Pathophysiology of protein aggregation and extended phenotyping in filaminopathy

Abstract

Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there is a noticeably diminished efficiency of both the ubiquitin-proteasome system and autophagy that impairs the muscle capacity to prevent the formation or mediate the degradation of aggregates. Transfection studies of cultured muscle cells imitate events observed in the patient's affected muscle and therefore provide a helpful model for testing future therapeutic strategies.

Figure 1

Pedigrees of three filaminopathy families. Affected members of the families from Macedonia (Family I) and China (Family II) harboured the p.W2710X mutation in FLNC. The German family with p.V930_T933del mutation in FLNC (Family III) was first described by Shatunov et al. (2009) (pedigree extended). Individuals with proven mutation and deceased family members who had suffered from muscle weakness are represented by filled symbols.

Figure 2

MRI (T₁-weighted) of lower extremities in filaminopathy patients (first row: proximal upper legs, second row: distal upper legs, third row: lower legs). (A and C) Patients with p.V930_T933del mutation in FLNC (A: a 48-year-old male, disease duration 10 years; C: a 45-year-old female, disease duration 10 years). (B and D) Patients harbouring the p.W2710X mutation in FLNC (B: a 58-year-old male, disease duration 13 years; D: a 54-year-old female, disease duration 5 years). Hyperintensities in muscles denote lipomatous alterations. The relative involvement of marked muscles is important for separation of filaminopathy from other myofibrillar myopathy subtypes. AM = adductor magnus; BF = biceps femoris; GR = gracilis; LG = lateral gastrocnemius; MG = medial gastrocnemius; PG = peroneal group muscles; SA = sartorius; SM = semimembranosus; ST = semitendinosus; TA = tibialis anterior.

Figure 3

Ultrastructural analysis of skeletal muscle from the proband of the Macedonian family. (A and B) Fine filaments emanating at the Z-line level that coalesced into electron-dense inclusions under the sarcolemma or between the myofibrils are surrounded by groups of mitochondria (B). (C–E) Collections of tubulofilaments (right upper corner in C), granulofilamentous material and small vacuoles (left lower corner in C); small rod bodies at the periphery of a fibre region harbouring granulofilamentous material (D), fine electron-dense granulofilamentous material, vacuoles and sparse mitochondria between normal myofibrils (E). (F) Abnormal fibre region containing remnants of filaments, small rod bodies, and prominent electrondense inclusions. Scale bars: C = 0.5 µm; B, D and E = 1 µm; A, F = 2 µm.

Figure 4

Immunolocalization of heat shock proteins in skeletal muscle cryosections from a filaminopathy patient from Family III (p.V930_T933del mutation). Serial cryosections were double-stained with antibodies recognizing the indicated heat shock proteins (Hsp) and either FLNC (Filamin C) or myotilin to localize protein aggregates. For comparison, trichrome and haematoxylin and eosin (H&E) stained sections are shown at the top. Scale bar = 50 µm.

Figure 5

Immunolocalization of the components of chaperone complexes involved in chaperone-assisted selective autophagy in skeletal muscle cryosections from a filaminopathy patient (p.V930_T933del mutation). Serial cryosections were double-stained with antibodies recognizing the indicated chaperone-assisted selective autophagy pathway markers and either FLNC or myotilin to localize protein aggregates. For comparison, trichrome and haematoxylin and eosin (H&E) stained sections are also shown. Scale bar = 50 µm.

Figure 6

Immunolocalization of markers of autophagy and associated proteins in skeletal muscle cryosections from a filaminopathy patient with p.V930_T933del mutation. Serial sections were double-stained with antibodies recognizing the indicated heat shock proteins and either FLNC or myotilin to localize protein aggregates. For comparison, trichrome and haematoxylin and eosin (H&E) stained sections are also shown. Scale bar = 50 µm.

Figure 7

Immunolocalization of markers of the ubiquitin proteasome system and associated proteins in skeletal muscle cryosections from a filaminopathy patient (p.V930_T933del mutation). Serial sections were double-stained with antibodies recognizing the indicated ubiquitin–proteasome system components and either FLNC or myotilin to localize protein aggregates. For comparison, trichrome and haematoxylin and eosin (H&E) stained sections are also shown. Scale bar = 50 µm.

Figure 8

Biophysical characterization and limited proteolysis of the FLNC p.V930_T933del mutation. (A) Circular dichroism spectroscopy performed with purified, bacterially expressed wild-type and deletion mutant FLNC d7-8ΔVKYT constructs. The mean residue ellipticity (ΔεMR) of the wild-type shows a minimum in the spectrum at 218 nm and a positive value at 205 nm, while the deletion mutant displays a blue shift with minimum at 208 nm and positive ellipticity at 200 nm, a characteristic sign of the presence of unstructured regions. Solid line = wild-type; dashed line = FLNC d7-8ΔVKYT construct. (B) Differential scanning fluorimetry. Temperature denaturation experiments for FLNC d7–8 and deletion mutant FLNC d7–8ΔVKYT. The melting temperature (T_m) is determined at the inflection point of the fluorescence signal, the first derivative of which is reported here. Solid line = wild-type; dashed line = deletion mutant, scaled to the wild-type. (C) Protein stability analysed by thermolysin digestion. Wild-type and mutant FLNC d5-9ΔVKYT were treated with the protease thermolysin for 1 to 60 min, as indicated above each lane. Samples were analysed by PAGE. Note that the mutant protein was completely digested after 30 min, while a significant portion of the wild-type variant was still intact after 60 min of incubation, indicating less stable folding of the mutant protein. (D) Ribbon diagram of homology model of immunoglobulin-like domain 7 of human FLNC. β-strands B, E and D forming a β-sheet are coloured orange, green and yellow, respectively. Main chain atoms of residues involved hydrogen bonds indicated by dashed lines, stabilizing the β-sheet are depicted in stick model, with N and O coloured in blue and red. Residues 930VKYT933 that are missing in the deletion mutant are shown as full-atom stick model and coloured in red.

Figure 9

Transient expression of full-length wild-type, p.W2710X or p.V930_T933del FLNC variants in C2C12 cells. C2C12 cells were transiently transfected with FLNC variants cloned in the pEGFPC2 (−C2) or pEGFPN3 (−N3) vectors and cells were photographed 12 h and 24 h after transfection. Scale bar = 100 µm; insert = 12.5 µm.