage-gated calcium channels / calcium channel 4 subunit / epilepsy / ataxia
Schlagwörter (GND)Epilepsie / Ataxie / Calciumkanal / Spannungskontrollierter Ionenkanal
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Zusammenfassung (Deutsch)

Voltage-gated calcium channel auxiliary 4 subunits, similar to other calcium channel subunits, enhance plasma membrane expression, modify the biophysical properties of CaV1 and CaV2 1 subunits, and consequently contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In this thesis, I report the discovery of a hitherto unknown 4 subunit, which is an alternatively spliced 4 subunit lacking the variable N-terminus, termed 4e. The novel 4e is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs). It modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons and is richly expressed in the distal axon. Recent literature suggested that 4 subunits are targeted to the nucleus and are involved in the epigenetic machinery. However, little is known about the roles of individual 4 splice variants in nuclear targeting and their involvement in the regulation of gene expression. In my thesis, I investigated the nuclear targeting properties of each individual full-length 4 splice variants, the two previously known 4a, 4b and the novel 4e, in primary cultured hippocampal neurons (HC) and in cerebellar granule cells (CGCs). To this end, I first established, optimized and characterized CGC cultures from wild-type and lethargic (lh) mouse. In cultured CGCs 4b and to a lesser degree 4a, displayed nuclear localization, but 4e did not show any nuclear targeting. To scrutinize the importance of 4 nuclear targeting for transcriptional regulation, whole genome expression profiling of lh-CGCs from 4-null mice individually reconstituted with either 4a, 4b, or 4e was performed. Up- or down-regulation of genes in lh-CGCs reconstituted with 4b and 4a correlated with their degree of nuclear targeting. In contrast no genes were regulated in CGCs expressing 4e, which is not targeted to the nucleus. Remarkably, gene ontology analysis revealed that 4b (and to a lesser degree 4a) directly regulates the expression of synaptic genes including the principal channel partner of 4 in cerebellar synapses, CaV2.1 and other ion channels, which have been linked to ataxia and epilepsy. Therefore, next I examined the nuclear localization of C-terminally truncated 4 subunit, which has a stop mutation at (R482X) and has been associated with juvenile myoclonic epilepsy in humans and ataxia and epileptic seizures in mice. The truncated 4 subunit exhibited identical nuclear targeting to the full-length 4, when compared in tsA201 cells, skeletal myotubes, and in hippocampal neurons. Thus the disease mechanism of the truncated 4 is far from being understood. Together my findings suggest isoform-specific functions of 4 splice variants in neurons, with 4b playing a dual role in channel modulation and gene regulation, while the newly detected 4e variant serves exclusively in calcium channel-dependent functions.

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