Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy

Abstract

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy-causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment-specific manner.

Figure 1

Microscopy of muscle biopsies from Finnish LGMD1D patients. (a) Confocal microscopy of LGMD1D muscle showed that the predominant localization of DNAJB6 in the Z-disc was preserved in the regions with unaltered sarcomeric structure. Scale bar, 10 μm. (b) Transmission electron microscopy showed early disruption of Z-disks (arrows; left panel) and authophagic pathology (right panel) in LGMD1D. For comparison, see region with normal ultrastructure (boxed in left panel). Scale bars, approx. 1 μm. (c) The autophagic pathology was highlighted by areas of LC3 accumulation within myofibers, revealed by brown precipitate in LC3 immunohistochemistry (left panel), and by the presence of rimmed vacuoles in the Herovici staining (arrows; right panel). Scale bars, approx. 50 μm. (d) DNAJB6, HSPA8, MLF1, desmin, myotilin, filamin, and KRT18 were found in protein accumulations. For individual channels, see Supplementary Fig. 4. Confocal images show single optical sections. Scale bars, 10 μm.

Figure 2

Muscle disintegration in DNAJB6b mutant and morphant zebrafish. (a–i) Lateral views of fish embryos 2 dpf subjected to whole-mount immunofluorescence staining of slow myosin heavy chain. Injected embryos expressing wild-type (wt) DNAJB6a/b showed slow myofibers spanning the somite normally between adjacent myosepta (c, f) and were indistinguishable from control embryos. Injection of DNAJB6b p.Phe93Leu and p.Phe89Ile mutant mRNAs resulted in detachment of fibers from the vertical myoseptum (a, b), whereas DNAJB6a mutants appeared normal (d, e). Similar muscular disintegration was observed in dnajb6b morphant embryos injected with sb-MO against the zebrafish ortholog of DNAJB6 (g) and in embryos expressing DNAJB6 p.Phe93Ala or p.Phe93Gly (h, i). The detachment of myofibers from the myoseptum can be partial (a, b, g, h, white arrowheads) or complete (i, white asterisk). Scale bar, approx. 50 μm. (j) Embryos injected with the indicated constructs were categorized phenotypically based on the presence of muscle fiber detachment affecting 1–2 somites (class I, mild) or multiple somites (class II, severe; see Supplementary Fig. 5 for an example). The phenotype in dnajb6b MO-injected embryos was rescued efficiently by wild-type human DNAJB6b mRNA (χ² test).

Figure 3

Dominant effect of mutant DNAJB6 proteins. (a) Co-injection of wild-type DNAJB6b mRNA with p.Phe93Leu or p.Phe89Ile mutant mRNA in zebrafish embryos led to a more severe muscle phenotype, with a statistically significant increase in the number of class II embryos (χ² test). (b) DNAJB6b constructs were expressed in 293FT cells and protein synthesis was blocked by cycloheximide (CHX). Whole-cell extracts were obtained at the indicated time points and analyzed by western blotting. DNAJB6 band intensities were quantified and normalized to α-tubulin (single transfections) or HSP90 (co-transfections) intensity to obtain the relative DNAJB6 levels. In single transfections (solid lines), cycloheximide treatment rapidly decreased wild-type DNAJB6b protein levels, while p.Phe89Ile and p.Phe93Leu mutant proteins showed reduced turnover. In co-transfections with p.Phe93Leu (dashed lines), the wild-type DNAJB6b level remained stable after 4 h of CHX treatment. Data from three independent experiments are presented as mean ± S.D. Relative DNAJB6 level at t = 0 h was set to 100 for each construct. Representative western blots are shown in Supplementary Fig. 7.

Figure 4

Impaired anti-aggregation activity of mutant DNAJB6b. GFP-tagged 120Q-huntingtin was expressed in T-REx 293 cells, with co-expression of V5-tagged wild-type or mutant (p.Phe89Ile, p.Phe93Leu) DNAJB6b, or wild-type DNAJB6a induced (+) or uninduced (−). (a) Aggregated huntingtin (aggr.) was detected in a filter trap assay, while SDS-soluble huntingtin (sol.), DNAJB6 (V5), and α-tubulin (α-tub.) were analyzed by western blotting. FTA and WB show representative samples. (b) Aggregated and soluble huntingtin were quantified, and anti-aggregation activity of DNAJB6 constructs was determined as the ratio of aggr./sol. ratios in induced vs. uninduced cells (aggregation score). Both mutant DNAJB6b constructs showed significantly impaired anti-aggregation activity (Mann-Whitney U test; P values adjusted for multiple testing with the Bonferroni correction). The nuclear DNAJB6a construct, used as a negative control, showed only weak activity. For each construct, n = 12 (3 experiments performed in quadruplicate). For supporting information, see Supplementary Fig. 8.

Figure 5

Association of DNAJB6 with the CASA complex. (a) Coimmunoprecipitation. Tested constructs were expressed in COS-1 cells, crosslinked, and immunoprecipitated with anti-V5 beads. V5-tagged wild-type and p.Phe93Leu mutant DNAJB6b pulled down endogenous BAG3 and HSPB8. No coimmunoprecipitation was observed with an untagged DNAJB6b construct or a non-related bait (V5-tagged titin is6–M9). (b) Proximity ligation assay (PLA) on rat muscle sections. PLA signals (red dots) indicated spatial proximity of DNAJB6 with its known interaction partner HSPA8, and with the CASA complex proteins BAG3, HSPB8, and STUB1. Negative control experiments were performed with DNAJB6 or BAG3 antibodies alone. Each image shows a representative maximum-intensity projection through a Z-stack of 6.4 μm, with the PLA signal (red) superimposed on the phalloidin counterstain (grey). Scale bar, 5 μm. (c) Co-injection of DNAJB6 and BAG3 in zebrafish. Zebrafish embryos were injected with DNAJB6b p.Phe93Leu mRNA and wild-type (wt) or p.Pro209Leu mutant BAG3 mRNA in indicated combinations. Co-injection of wild-type BAG3 with mutant DNAJB6 resulted in a more severe phenotype than mutant DNAJB6 alone, reflected by a significant increase in class II embryos. In contrast, co-injection of p.Pro209Leu BAG3 with mutant DNAJB6b did not alter the phenotype distribution. Statistical significance (χ² test) is denoted by P value or n.s. (not significant). Scale bar, approx. 50 μm.