Virchows Arch (2007) 451:1047–1055 DOI 10.1007/s00428-007-0515-3
ORIGINAL ARTICLE
Biochemical and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I Chiara A. Boito & Marina Fanin & Bruno F. Gavassini & Giovanna Cenacchi & Corrado Angelini & Elena Pegoraro
Received: 25 June 2007 / Revised: 2 August 2007 / Accepted: 14 September 2007 / Published online: 20 October 2007 # Springer-Verlag 2007
Abstract Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in α-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of α-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and α-dystroglycan metabolism and malfunctioning. C. A. Boito : M. Fanin : B. F. Gavassini : C. Angelini : E. Pegoraro (*) Department of Neurosciences, University of Padova, via Giustiniani 5, 35128 Padova, Italy e-mail:
[email protected]
G. Cenacchi Department of Pathology, University of Bologna, Bologna, Italy
Keywords FKRP . Glycosylation . LGMD2I . Muscular dystrophy . UPR
Introduction Limb girdle muscular dystrophy type 2I (LGMD2I) is an autosomal recessive muscular dystrophy caused by mutations in the gene coding for fukutin-related protein (FKRP) [6]. LGMD2I is clinically heterogeneous [4, 6, 9, 22, 28], and it is allelic to MDC1C (congenital muscular dystrophy type 1C) with onset in the neonatal period [5]. The function of FKRP in skeletal muscle is largely unknown; however, indirect evidence suggests that FKRP may be a putative glycosyltransferase involved in αdystroglycan (α-DG) processing [6, 13]. Muscle biopsies of patients affected by either LGMD2I or MDC1C show a variable reduction of α-DG glycosylation [4–6]. α-DG glycosylation is essential for proper interaction of dystroglycan with its extracellular matrix ligands (such as laminin α2) [16] and thus to anchor the muscle fibers to the basement membrane [17]. In the literature, there is a large debate about the subcellular localization of FKRP. Initially, it was hypothesized that FKRP was a Golgi-resident protein required for post-translational modification of dystroglycan and that mislocalization of the mutant protein may underlie MDC1C [13]. Later, it was suggested that FKRP localizes in rough endoplasmic reticulum (ER) [14, 21, 27] and that protein mislocalization is not a common mechanism of disease in LGMD2I/MDC1C [10, 27]. More recently, data indicate that FKRP is present at the muscle cell surface, associates with the dystrophin-glycoprotein complex, and has a unique role in α-DG processing pathway [2].
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Here, we have analyzed the histopathological and ultrastructural changes in LGMD2I skeletal muscles in order to further characterize the pathogenesis of LGMD2I. Material and methods Patients Muscle biopsies from ten patients affected by LGMD2I were selected from our muscle biopsy database. Both FKRP-mutated alleles were identified in all patients (Table 1). Clinical and genetic findings in all but one patient have been described in detail [4]. All, but patient number 5857, who was asymptomatic, showed proximal muscle weakness. The Gardner-Medwin and Walton functional scale was used to grade muscle function in the patients [15]. At the time of diagnosis, open muscle biopsy was obtained after written informed consent. Muscle pathology and immunohistochemical studies Thirteen serial sections, 10 μm thick, were obtained from each muscle biopsy. The first section was stained with ATPase at pH 4.3 and the total number of fibers (at least 1,000) counted on photographic enlargement and used to calculate the percentage of fibers immunolabelled with the different antibodies. The fibers area was estimated using the Scion Image Beta 4.03 software. Twelve different monoclonal antibodies were used as follows: collagen IV (1:50) (Chemicon, Temecula, CA),
perlecan (1:100) (MAB1948, Chemicon), laminin α2 directed against the 80-kDa fragment (1:1,000; MAB1922, Chemicon) and against the entire molecule (1:500; NCL-merosin, Novacastra, Newcastle upon Tyne, UK), α-dystroglycan (1:200; IIH6) [12], α-sarcoglycan (1:100; Novocastra), dystrophin (1:100; Novocastra), neonatal myosin heavy chain (1:100; NCL-MHCn; Novocastra), macrophages (CD68; clone EBM11; Dako, Carpinteria, California, USA), CD4 positive helper/inducer T cells (clone MT310; Dako), CD8 positive cytotoxic/ suppressor T cells (clone DK25; Dako), and major histocompatibility complex (MHC) class I molecules (W6/32; Dako). The intensity of reaction was examined by visual inspection by the same observer and graded as follows: absent or normal (−), slight increase or reduction (+), moderate increase or reduction (++), severe increase or reduction (+++). The severity of the dystrophic process and muscle histopathology was scored as previously described [7]. α-DG immunoblot was done as previously described using the IIH6 antibody [4]. Electron microscopy Small muscle specimens were fixed after surgery in 2.5% glutaraldehyde and postfixed in 1% OsO4. After dehydration in graded ethanol, the specimens were embedded in Araldite. Thin sections were stained in uranyl acetate and lead citrate and observed in a Philips 410T transmission electron microscope.
