Journal of Clinical Neuroscience (2005) 12(8), 927–929 0967-5868/$ - see front matter ª 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2005.04.005
Laboratory study
Parkin expression in human skeletal muscle P. Serdaroglu MD, H. Tasli BS, H. Hanagasi MD, M. Emre MD Department of Neurology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
Summary Parkin is known to be present in human neurons and peripheral nerves. Using an antibody against parkin protein we have now demonstrated that parkin is also expressed in the sarcoplasm and sarcolemmal region of human skeletal muscle fibres. We have also found different age-related patterns of expression with increase in intensity and organization of distribution at older ages. These findings suggest a change in the functional role of parkin in skeletal muscle with ageing and may contribute to understanding the mechanisms of muscle aging. ª 2005 Elsevier Ltd. All rights reserved. Keywords: parkin, muscle, ageing
INTRODUCTION Parkin is a 52kDa, RING-finger-containing protein which has a high degree of homology with ubiquitin at its N- terminal.1–4 The C- terminal consists of two ‘really interesting new gene’ (RING) finger motifs (RING1, RING2), separated by an inbetween-RING(IBR) domain.4,6,7 It is encoded by the parkin gene, which is mapped to 6q25.2-27.4,5 Mutations in this gene have been reported to cause autosomal recessive Parkinson’s disease (AR-PD; PARK2).4,6 Like many RING finger proteins, parkin possesses Ub-ligase activity and acts as an E3 Ub-ligase on a substrate specific basis, executing its function through its RING1-IBR-RING2 domain, in concert with Ub-activating (E1) and Ub-conjugating (E2) enzymes.4,6,7 Therefore, parkin has a role in the ubiquitination process, through which a large variety of cellular proteins that are damaged or misfolded are exposed to proteasomal degradation to maintain cell functioning and survival.6,7,8 Disease causing missense mutations abolish this ubiquitylation function of parkin.4,6 A wide variety of proteins, including the brain specific glycosylated form of a-synuclein (a-Sp22), are known to interact with parkin.9 Parkin has been shown to be expressed at different levels in different tissues. It is widely expressed in central nervous system neurons of humans in the cytosol, endoplasmic reticulum, Golgi complex, synaptic vesicles, postsynaptic densities, nuclear matrix and the outer mitochondrial membrane.7 Studies on extracerebral expression and functions of parkin in parkin mutant animals showed that parkin and splice variants of parkin are present in mouse lung, liver, kidney and testis as well as in bovine peripheral nerve.10,11 In humans, peripheral nerve is the only extra-cerebral tissue in which parkin has been shown to be transcribed.12 Studies in parkin mutant drosophila showed that these flies exhibited age dependent expression of parkin and that they showed flight and climbing deficits due to a drooped wing phenotype.13,14 The constantly vibrating indirect flight muscles (IFM) of these mutant flies showed progressive degeneration of myofibrils and mitochondria.13,14 The degeneration in these selected and more energy requiring muscles in parkin mutant drosophila sup-
ported the previous suggestion of a mitochondria-related oxidative stress reaction in the absence of parkin.14 The presence and age-dependent expression of parkin in drosophila FIM muscles, the link between deficiency of parkin and mitochondrial pathology and the occurrence of mitochondrial changes in elderly muscle of humans, together with the fact that human skeletal muscle is a tissue which can develop some myopathies exclusively in late life, prompted us to search for the presence of parkin in human skeletal muscle. Here we report the results of a preliminary study on the immunolocalization of parkin in normal muscle biopsies from individuals of different ages. MATERIALS AND METHODS Muscle biopsy specimens from 12 individuals ranging from 3.5 months to 65 years of age, which had been freshly frozen previously and interpreted as normal, were used in the study. Five of the cases were over 45 years (range 46–65) (group I), while 7 were below 10 years of age (range 3.5–7 years) (group II). Eight mm cryostat sections were dried in air and were incubated with undiluted normal rabbit serum, then with 1:150 diluted antirabbit primary antibody raised against a peptide corresponding to sequence number 305–323 of the human parkin molecule (Chemicon) for 1 hour each at room temperature. After being repeatedly rinsed in PBS the sections were incubated with RITC labeled goat anti-rabbit secondary antibody. Control staining was performed by omitting the primary antibody in each staining session. Double labeling studies were performed with anti-parkin and anti-dystrophin (DYS-2), anti-a-sarcoglycan (50-DAG) and anti-merosin antibodies separately. The procedure for the double labeling studies was the same as above, except that mouse monoclonal DYS-2, anti-50-DAG or anti-merosin antibodies were added to the primary antibody and that FITC labeled goat anti-mouse antibody was added to the secondary antibody steps. Visualization was made on Olympus BH2 fluorescent microscope and 35mm slides were taken with Kodak 400ASA film. Sections were evaluated for the pattern of staining in the sarcolemma, sarcoplasm, and myonuclei.
