Date Published: March 9, 2016
Publisher: Public Library of Science
Author(s): Ainhoa Martínez-Pizarro, Lourdes R. Desviat, Magdalena Ugarte, Belén Pérez, Eva Richard, Dong-Yan Jin.
Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP3R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca2+ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.
Homocysteine is an amino acid located at a branch-point of metabolic pathways: either it is irreversibly degraded via the transsulphuration pathway to cysteine or it is remethylated back to methionine. Remethylation disorders include defects in methionine synthase (MTR, OMIM ID: 156570), methionine synthase reductase (MTRR, OMIM ID: 602568), MMADHC (OMIM ID: 611935) proteins corresponding to cblG, cblE, and cblD-variant 1 cobalamin complementation groups, respectively; and in 5,10-methylene tetrahydrofolate reductase enzyme (MTHFR, OMIM ID: 236250) . Folate derivatives maintain homocysteine at non-toxic levels, via the donation of a carbon group from methyltetrahydrofolate (synthesized by MTHFR) for homocysteine remethylation to methionine. This reaction is catalyzed by MTR that transfers a methyl group from 5-methyltetrahydrofolate to the cob(I)alamin form of the cofactor and from methylcobalamin to homocysteine to form methionine and tetrahydrofolate as products. Optimal activity of MTR requires vitamin B12 and MTRR for reductive reactivation of the cobalamin moiety of the vitamin cofactor using S-adenosylmethionine (SAM) as methyl donor to regenerate methylcobalamin. SAM is the primary methyl donor in numerous methylation reactions including DNA methylation and phospholipid biosynthesis .
The elevation of homocysteine has been correlated with complex and multifactorial diseases, including cardiovascular diseases, neurodegenerative diseases, and neural tube defects; and its toxic effects have been frequently attributed to direct or indirect perturbation of redox homeostasis . Since homocystinuria patients-derived fibroblasts with remethylation defects previously showed an increase in ROS and apoptosis levels , this work aims to deepen our understanding of ROS effects at the cellular level analysing ER stress, ER-mitochondria connectivity and autophagy.