NAD+ enhances ribitol and ribose rescue of α-dystroglycan functional glycosylation in human FKRP-mutant myotubes

  1. Carolina Ortiz-Cordero
  2. Alessandro Magli
  3. Neha R Dhoke
  4. Taylor Kuebler
  5. Sridhar Selvaraj
  6. Nelio AJ Oliveira
  7. Haowen Zhou
  8. Yuk Y Sham
  9. Anne G Bang
  10. Rita CR Perlingeiro  Is a corresponding author
  1. Department of Integrative Biology and Physiology, University of Minnesota, United States
  2. Lillehei Heart Institute, Department of Medicine, University of Minnesota, United States
  3. Stem Cell Institute, University of Minnesota, United States
  4. Bioinformatics and Computational Biology Program, University of Minnesota, United States
  5. Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Patient-specific Walker-Warburg syndrome (WWS) induced pluripotent stem (iPS) cell-derived myotubes display reduced functional glycosylation of α-dystroglycan (α-DG).

(A) Representative immunostaining of wild type (WT) and FP4 iPS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stains nuclei (in blue). Scale bar, 200 μm. (B) …

Figure 1—figure supplement 1
Pluripotency characterization of reprogrammed FP4 induced pluripotent stem (iPS) cell line.

(A–C) Representative images show typical (A) pluripotent colony morphology, (B) alkaline phosphatase activity, and (C) immunostaining for OCT3/4, SOX2, and NANOG (red). DAPI stains nuclei (in blue). …

Figure 1—figure supplement 2
Fukutin-related protein (FKRP) overexpression rescues functional glycosylation of α-dystroglycan (α-DG) in Walker-Warburg syndrome (WWS) FP4 induced pluripotent stem (iPS) cell-derived myotubes.

(A) Representative immunofluorescence staining of control (empty vector LV) and FKRP-LV FP4 iPS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). Wild type (WT) myotubes were …

Figure 2 with 1 supplement
Ribitol and ribose rescue α-dystroglycan (α-DG) functional glycosylation in patient-specific Walker-Warburg syndrome (WWS) myotubes.

(A) Scheme denoting the role of ribose and ribitol in the generation of Rbo5P, which is required by fukutin (FKTN) and fukutin-related protein (FKRP) for glycosylation of α-DG. (B) Representative …

Figure 2—figure supplement 1
Ribitol and ribose dose-response studies in induced pluripotent stem (iPS) cell-derived myotubes.

(A–B) Phase contrast images of FP4 myogenic progenitors that upon induction of differentiation were treated with the indicated concentrations of ribitol for 5 days. (B) Western blot shows enhanced …

Figure 3 with 1 supplement
Ribitol and ribose increase the levels of ribitol-5-P and cytidine diphosphate (CDP)-ribitol in patient-specific Walker-Warburg syndrome (WWS) myotubes.

(A) Detection of ribitol, ribose, ribitol-5-P, and CDP-ribitol in WWS FP4 myotubes that had been treated or not with ribitol or ribose for 5 days (n = 3 for each cohort). Error bars represent …

Figure 3—figure supplement 1
Liquid chromatography with tandem mass spectrometry (LC/MS-MS) standard curves.

(A–D) Standard curves from serial dilution of metabolites (A) ribitol, (B) ribose, (C) ribitol-5-P, and (D) cytidine diphosphate (CDP)-ribitol.

Figure 4 with 2 supplements
NAD+ potentiates the capacity of ribitol and ribose to rescue α-dystroglycan (α-DG) functional glycosylation.

(A–B) Western blots show increased IIH6 staining in Walker-Warburg syndrome (WWS) FP4 myotubes that had been supplemented with ribitol and NAD+ compared to ribitol alone (A) or with ribose and NAD+ …

Figure 4—figure supplement 1
NAD+ rescue functional glycosylation of α-dystroglycan (α-DG) in FP4 induced pluripotent stem (iPS) cell-derived myotubes.

(A) Representative immunostaining of FP4 myotubes treated with NAD+ and respective control wild type (WT) myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stained nuclei (in blue). …

Figure 4—figure supplement 2
NAD+, ribitol/NAD+, and ribose/NAD+ do not increase ribitol-5-P or cytidine diphosphate (CDP)-ribitol in FP4 myotubes.

(A) Liquid chromatography with tandem mass spectrometry (LC/MS-MS) detection of ribitol, ribose, ribitol-5-P, and cytidine diphosphate (CDP)-ribitol from FP4 myotubes untreated or supplemented with …

Figure 5 with 1 supplement
Functional fukutin-related protein (FKRP) is required for NAD+ ribitol/ribose-mediated rescue of α-dystroglycan (α-DG) functional glycosylation.

(A) Representative immunostaining of FKRP knockout (KO) and respective control wild type (WT) isogenic PS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stained nuclei …

Figure 5—figure supplement 1
NAD+ treatment in FKRP C318Y myotubes increases α-dystroglycan (α-DG) levels.