RESULTS Received 17 January 2005 Accepted 11 April 2005 Correspondence to: P. Serdaroglu MD, Department of Neurology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey. Tel.: +90 212 414 2000Ext32571; Fax: +90 212 533 4393; E-mail:
[email protected]
All muscles in both groups showed immunoreactivity with an antiparkin antibody raised against a peptide corresponding to sequence number 305–323 of the human parkin molecule. This peptide recognizes a doublet at 44 and 52 kDa on Western blots from rat brain, and as it is located in the midportion of the parkin 927
928 Serdaroglu et al.
Fig. 1 Parkin (red) and merosin (green) double immunostaining in a 3.5 year old child (b, c) and 53 year old elderly subject (e, f). Immunostaining of parkin and merosin on the same section shows that parkin staining is punctate and is limited to sarcoplasm in children (c) as compared to negative controls (a). Merosin immunostaining on the same section (b). In the elderly subject the parkin immunostaining shows more organized pattern in sarcoplasm and also at the sarcolemmal area (e). (d) is negative control in this group. (a, b and c X200, d, e and f X400).
molecule it does not show cross immunoreactivity with ubiquitin.15 Negative control sections did not show any staining. Group I showed a clear pattern of staining within the sarcoplasm and also at the periphery of muscle fibers when compared with control sections (Fig. 1 d, e). The staining within the sarco-
plasm had a roughly reticular pattern which formed regular arrays. In longitudinal sections these were seen to be arranged in linear configurations (Fig. 2a). In this group staining at the periphery of muscle fibres was slightly stronger than in the sarcoplasm and co-localized with dystrophin, a-sarcoglycan and merosin (Fig. 2b). The samples from children below 10 years of age (group II) showed less prominent sarcoplasmic staining. The pattern of this staining was punctate and was more irregular compared to samples from the older individuals. No sarcolemmal staining was observed in children (Fig. 1a, b). DISCUSSION
Fig. 2 Parkin (a) and parkin-merosin (b) double immunolabelling patterns in a 58 year old subject. Parkin staining is red and and merosin staining is green. Sarcolemmal staining for parkin is internal to merosin staining in the sarcolemmal/basal lamina area (X1000).
Journal of Clinical Neuroscience (2005) 12(8), 927–929
We demonstrated, for the first time, the presence of parkin protein, as stained with an anti-parkin antibody in human skeletal muscle fibres at all ages. As this antibody recognizes a doublet at 44 and 52 kDa on Western blots, it is very likely that the protein it recognized in muscle is also parkin, or an isoform of parkin which includes the 305–323 sequence.11,15 This immunoreactivity showed age-dependent differences in the intensity and patterns of immunostaining. Thus, children had a weaker and more irregular punctate pattern of immunostaining in the sarcoplasm than adults who had a more intense expression and a reticular, longitudinal array ª 2005 Elsevier Ltd. All rights reserved.
Parkin expression in muscle 929
pattern of staining in the sarcoplasm and more so at the sarcolemma. The ontogenesis of parkin expression in human skeletal muscle is not known. A parkin expression study in the mouse demonstrated that parkin appeared in some tissues at embryonic days 10–12 and showed an increase during development, leading to the conclusion that parkin expression is maturationally regulated.9 In another study in drosophila, parkin first appeared at the late larval stage and was present during pupal and adult stages, which suggested that parkin is required later in development from the pupal stage onwards.14 In accordance with this finding, the same study showed that mutation in the parkin gene did not result in abnormality at the larval stage but that the flight muscles did degenerate after eclosion, further confirming the age-dependent requirement for parkin in drosophila.14 The stage of development when parkin first appears in human tissues and its significance in muscle ubiquitination is still not known. Furthermore, no myopathy related to parkin deficiency has yet been described. However, in the light of the observations in drosophila, the present findings indicate that a parkin molecule, or at least an isoform corresponding to sequence number 305–323 is expressed in human muscle and that its level of expression and distribution in the muscle fibre is age-dependent. The role of parkin in human skeletal muscle is yet to be understood. However, regardless of the pattern, the diffuse distribution of parkin in the sarcoplasm suggests that it may be essential for certain intracellular processes. It has previously been shown that the absence of parkin in mutant drosophila causes disruption in myofibril integrity and swelling of mitochondria in the IFMs, demonstrating that parkin is important in the maintenance of muscle cells.13,14 In these reports it was shown that this degeneration was via mitochondria-related oxidative stress reaction leading to apoptotic cell death. As the muscle fibre is a highly energy dependent tissue it is exposed to the cumulative effects of oxidative stress with increasing age. Moreover, it is known that subclinical mitochondrial changes are present in normal elderly human skeletal muscles. It may be possible therefore that parkin acts as part of a protective system against lethal stresses within the muscle fibre. Thus, it can be hypothesized that the upregulation and rearrangement in the distribution of parkin in older age may be an attempt to compensate for the cumulative effects of age-related processes such as oxidative stress. It has been shown previously that parkin co-localises with actin filaments in cultured kidney cells and neurons which suggested that parkin was a cytoskeletal-associated protein.16 The diffuse immunolabelling pattern which we have found suggests that the protein is present within the cytosol and may be associated with the cytoskeleton and that the protein may either perform a general function or may more specifically play a part in maintaining the integrity of some subcellular structures, such as myofibrils. Another finding in this study was the immunolabelling of parkin at the periphery of skeletal muscle fibres where it co-localized with dystrophin, a-sarcoglycan and merosin. The precise localisation of parkin in the sarcolemmal region, and its relationship to the plasma membrane and cytoskeleton, requires further clarification as does the functional significance of this localisation. It is possible that there are different isoforms of parkin with different functions in human skeletal muscle fibres. This observation might be in accord with a similar finding in a study of parkin distribution in bovine peripheral nerve.11 In this study the authors reported that
ª 2005 Elsevier Ltd. All rights reserved.
