Cellular prion protein dysfunction in a prototypical inherited metabolic myopathy

Abstract

Inherited fatty acid oxidation diseases in their mild forms often present as metabolic myopathies. Carnitine Palmitoyl Transferase 2 (CPT2) deficiency, one such prototypical disorder is associated with compromised myotube differentiation. Here, we show that CPT2-deficient myotubes exhibit defects in focal adhesions and redox balance, exemplified by increased SOD2 expression. We document unprecedented alterations in the cellular prion protein PrP^C, which directly arise from the failure in CPT2 enzymatic activity. We also demonstrate that the loss of PrP^C function in normal myotubes recapitulates the defects in focal adhesion, redox balance and differentiation hallmarks monitored in CPT2-deficient cells. These results are further corroborated by studies performed in muscles from Prnp^-/- mice. Altogether, our results unveil a molecular scenario, whereby PrP^C dysfunction governed by faulty CPT2 activity may drive aberrant focal adhesion turnover and hinder proper myotube differentiation. Our study adds a novel facet to the involvement of PrP^C in diverse physiopathological situations.

Keywords: Cellular prion protein; Focal adhesions; Inherited fatty acid oxidation disorders; Inherited metabolic myopathy; Muscle differentiation; Redox balance.

Fig. 1

CPT2-deficient myotubes exhibit FA alterations and increased SOD2 expression. a Representative images of myotubes from healthy subject (top panel) or CPT2-deficient patient (bottom panel) stained with MHC-I and PAX antibodies. Nuclei were stained with TO-PRO-3. Scale bar: 20 µM. b Representative western blot and quantification of PAX protein from control or CPT2-deficient myotubes. c Representative images of myotubes from healthy subject (top panel) or CPT2-deficient patient (bottom panel) stained with MHC-I and FAK antibodies. Nuclei were stained with TO-PRO-3. Scale bar: 20 µM. d Representative western blot and quantification of FAK protein from control or CPT2-deficient myotubes. e SOD2 mRNA expression in control and CPT2-deficient myotubes (left panel) and representative western blot and quantification of SOD2 protein from control or CPT2-deficient myotubes (right panel). a and c Images are representative of three independent experiments carried out on 2 controls and 2 patients. In b, d and e results are means of at least three independent experiments, each carried out on 3 controls and 4 patients. For b, statistical significance was assessed by Mann–Whitney test, for d and e, statistical significance was assessed by unpaired two-tailed Student’s t test. The P values are indicated in the figure. Data are represented as mean ± SEM

Fig. 2

CPT2-deficient myotubes display defects in PrP^C. a PRNP mRNA expression in control and CPT2-deficient myotubes. b Representative western blot and quantification of the various PrP^C protein isoforms from control or CPT2-deficient myotubes. c Representative images of myotubes from healthy subject (top panel) or CPT2-deficient patient (bottom panel) stained with PrP^C and PAX antibodies. Nuclei were stained with TO-PRO-3. Scale bar: 20 µM. d Representative western blot and quantification of the various PrP^C protein isoforms in the cytoplasmic (C) or nuclear (N) fraction of control or CPT2-deficient myotubes. In a, b and d results are means of at least three independent experiments, each carried out on 3 controls and 4 patients. c Images are representative of two independent experiments carried out on 2 controls and 2 patients. For a and b statistical significance was assessed by unpaired two-tailed Student’s t test; for d statistical significance was assessed by Two-way ANOVA and the Tuckey test. The P values are indicated in the figure. Data are represented as mean ± SEM

Fig. 3

Inhibition of CPT2 enzymatic activity phenocopies CPT2 genetic deficit. a Representative western blot and quantification of MHC-I, SOD2, MYF5 and FAK proteins from vehicle or l-amino-Car-treated (2 mM for 6 days) myotubes from healthy subject. b Representative western blot and quantification of the various PrP^C protein isoforms from vehicle or l-amino-Car-treated myotubes from healthy subject. Results are means of at least three independent experiments carried out on 3 controls. Statistical significance was assessed by paired two-tailed Student’s t test. The P values are indicated in the figure. Data are represented as mean ± SEM

Fig. 4

PrP^C silencing recapitulates the phenotypes of CPT2-deficiency. a Representative western blot of PrP^C protein in myotubes from healthy subject treated with siRNA Non target (NT) or against PRNP (siPrPc). b Representative western blot and quantification of MHC-I, SOD2, MYF5 and FAK proteins in myotubes from healthy subject treated with siRNA Non target (NT) or against PRNP (siPrPc). c PRNP, MYH7, SOD2 and MYF5 mRNA expression in myotubes from healthy subject treated with siRNA Non target (NT) or against PRNP (siPrPc). d Representative images of myotubes from healthy subject treated with siRNA Non target (NT) (top panel) or against PRNP (siPrPc) (bottom panel) stained with PAX and MHC-I antibodies. nuclei were stained with TO-PRO-3. Scale bar: 20 µM. e Calculation of the fusion index in myotubes from healthy subject treated with siRNA Non target (NT) or against PRNP (siPrPc). f Representative western blot of PrP^C protein in muscles from wild-type (WT) and Prnp^−/− mice. g Representative western blot and quantification of MHC-I, SOD2, and FAK proteins in muscles from WT controls and Prnp^−/− mice. h Proposed schematic model explaining the relationship between CPT2 deficiency, ROS imbalance, PrP^C cleavage, altered FA dynamics and impaired myotubes differentiation. a–c Results are means of at least three independent experiments carried out on 3 controls. d Images are representative of two independent experiments on 2 controls. e Four fields of each condition were counted at × 20 magnification. f–g Results are means of at least three independent experiments performed on 4 WT and 4 Prnp^−/− mice. Data are represented as mean ± SEM. For b, c and e, statistical significance was assessed by paired two-tailed Student’s t test. For g, statistical significance was assessed by unpaired two-tailed Student’s t test. The P values are indicated in the figure. Data are represented as mean ± SEM