Abstract
AIM: Huntington's disease (HD) is an inherited disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene (HTT) that ultimately leads to neurodegeneration. To study the molecular basis of this disease, induced pluripotent stem cells (iPSCs) generated from patients' fibroblasts were used to investigate axonal mitochondrial trafficking and the nature of nuclear indentations. METHODS: Pathological and control iPSCs generated from patients with a low number of repeats were differentiated in striatal neurons of the brain. Mitochondrial density was measured along the axon using tubulin beta 3 co-staining in pathological and control neurons. To investigate the connection of nuclear roundness with calcium dysregulation, several calcium inhibitors were used. Proteasome system inhibition was applied to mimic premature neuronal ageing. RESULTS: We found that the mitochondrial density was approximately 7.6 ± 0.2 in neurites in control neurons but was only 5.3 ± 0.2 in mutant neurons with 40-44 CAG repeats (p-value <0.005). Neuronal ageing induced by proteasome inhibitor MG132 significantly decreased the mitochondrial density by 15% and 25% in control and mutant neurons to 6.5 ± 0.1 (p-value < 0.005) and 4.0 ± 0.3 (p-value < 0.005), respectively. Thus, for the first time, an impairment of mitochondrial trafficking in pathological neurons with endogenous mutant huntingtin was demonstrated. We found that inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), the ryanodine-receptor (RyR) or the inositol 1,4,5-trisphosphate receptor (IP3R) by specific inhibitors did not specifically affect the nuclear roundness or survival of pathological neurons differentiated from patient iPSCs. Therefore, nuclear calcium homeostasis is not directly associated with HD pathology. CONCLUSION: Identifying HD iPSCs and differentiating from them neurons provide a unique system for modelling the disease in vitro. Impairments of mitochondrial trafficking and nuclear roundness manifest long before the disease onset, while premature neuronal ageing enhances differences in mitochondrial distribution.
Highlights
Huntington's disease (HD) is an incurable hereditary neurodegenerative disease that occurs at a frequency of 1-20 per 100,000 people and typically manifests between 35–55 years of age
Neurons that were differentiated from WT and HD induced pluripotent stem cells (iPSCs) were treated with 2-APB or ruthenium red (RR), and the mean value of the nuclear roundness was analysed
The decreased mitochondrial density observed in axons of human spiny neurons differentiated from HD patient iPSCs indicates that mitochondria trafficking was affected long before disease onset
Summary
Huntington's disease (HD) is an incurable hereditary neurodegenerative disease that occurs at a frequency of 1-20 per 100,000 people and typically manifests between 35–55 years of age. HD causes a combination of steadily progressing movement, cognitive and psychiatric disorders with a wide spectrum of signs and symptoms. HD is caused by an expansion of cytosine-adenineguanine (CAG) repeats in the huntingtin gene (HTT) that leads to a pathological elongation of polyglutamine repeats in the huntingtin protein[1]. Normal alleles of the HTT gene contain 1035 CAG repeats, and when the number of repeats exceeds 35, the disease develops. The number of CAG repeats does not exceed 50. A higher number of CAG repeats leads to the juvenile form of HD, which begins in childhood or adolescence and tends to progress more quickly than the adult-onset form[2,3,4]. HD pathogenesis is incredibly complex, and despite a large number of studies using various transgenic models, the mechanisms leading to neuronal loss in HD patients are still unknown
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