Dysferlin-peptides reallocate mutated dysferlin thereby restoring function

Abstract

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca(2+)dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients' myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.

Figure 1. Dysferlin-peptides redirect mutant dysferlin in transfected C2C12 cells.

C2C12 cells were transfected with either GFP-tagged wildtype human dysferlin cDNA or missense-mutated dysferlin cDNA DYSF p.G299R or p.L1341P. Transfected cells were treated with TAT-labeled dysferlin-peptides corresponding to the mutation. (A–C) Wildtype human dysferlin-GFP tagged. (A) Dysferlin-GFP. (B) Localization of dysferlin is confirmed by immunostaining using an anti-dysferlin ab. (C) merge. (D–F) DYSF p.G299R-GFP tagged. Experiment performed 14×. (D) No peptides added. GFP-dysferlin is not expressed at the plasma membrane. (E) Peptide A2 (10-mer) added corresponding to DYSF p.G299R. GFP-dysferlin relocalizes to sarcolemmal sites. (F) Peptide A1 corresponding to WT dysferlin added. Distribution of dysferlin is granular but not expressed at the sarcolemma. (G–I) DYSF p.L1341P-GFP tagged. Experiment performed 12×. (G) No peptides added. (H) Peptide B2 (10-mer) corresponding to DYSF p.L1341P supports reallocation of dysferlin to the sarcolemma. (I) Peptide B4 (15-mer) corresponding to DYSF p.L1341P. 10mer peptides carrying the corresponding mutation were most effective. Bar: 10 µm. Inserts represent enlarged boxed areas.

Figure 2. Dysferlin-peptides redirect mutant dysferlin to the sarcolemma in primary human myotubes.

Primary human myotubes carrying dysferlin missense mutations were treated with the TAT-labeled dysferlin-peptides. Dysferlin was detected by anti-dysferlin ab. Nuclei are stained with Hoechst. Missense mutated dysferlin aggregates within the myotubes (A, D). After treating human myotubes with 10mer peptides (B, E) harboring the missense mutation mutant dysferlin can be localized at sarcolemmal sites whereas nonsense peptides (C) do not elicit this effect. (A–C) Primary human myotubes expressing DYSF p.G299R. Experiment performed 9x. (A) No peptides added. (B) Peptide A2 (10-mer) added corresponding to DYSF p.G299R. (C) Nonsense peptide added (control). (D–E) Primary human myotubes harboring the dysferlin mutation p.L1341P. Experiment performed 7x. (D) No peptides added. (E) Peptide B2 (10-mer) added corresponding to DYSF p.L1341P. (F) Sarcolemmal dysferlin localization in a normal human myotube. Bar: 10 µm. Arrows indicate reallocated dysferlin to sarcolemmal sites.

Figure 3. Impaired membrane repair in dysferlin deficient myotubes was rescued by specific peptides: Laser wounding.

Fluorescent dye influx (FM1-43) in human myotubes harboring DYSF p.G299R or in normal human myotubes is shown at the time of laser wounding (0 seconds) and 40, 60 and 120 seconds thereafter. Myotubes were wounded by irradiating a 2.5×2.5 µm boundary area of the plasma membrane. (A) DYSF p.G299R, no peptide. (B) DYSF p.G299R, nonsense peptide added. (C) DYSF p.G299R, corresponding mutant peptide A2 added. (D) Normal human myotubes, no peptides. Without peptides or after incubation with nonsense peptide FM1-43 dye rapidly spreads within the DYSF p.G299R human myotubes. After treatment with specific peptide A2 distribution of FM1-43 is similar to normal myotubes. Arrows indicate the wounded area of the plasma membrane. Scale bar: 10 µm. (E) Quantification of fluorescence intensity (FM1-43) before (0 seconds) and after laser wounding. Data represent mean ± SEM, n = 6/group. See also video S1, S2, S3, S4.

Figure 4. Specific peptides cause functional recovery in dysferlin-deficient myotubes: Interventional atomic force microscopy.

Mechanical sarcolemmal wounding was induced by atomic force microscopy. In all experiments 2 µm longitudinal lesions were set. Arrows indicate the lesion site. The instrument shown is the cantilever used for membrane wounding. (A–C) Normal human myotube 5, 19 and 30 seconds after wounding. The lesion is hardly detectable and closes rapidly. (D–F) DYSF p.L1341P primary human myotube 6, 20 and 90 sec after wounding. The lesion continuously increases in size. (G–I) DYSF p.L1341P primary human myotube treated with corresponding B2 peptide 6, 22 and 30 seconds after wounding. The lesion disappears rapidly. Scale bar: 10 µm. See also video S5, S6, S7.

Figure 5. TAT-labeled dysferlin-peptides in primary human myotubes localize to the ER.

Peptide B2 corresponding to DYSF p.L1341P was labeled with ATTO-495-ME fluorescent dye (red) and added to DYSF p.L1341P human myotubes. (A) DYSF p.L1341P human myotube immediately after addition of ATTO-495-ME-peptide B2 to DYSF p.L1341P human myotubes, (B) after 8 minutes and (C) after 4 hours. (D) Immunostain using anti-calnexin ab (blue). Perfect co-localization of ATTO-495-ME-peptide B2 and ER marker calnexin. No dysferlin-peptide detected at the sarcolemma. Bar: 10 µm. See also video S8.

Figure 6. TAT-labeled dysferlin-peptides reduce the expression of the ER stress sensor BiP.

(A) In DYSF p.G299R human myotubes HSPA5 gene expression is significantly up-regulated. Data represent median + SEM, n = 9/group. (B and C) Mutant peptides decrease HSPA5 gene expression in DYSF p.G299R human myotubes most effectively (RT-PCR). Consequently, treatment with specific peptide A2 reduces BiP protein expression. C1: no peptides added; C2: addition of nonsense peptide; C3: treatment with specific peptide A2. The nonsense peptide serves as a control and its amino-acid sequence has no analogy to dysferlin. Data represent median + SEM, n = 3/group. Fig. S5 provides additional information on statistics.