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Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

ABSTRACT: Dominant mutations in CACNA1A, encoding the ?-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function.

SUBMITTER: Luo X

PROVIDER: S-EPMC5557584 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

Luo Xi X Rosenfeld Jill A JA Yamamoto Shinya S Harel Tamar T Zuo Zhongyuan Z Hall Melissa M Wierenga Klaas J KJ Pastore Matthew T MT Bartholomew Dennis D Delgado Mauricio R MR Rotenberg Joshua J Lewis Richard Alan RA Emrick Lisa L Bacino Carlos A CA Eldomery Mohammad K MK Coban Akdemir Zeynep Z Xia Fan F Yang Yaping Y Lalani Seema R SR Lotze Timothy T Lupski James R JR Lee Brendan B Bellen Hugo J HJ Wangler Michael F MF

PLoS genetics 20170724 7

Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel v ...[more]

PMID: 28742085

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Project description:T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

Dataset Information

Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

Publications

Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

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