Table 1 Clinical and molecular data in LGMD21 patients Patient, sex
FKRP protein change
Age at onset (years)
Age at biopsy (years)
Disease duration at biopsy
Clinical grading at biopsy/at last evaluation (years)
960, M
Leu276Ile Pro462Ser Pro358Leu (homozygous) Leu276Ile (homozygous) Leu276Ile (homozygous) Pro358Leu Pro462Ser Leu276Ile (homozygous) Val160Phe Pro358Leu Leu276Ile (homozygous) Arg244His (homozygous) Leu276Ile (homozygous)
2
2
0
4/8 (17a)
6
6
0
1/1 (8)
7
42
35
5/5 (42)
13
26
13
4/8 (42)
14 (at 4 years high CK) 22
4
–
0/8 (24)
23
1
1/5 (42)
35
39
4
4/6 (61)
49
54
5
3/4 (63)
50
58
8
1/4 (64)
Asymptomatic
48
–
0/0 (48)
6661, F 5342, M 1191, M 888, F 899, M 879, F 3285, F 1475, F 5857, M a
deceased
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Table 2 Muscle pathology and immunohistochemical data in LGMD2I Patient
Muscle type
Type I fibers (%)
Fibrofatty tissue
Degenerating fibers (%)
Neonatal myosin (%)
IIH6
MHC class Ia
CD68
CD4+/ CD8+
Pathology severity score
960 6661 5342 1191 888 899 879 3285 1475 5857
Quadriceps Quadriceps Quadriceps Quadriceps Quadriceps Quadriceps Biceps Triceps Biceps Quadriceps
80 68 75 41 65 48 51 35 48 34
+++ + ++ + + ± ± + ± –
0.7 0.1 0.1 0.8 0.7 0 0.1 0 0 0.4
8 0.1 4 4 2 0.7 0 0 0 0
– + + + – ++ + ++ ++ ++
± ± ± + + + ± + ++ ++
+++ ++ ++ ++ + ++ ± + ± ++
++/± −/− +/+ N.D./± +/± −/+ ±/± ±/− ++/+ ++/+
Active Mild Severe Moderate Moderate Moderate Mild Moderate Mild Mild
N.D. Not determined Muscle membrane
a
Unfolded Protein Response studies RNA was extracted from frozen muscle biopsies from 10 patients and 11 normal controls using SV Total RNA Isolation (Promega, Madison, WI, USA) and reverse transcribed using an ImProm-II Reverse Transcription System (Promega) according to the manufacturer’s instructions. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed using an ABI Prism 7900 (Applied Biosystems, Foster City, CA, USA) and SYBR Green detection (Invitrogen, Carlsbad, CA, USA). The sequence of primers for GRP78 and CHOP were as published [1, 29]. As an internal control, the extent of glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression in the same tissue was determined. Relative GRP78 and CHOP expressions were given as GRP78/GADPH, and CHOP/GADPH ratios and normalized values were subjected to a 2-ΔΔCt formula to calculate the fold change between controls and patients. All reactions were carried out in triplicate to reduce variation.
Results Patients Five male and five female patients affected by LGMD2I were studied. Age at onset was on average 22 years (range 2–50 years), and average age at biopsy 30 years (range 2–58 years). Mean disease duration at biopsy ranged from 0 to 35 years. Clinical severity at biopsy ranged from 0 (asymptomatic) to 5 (unable to rise from the floor) according to the Gardner Medwin and Walton scale (Table 1). α-DG studies Since mutations in the FKRP gene result in α-DG hypoglycosylation, we used an antibody directed against a glycosylated epitope of the protein (IIH6) to
study muscles biopsies from ten LGMD2I patients and found a depletion in α-DG glycosylation in all biopsies studied (Table 2; Fig. 1). However, comparison between patients showed a complete deficiency in only two of them (numbers 960 and 888) (Fig. 1b), while a variable reduction of α-DG glycosylation ranging from slight to moderate was observed in the remaining patients (Fig. 1c, d). Interestingly, a mosaic-like pattern was also noted in most cases, with some fibers showing clear, strong α-DG labeling at the sarcolemma and others a marked depletion of the protein (Fig. 1c). α-DG immunoblotting using the IIH6 antibody was done in seven of the ten LGMD2I patients studied and confirmed a depletion in glycosylated α-DG in all. In five patients (patients 960, 1191, 888, 899, 979) only traces of glycosylated α-DG, in patient #5857 about 5%, and in patient #1475 about 20% of controls glycosylated α-DG were detected. Extracellular matrix and dystrophin-associated protein studies To determine whether modifications of α-DG glycosylation may influence the expression of extracellular matrix proteins, laminin α2, perlecan, and collagen IV were studied in patients’ muscle. No significant differences were observed in patient compared with control muscle (Fig. 1). Moreover, to investigate the dystrophin-associated complex, dystrophin and α-sarcoglycan were also studied; their expression in LGMD2I muscle biopsies proved normal compared to controls (Fig. 1). Muscle pathology According to the severity of the pathological pictures (regeneration, degeneration, and fibrofatty replacement) muscle biopsies were graded as mild, moderate, severe, and active muscular dystrophy (Table 2).
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Fig.
1 α-Dystroglycan immunofluorescence in LGMD2I patients’ muscle biopsies. a Control, b patient number 888, c patient number 5342, and d patient number 1475 muscle biopsy stained with the IIH6 antibody directed against a glycosylated epitope of α-dystroglycan. αDystroglycan was normal in the control and completely negative in patient number 888. Muscle biopsy of patient number 5342 shows a marked reduction of immunostaining but a few scattered fibers are αdystroglycan-positive in a mosaic-like pattern. α-Dystroglycan of patient number 1475 is mildly reduced. e–j Muscle biopsy of patient number 888 stained with antibodies directed against various membrane and extracellular matrix proteins. All proteins showed normal reaction in the patient’s muscle biopsy
To study muscle regeneration, neonatal myosin was immunostained in patients and controls (Table 2). Neonatal myosin-positive muscle fibers were observed in six patients’ biopsies, ranging from
