Authors :
Presenting Author: Kelvin De Leon, BA – Brown University
Li-Jin Chew, Ph.D. – Department of Molecular Biology, Cell Biology, and Biochemistry – Brown University; Haruki Higashimori, Ph.D. – Department of Molecular Biology, Cell Biology, and Biochemistry – Brown University; Kirsten Whitley, Sc.B. – Department of Molecular Biology, Cell Biology, and Biochemistry – Brown University; Sihan Chen, Sc.B. Candidate – Department of Life Sciences – University of Toronto; Alex Zeng, Sc.B. Candidate – Brown University; Judy Liu, M.D./Ph.D. – Department of Molecular Biology, Cell Biology, and Biochemistry – Brown University
Rationale:
Mutations in the SLC13A5 gene, which encodes a plasma membrane citrate transporter, result in early infantile epileptic encephalopathy (EIEE25), a neonatal neurological disorder characterized by multi-focal seizures, severe hypotonia and intellectual delay. Human genetics has identified commonly occurring SLC13A5 deletion and missense mutations that abolish citrate transport, but it is not known how distinct mutations cause disease.
Methods:
To determine whether missense mutants show features of SLC13A5 deficiency, we embarked on the characterization of an array of SLC13A5 mutant mice: i) ablation of endogenous Slc13a5 gene (null) as well as ii) G222R mutation (equivalent to human G219R mutation), and iii) T230M (mouse equivalent of human T227M). These are two of the most frequently occurring patient missense mutations.
Results:
Our epileptiform electroencephalogram (EEG) analysis indicates that the Slc13a5 null mouse shows few seizures with abnormal spike activity that is not statistically different from wild type (WT) control. G222R and T227M mice however display significantly more interictal spiking activity, with >50% mice showing status epilepticus at two and three months. Hippocampal pyramidal neurons in the null mouse have a reduction in rheobase compared to the WT which leads to a lower action potential firing threshold. G222R mutation further lowers the firing threshold. These functional changes are associated with cellular abnormalities that are most significant in G222R, followed by T230M, including reduced parvalbumin interneurons in CA1 compared with WT and null. Doublecortin-expressing immature neurons of the dentate gyrus are increased in missense mutants over WT and null, suggesting aberrant neurogenesis. MAG and CC1 oligodendrocytes in G222R and T227M white matter are also reduced compared with WT and SLC13A5 null, while astrocytes and CA1 CAMKII excitatory neurons appear unaffected across SLC13A5 mutants. Preliminary morphological analysis of cortical neurons cultured from G222R mice revealed reduced neurite length and branching, suggesting aberrant development. Recombinant human WT and SLC13A5 T227M mutant proteins are localized at the plasma membrane of cultured mouse cortical and hippocampal neurons while G219R mutant protein is primarily detected with GP96 chaperone in endoplasmic reticulum. Similar observations were obtained in mouse astrocytes and HEK293T cells. These results indicate that neuronal hyperactivity is differentially regulated by SLC13A5 mutations.
Conclusions:
While the phenotypes of
Slc13a5 missense mutants are distinguishable from the null, the intracellular distribution of the G219R mutant protein may further enhance pathogenicity. Our observations support the notion that loss of SLC13A5 underlies epileptiform activity, but specific missense mutations may increase neuronal vulnerability through additional mechanisms that exacerbate hyperexcitability.
Funding: NIH Grant 5R01NS104428-03
NIH Grant 1R01NS131865-01
NIH Grant 1F99NS129126-01A1