CCN6 REGULATES MITOCHONDRIAL RESPIRATORY COMPLEX ASSEMBLY AND ACTIVITY

Abstract

BackgroundCCN6 is a multidomain protein of mesenchymal origin. Mutations in CCN6 lead to PPRD (Progressive Pseudo Rheumatoid Dysplasia), which is associated with cartilage loss, muscle wasting and restricted skeletal development. Mechanism of PPRD pathogenesis and its connection with CCN6 is unclear. Previously we documented that CCN6 localizes to mitochondria and regulates ATP production. Here we have investigated the molecular mechanism of this regulation in context of PPRD.Objective To check whether CCN6 regulates mitochondrial respiratory complex assembly and activity. To check if engineering PPRD causing CCN6 mutations alters the function of the mitochondrial respiratory complex.MethodsCCN6 expression was depleted by siRNA transfection in C28/I2 chondrocyte cell line. CCN6 mutants were generated by CRISPR‐Cas9 technology. CCN6 association with respiratory complex was assessed by 2D BN/SDS PAGE & size‐exclusion chromatography. Mitochondrial respiratory complex activity/assembly was assessed spectrophotometrically and by BN‐PAGE in‐gel assay. Transmission electron microscopy (TEM) was used to study mitochondrial morphology.ResultsSize‐exclusion chromatography and 2D BN/SDS PAGE of mitochondria reveals that CCN6 is associated with Complex I/Super complexes of mitochondrial electron transport chain. Partial depletion of CCN6 by siRNA shows close association of RER with mitochondria and alteration of its mitochondrial distribution relative to cytosol, thereby resulting in increased Complex I activity and assembly. CCN6 mutation (truncation in exon 5, similar to PPRD linked mutation), however, results in disrupted complex I activity and assembly. CCN6 mutation also correlates with distorted mitochondria and mitophagy.ConclusionCCN6 is required for the regulation of mitochondrial respiratory complex activity/assembly in chondrocytes, which involves controlled juxtaposition of RER‐mitochondria. Mutations in CCN6 disrupt respiratory complex activity/assembly and cause mitophagy.Support or Funding InformationThis work is supported by a grant from the Department of Science and Technology, Grant No‐SB/SO/BB‐016/2014 and Department of Biotechnology, Grant No‐BT/PR24244/MED/12/764/2017, Ministry of Science and Technology, Government of India. D.K.P. is supported by the Council of Scientific and Industrial Research (CSIR) fellowship, A.S is supported by DBT‐RA programme and A.G is supported by University Grant Commission (UGC), Government of India.CCN6 migrates as a high molecular weight complex with mitochondrial respiratory complex proteins.CCN6 remains as high molecular weight complex in mitochondria along with subunits of mitochondrial electron transport chain including complex I subunit NDUFB8 as shown by 2D BN‐PAGE/SDS‐PAGE (A) and size exclusion chromatography (B) of mitochondrial lysate from C28/I2 cells (chondrocyte line).Figure 1CCN6 regulates mitochondrial respiratory complex assembly and activity.(A) Increased Complex I assembly and activity was documented by BN‐PAGE western blot with NDUFB8 (Complex I subunit) antibody and in‐gel Complex I activity assay in CCN6 siRNA transfected (CCN6 partially depleted) C‐28/I2 cells (CCN6 si) as compared to control cells (Cont.si). (B) Transmission Electron Microscopy (TEM) micrographs showing decreased RER‐Mitochondria distance in CCN6 siRNA transfected cells. (C) BN‐PAGE western blot with NDUFB8 antibody and in‐gel Complex I activity of CRISPR Cas9 mediated PPRD causing CCN6 mutants shows reduced Complex I/Super complex assembly and activity. (D) TEM micrographs shows presence of mitophagy in CCN6 mutant cells.. RER: Rough endoplasmic reticulum. M: MitochondriaFigure 2