Misfolding of cystathionine beta-synthase impairs mitochondrial homeostasis and cellular bioenergetics.

Homocystinuria (HCU) is an inborn error of metabolism and a conformational disorder chiefly caused by missense mutations in the cystathionine beta-synthase (CBS) gene. These mutations often cause CBS destabilization, misfolding and dysfunction resulting in CBS deficiency and pathological accumulation of homocysteine. Morphological changes in mitochondria were described in HCU patients and mouse models; however, their functional significance has remained unknown. Here, we characterized the impact of CBS deficiency due to expression of the most common HCU-causing variant CBS I278T on mitochondrial function using three cellular models of HCU: mouse hepatocytes, human fibroblasts and newly developed CRISPR/Cas9-modified HEK293 cells. We found that the expression of the CBS I278T variant resulted in the unfolded protein response, oxidative stress and impaired cellular energy metabolism in all three cellular models of HCU. Mitochondrial respiration and ATP production were substantially impaired. Bioenergetic deficit correlated morphologically with mitochondrial swelling and loss of cristae and functionally with decreased membrane potential and cytosolic mitochondrial DNA release. Impaired clearance of damaged, non-functional mitochondria was caused by compromised mitophagy activation and dysfunctional lysosomes. Methionine restriction substantially reduced plasma total homocysteine and rescued mitochondrial function of hepatocytes isolated from treated Tg-I278T HCU mice. Importantly, CBS-knockout HEK293 cells showed normal proteostasis and mitochondrial function indicating that CBS I278T misfolding is the main cause and trigger of the described pathological phenotype. These findings provide the first mechanistic insight into the impaired cellular bioenergetics in HCU.