Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the central nervous system is formed. Its encoded GABA transporter 1 is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GABA transporter 1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GABA transporter 1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the patho-mechanisms associated with these phenotypes remain unclear. The presence of GABA transporter 1 in both neurons and astrocytes further obscures the role of abnormal GABA transporter 1 in the heterogenous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of twenty-two SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GABA transporter 1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss of function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy vs developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacologic approaches would be beneficial.
Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.
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