parkin was present both in the axoplasm of myelinated fibres and in the cytoplasm as well as the outer membrane of Schwann cells and that the membrane-bound and cytosolic parkin fractions were different from each other. In conclusion, although this is a preliminary study using only one antibody, we have shown that a 305–323 peptide recognized by this anti-parkin antibody is expressed in human skeletal muscle from an early age and that the degree of expression and distribution of the protein change in an age-dependent fashion. These findings may help to understand the mechanisms of the ageing process in human muscle as well as some degenerative muscle diseases which are seen selectively in the elderly population. Further studies are required to clarify the subcellular distribution of parkin in muscle fibres and whether different isoforms of the protein exist in muscle.
ACKNOWLEDGEMENT We would like to thank Dr. F. Deymeer for her critical reading of the manuscript. Rezan Fahrioglu is thanked for her help with the photography.
REFERENCES 1. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998; 392: 605–608. 2. Imai Y, Takahashi R. How do parkin mutations result in neurodegeneration? Curr Opin Neurobiol 2004; 14: 384–389. 3. West AB, Lockhart PJ, O’Farell C, Farrer MJ. Identification of a novel gene linked to parkin via a bi-directional promoter. J Mol Biol 2003; 326: 11–19. 4. von Bohlen und Halbach O, Schober A, Krieglstein K. Genes, proteins, and neurotoxins involved in Parkinson’s disease. Prog Neurobiol 2004; 73: 151–177. 5. Cordato DJ, Chan DKY. Genetics and Parkinson’s disease. J Clin Neurosci 2004; 11: 119–123. 6. Shimura H, Schlossmacher MG, Hattori N, et al. Ubiquitination of a new form of a–synuclein by parkin from human brain: Implications for Parkinson’s disease. Science 2001; 293: 263–269. 7. Bonifati V, Oostra BA, Heutink P. Unraveling the pathogenesis of Parkinson’s disease – the contribution of monogenic forms. Cel Mol Life Sci 2004; 61: 1729–1750. 8. Chung KKK, Dawson VL, Dawson TM. The role of the ubiquitin-proteasomal pathway in Parkinson’s disease and other neurodegenerative disorders. TINS 2001; 24: S7–S14. 9. Huynh DP, Dy M, Nguyen D, Kiehl T-R, Pulst SM. Differential expression and tissue distribution of parkin isoforms during mouse development. Dev Brain Res 2001; 130: 173–181. 10. Khn K, Zhu X-R, Lbbert H, Stichel CC. Parkin expression in the developing mouse. Dev Brain Res 2004; 149: 131–142. 11. Hase A, Yamada H, Arai K, Arai K, Sunada Y, Shimizu T, Matsumura K. Characterization of parkin in bovine peripheral nerve. Brain Res 2002; 930: 143–149. 12. Abbruzzese G, Piguillo S, Schenone A, et al. Does parkin play a role in the peripheral nervous system? A family report. Mov Disord 2004; 19: 978–981. 13. Greene JC, Whitworth AJ, Kuo I, Andrews LA, Feany MB, Pallanck LJ, et al. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc Natl Acad Sci 2003; 100: 4078–4083. 14. Pesah Y, Pham T, Burgess H, et al. Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stres. Development 2004; 131: 2183–2194. 15. Horowitz JM, Myers J, Stachowiak MK, Torres G. Identification and distribution of parkin in rat brain. NeuroReport 1999; 10: 3393–3397. 16. Huynh DP, Scoles DR, Ho TH, Del Bigio MR, Pulst S-M. Parkin is associated with actin filaments in neuronal and nonneural cells. Ann Neurol 2000; 48: 737–744.
Journal of Clinical Neuroscience (2005) 12(8), 927–929
