Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F

Abstract

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurologic disorder, but its molecular mechanisms remain unclear. One variant of CMT, 2F, is characterized by mutations in heat shock protein 27 (Hsp27). As bioactive sphingolipids have been implicated in neurodegenerative diseases, we sought to determine if their dysregulation is involved in CMT. Here, we show that Hsp27 knockout mice demonstrated decreases in ceramide in peripheral nerve tissue and that the disease-associated Hsp27 S135F mutant demonstrated decreases in mitochondrial ceramide. Given that Hsp27 is a chaperone protein, we examined its role in regulating ceramide synthases (CerSs), an enzyme family responsible for catalyzing generation of the sphingolipid ceramide. We determined that CerSs colocalized with Hsp27, and upon the presence of S135F mutants, CerS1 lost its colocalization with mitochondria suggesting that decreased mitochondrial ceramides result from reduced mitochondrial CerS localization rather than decreased CerS activity. Mitochondria in mutant cells appeared larger with increased interconnectivity. Furthermore, mutant cell lines demonstrated decreased mitochondrial respiratory function and increased autophagic flux. Mitochondrial structural and functional changes were recapitulated by blocking ceramide generation pharmacologically. These results suggest that mutant Hsp27 decreases mitochondrial ceramide levels, producing structural and functional changes in mitochondria leading to neuronal degeneration.-Schwartz, N. U., Linzer, R. W., Truman, J.-P., Gurevich, M., Hannun, Y. A., Senkal, C. E., Obeid, L. M. Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F.

Keywords: CMT2F; CerS; mitochondria; neuropathy; sphingolipid.

Figure 1.

Decreased ceramides in Hsp27 KO sciatic nerve tissue. A–J) Sciatic nerve tissue from mice was homogenized and used for lipid measurements with LC/MS. C14, C16, C18, C20, C22, C24, C26, C18:1, C22:1, and C24:1 ceramides, are shown, respectively (n = 3). K, L) Total ceramide levels from sciatic nerve (SN) and brain tissue (n = 3). HSPB1, Hsp-β1. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Decreased mitochondrial ceramides in S135F mutant HT-22 cells. Mitochondrial (Mito.; A), cytoplasmic (Cyto.; B), and nuclear (Nuc.; C) fractions of HT-22 cells were isolated and used for lipid measurements with LC/MS. Mitochondrial fractions demonstrate reduced total ceramide (*P < 0.05, n = 4) and cytoplasmic fractions with increased total ceramide (**P < 0.01, n = 4), and there is no change in nuclear ceramides (P = 0.95, n = 4).

Figure 3.

Hsp27:CerS1 interaction localizes to the ER. A–C) WT Hsp27 (red) colocalizes with FLAG-tagged CerS1, CerS2, and CerS5, respectively, (green) in HT-22 cells by confocal microscopy. D) Ceramide profile from DRGs cultured for 4 d from 7 mo female mice (n = 3). E, F) WT and S135F Hsp27 (red), respectively, colocalize with FLAG-tagged CerS1 (green) in HT-22 cells by SIM microscopy. G) FLAG-tagged CerS1 (green) and calnexin (pink) strongly colocalize. H) Endogenous WT-Hsp27 (red) and anti-CerS1 (green) colocalize in mouse DRG. I) Electroporated S135F Hsp27 (red) and FLAG-tagged CerS1 (green) colocalize in mouse DRG cells. Confirmation of neuronal identity with TrpV1 (pink).

Figure 4.

S135F mutant decreases CerS1 localization to mitochondria. A) CerS1 localizes to mitochondria in the presence of WT Hsp27 more than S135F in HT-22 cells. Quantification of super-resolution SIM colocalization of FLAG (CerS1) and MitoTracker Deep Red (mitochondrial marker) used PCC and MOC (*P < 0.05 for PCC, *P < 0.05 for MOC, n = 31 and n = 35). B) SIM quantification of FLAG and calnexin (ER marker) colocalization (P = 0.797 for PCC, P = 0.188 for MOC, n = 24 and n = 27). C) CerS1 localizes to the ER in the presence of WT and S135F Hsp27 in HT-22 cells. SIM quantification of FLAG and calnexin colocalization using PCC and MOC (P = 0.131 for PCC, P = 0.70 for MOC, n = 15 and n = 18). D) SIM quantification of FLAG and calnexin colocalization was quantified using PCC and MOC (P = 0.324 for PCC, P = 0.287 for MOC, n = 9 and n = 10). E) SIM quantification of calnexin and MitoTracker Deep Red (P = 0.324 for PCC, P = 0.287 for MOC, n = 9 and n = 10).

Figure 5.

S135F mutant produces enlarged mitochondria consistent with decreased CerS function. A, B) Representative images of mitochondria in HT-22 cells transfected with WT and S135F Hsp27, respectively. C) Increased area is observed in S135F mutant-transfected HT-22 cells (n = 41, 44) and cells treated with FB1 (n = 15, 13). D) Increased area/perimeter ratio is observed in S135F mutant-transfected HT-22 cells (n = 41, 44) and cells treated with FB1 (n = 15, 13). **P < 0.01, ***P < 0.001.

Figure 6.

S135F and P182L mutants display decreased mitochondrial function consistent with decreased CerS function. A) Mutants (S135F and P182L) and FB1 display decreased basal respiration (Resp.) Oxygen Consumption Rate (OCR) compared with WT and V, respectively. B) Mutants and FB1 display decreased maximal respiration compared with WT and V, respectively. C) Mutants and FB1 display decreased spare respiratory capacity (Capac.) compared with WT and V, respectively. All experiments were repeated at least twice with similar results; error bars represent sd of a single experiment. ***P < 0.001.

Figure 7.

Increased autophagy but no change in growth of S135F mutant cells. A) The S135F mutation does not affect cell proliferation. B) Representative Western blot showing a mildly increased LC3B signal in S135F compared with WT-transfected HT-22 cells. C) There is an ∼1.3-fold increase in autophagy in S135F Hsp27, as indicated by the LC3B-II signal by immunoblotting (n = 6). D) Quantification of confocal images reveals an increase in autophagy, as indicated by LC3B:Hsp27 colocalization (n = 22, 23). E) Quantification of intensity of LC3B staining as a measure of autophagy reveals increased autophagy in S135F (n = 12, 19). *P < 0.05, **P < 0.01.