Mitochondrial and lysosomal dysfunctions might be involved in the pathogenesis of the CACNA1A-related neurodevelopmental disorders according to in vitro studies.

BACKGROUND: CACNA1A variants are associated with severe neurodevelopmental disorders (NDDs), but the underlying mechanisms remain unclear. Our goal was to investigate the molecular mechanisms through which these variants lead to intellectual disability (ID), autism spectrum disorder (ASD), epilepsy, and ataxia. METHODS: Clinical information was collected from six pediatric patients. Molecular experiments were performed on transfected human embryonic kidney and Chinese hamster ovary cells to study the effect of these variants on mitochondrial and lysosomal function. RT-qPCR, Western blot, apoptosis assay, mitochondrial and lysosomal tracker fluorescence intensity, and mitochondrial calcium concentration tests were performed. Additionally, we examined the levels of reactive oxygen species (ROS), adenosine triphosphate (ATP), and mitochondrial enzymes and copy numbers. RESULTS: We identified six variants that downregulated CACNA1A mRNA: p.D1644N, p.Y62C, p.G701R, p.R279C, p.R1664Q, and p.L1422Sfs*8. Five variants down-regulated Ca(v)2.1 protein expression, whereas, the p.R279C variant up-regulated it. All variants led to dysfunctions in the autophagy-lysosomal system: p.D1644N, p.R279C, and p.G701R variants blocked the fusion of autophagosomes and lysosomes while p.Y62C, p.R1664Q, and p.L1422Sfs*8 variants displayed increased lysosomal expression. The p.Y62C, p.G701R, p.R279C, p.R1664Q, and p.L1422Sfs*8 variants exhibited defective autophagy. The p.Y62C and p.D1644N variants disrupted mitochondrial function by downregulating mitochondrial enzyme activities and ATP levels, as well as by upregulating mitochondrial copy numbers, calcium levels, and ROS levels. Furthermore, the p.Y62C variant increased mitochondrial expression, fusion, and fission. In contrast, the p.D1644N variant decreased mitochondrial expression, fusion, fission, and mitophagy. The p.G701R, p.R279C, and p.R1664Q variants also interrupted mitochondrial function. These variants down-regulated mitochondrial enzyme activities, fusion and fission, the mitophagy process, and ATP levels while up-regulating mitochondrial copy numbers and ROS levels. The p.L1422Sfs*8 variant increased the expression, fusion and fission of mitochondrial proteins, while decreasing mitochondrial calcium levels and the mitophagy process. The p.R279C variant increased mitochondrial expression and calcium levels while enhancing apoptosis. The p.G701R variant decreased mitochondrial expression and calcium levels while enhancing apoptosis. The p.R1664Q variant increased mitochondrial calcium levels and enhanced apoptosis without changing mitochondrial expression. CONCLUSIONS: CACNA1A variants may alter mitochondrial and lysosomal function, resulting in the development of NDDs. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40659-025-00655-w.