In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics

Abstract

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).

Keywords: ALS; Frontotemporal dementia; Inclusion body myopathy; Mitochondrial bioenergetics; Paget's disease of the bone; Valosin containing protein.

Copyright © 2015 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1.

Immunocytochemical analyses of autophagy markers in human fibroblasts affected with VCP disease and controls. Immunocytochemical staining of control and patient VCP fibroblasts with (A) LC3-I/II, (B) p62/SQSTM1, (C) Ubiquitin and (D) TDP-43 showing increased staining suggestive of disrupted autophagy pathways. (E) Staining of human fibroblasts with JC-1, a mitochondrial membrane potential marker. (Magnification 630×) White arrows pointing to increased protein expression levels of LC3, p62/SQSTM1, ubiquitin and TDP-43 and decreased expression levels of JC-1 in patient fibroblasts. (F) Western blot protein expression analysis of LC3, p62/SQSTM1, ubiquitin and TDP-43 antibodies in patient fibroblasts. Alpha tubulin was used as a loading control. (G) Levels of ATP (nmol/mg soluble protein) were determined in the control and patient fibroblasts. Statistical significance is denoted by *p ≤ 0.05.

Fig. 2.

Seahorse XF-24 metabolic flux analyses in fibroblasts from patients affected with VCP disease and controls. (A) Seahorse Bioscience analysis of cellular bioenergetics assay approach (measurements of glycolysis and mitochondrial function). The Seahorse Bioscience analysis comprised of measuring (B) % oxygen consumption rate (OCR), (C) % extracellular acidification rate (ECAR), (D) spare respiratory capacity and (E) proton leak in controls 1 and 2 and VCP patient fibroblast samples 1–4.

Fig. 3.

Measurements of mitochondrial membrane potential, enzyme complex activities, and ATP levels in VCP patient myoblasts. Immunocytochemical staining of control myoblasts versus patient myoblasts (421/07) stained with (A) ubiquitin and p62/SQSTM1, (B) VCP and LC3, (C) VCP and TDP-43 (magnification 400×), and (D) JC-1 (mitochondrial membrane potential marker) (magnification 630×). White arrows pointing to increased protein expression levels of ubiquitin and p62/SQSTM1, VCP and LC3, VCP and TDP-43, and decreased expression levels of JC-1 in patient myoblasts. (E) Western blot protein expression analyses of LC3, p62/SQSTM1, TDP-43, and complexes I–V in VCP patient and control myoblasts. Alpha tubulin was used as a loading control. (F) Complex I activity levels in VCP patient and control myoblasts. (G) Comparison of mitochondrial ΔΨm in the control and patient myoblasts after the maximal loss of the mitochondrial ΔΨm produced by FCCP. The peak/basal (Fx/Fo) ratio of rhodamine 123 fluorescence was measured at baseline (before adding FCCP) and peak (upon addition of FCCP). Results are presented as relative values compared to the values of control A. Data are normalized by the citrate synthase activity. (H) Levels of ATP (nmol/mg soluble protein) were determined in the control and VCP patient myoblasts. Statistical significance is denoted by *p ≤ 0.05.

Fig. 4.

DIC images and immunocytochemical analyses of autophagy markers in Wild Type, VCPR155H/+, and VCPR155H/R155H myoblasts. (A) DIC images of myoblasts from WT, VCPR155H/+ and VCPR155H/R155H mice. Immunocytochemical staining of WT, VCPR155H/+ and VCPR155H/R155H fibroblasts with (B) LC3-I/II, (C) p62/SQSTM1, (D) TDP-43, and (E) JC-1 mitochondrial membrane potential marker (magnification 630×). White arrows pointing to increased protein expression levels of LC3-I/II, p62/SQSTM1, ubiquitin and TDP-43, and decreased expression levels of JC-1 in mouse myoblasts. (F) Western blot expression analyses of mouse myoblasts with LC3, p62/SQSTM1, ubiquitin and TDP-43 antibodies. Alpha tubulin was used as a loading control. (G) Levels of ATP (nmol/mg soluble protein) were determined in the control and VCP mouse myoblasts.