(A–B) Western blot for core α-DG and β-DG in FKRP C318Y myotubes treated with NAD+. (B) Total protein stain was used as loading control. (C) Bar graphs shows quantification of α-DG and β-DG …

Model depicting the Walker-Warburg syndrome (WWS)-associated mutations in the fukutin-related protein (FKRP) catalytic domain.

(A) Schematic representation of FKRP protein highlighting the N-terminal stem domain and the localization of C318Y and F473C mutations in the C-terminal catalytic domain. (B) Crystal structure of …

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens, male)FP4This studyAvailable from the Anne Bang lab
Genetic reagent (Homo sapiens, male)FKRP C318YThis studyTC1133 FKRP C318YAvailable from the Rita Perlingeiro lab
Genetic reagent (Homo sapiens, male)FKRP KOThis studyH9 FKRP KOAvailable from the Rita Perlingeiro lab
Cell line (Homo sapiens, male)WTPMID:22560081PLZControl line, available from the Rita Perlingeiro lab
Cell line (Homo sapiens, male)Parental WT FKRP KOWiCellH9ESC control line (WA09)
Cell line (Homo sapiens, male)Parental WT FKRP C318YPMID:26411904TC-1133Control line, available with RUCDR Infinite Biologics
Chemical compound, drugCHIR99021TocrisCat# 442310 µM
Chemical compound, drugLDN193189Cayman chemicalCat# 19396200 nM
Chemical compound, drugSB431542Cayman chemicalCat# 1303110 µM
Chemical compound, drugDAPTCayman chemicalCat# 1319710 µM
Chemical compound, drugDexamethasoneCayman chemicalCat# 1101510 µM
Chemical compound, drugForskolinCayman chemicalCat# 1101810 µM
Chemical compound, drugRibitolSigma-AldrichCat# A550250 mM
Chemical compound, drugD-(−)-riboseSigma-AldrichCat# R962910 mM
Chemical compound, drugNAD+Sigma-AldrichCat# N0632100 µM
Chemical compound, drugSorbinilSigma-AldrichCat# S7701100 μM
Chemical compound, drugDoxycyclineSigma-AldrichCat# D98911 µg/ml
Recombinant proteinRecombinant human FGF-basicPeprotechCat# 100-18B5 ng/ml
AntibodyAnti-alpha dystroglycan (mouse monoclonal)MilliporeCat# 05–593, RRID:AB_309828Dilution
1:1000 (WB)
AntibodyAnti-alpha dystroglycan
(mouse monoclonal)
DSHBCat# IIH6 C4, RRID:AB_2617216Dilution
1:50 (IF)
AntibodyAnti-human dystroglycan (sheep polyclonal)R and D SystemsCat# AF6868, RRID:AB_10891298Dilution
1:1000 (WB)
AntibodyAnti-MHC (mouse monoclonal)DSHBCat# MF20, RRID: AB_2147781Dilution
1:50 (IF)
1:200 (WB)
AntibodyAnti-laminin (rabbit polyclonal)Sigma-AldrichCat# L9393, RRID:AB_477163Dilution
1:1000 (WB)
AntibodyAnti-beta dystroglycan, concentrated (mouse monoclonal)DSHBCat# MANDAG2 clone 7D11, RRID:AB_2211772Dilution
1:1500 (WB)
AntibodyAnti-OCT3/4 (mouse monoclonal)SCBTCat# C-10, RRID: AB_628051Dilution
1:50 (IF)
AntibodyAnti-SOX2
(goat polyclonal)
SCBTCat# Y-17, RRID: AB_2286684Dilution
1:50 (IF)
AntibodyAnti-NANOG (mouse monoclonal)SCBTCat# H-2, RRID: AB_10918255Dilution
1:50 (IF)
AntibodyAnti-SSEA4 (mouse monoclonal)SCBTCat# sc-21704, RRID: AB_628289Dilution
1:50 (IF)
AntibodyAlexa fluor 555 goat anti-mouse IgG and IgM
(goat polyclonal)
Thermo Fisher ScientificCat# A-21424, RRID: AB_141780
Cat# A-21042, RRID:AB_2535711
Dilution
1:500 (IF)
AntibodyDyLight 680 anti-rabbit IgG
(goat polyclonal)
Thermo Fisher ScientificCat# 35568, RRID:AB_614946Dilution
1:10000 (WB)
AntibodyDyLight 680 anti-mouse IgM
(goat polyclonal)
Thermo Fisher ScientificCat# SA5-10154, RRID:AB_2556734Dilution
1:10000 (WB)
AntibodyDyLight 680 anti-sheep IgG
(rabbit polyclonal)
Thermo Fisher ScientificCat# SA5-10058, RRID:AB_2556638Dilution
1:10000 (WB)
AntibodyDyLight 800 anti-mouse IgG
(goat polyclonal)
Thermo Fisher ScientificCat# SA5-10176, RRID:AB_2556756Dilution
1:10000 (WB)
OtherDAPI stainSCBTsc-3598(1.5 µg/ml)
OtherLaminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneMillipore SigmaCat# L20201 µg/µl

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