Journal of Neuro-Ophthalmology, March 2004, Volume 24, Issue 1

Continuous Remodeling of Adult Extraocular Muscles in Oculopharyngeal Muscular Dystrophy

Oculopharyngeal muscular mystrophy (OPMD) is an inherited disorder caused by mutations of the polyadenylate binding protein nuclear 1 (PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormalities, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous remodeling of normal adult extraocular muscles (EOMs). Unlike the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Continuous myofiber remodeling most likely requires upregulation of genes involved in cell cycling, and in protein degradation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenylation of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood.

VIEWPOINT Continuous Remodeling of Adult Extraocular Muscles as an Explanation for Selective Craniofacial Vulnerability in Oculopharyngeal Muscular Dystrophy Jonathan D. Wirtschafter, MD, Deborah A. Ferrington, PhD, and Linda K. McLoon, PhD Abstract: Oculopharyngeal muscular mystrophy ( OPMD) is an inherited disorder caused by mutations of the polyade-nylate binding protein nuclear 1 ( PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormali­ties, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous re­modeling of normal adult extraocular muscles ( EOMs). Un­like the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Con­tinuous myofiber remodeling most likely requires upregu-lation of genes involved in cell cycling, and in protein deg­radation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenyla-tion of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood. ( JNeuro- Ophthalmol 2004; 24: 62- 67) This paper presents a hypothesis regarding the predilec­tion of oculopharyngeal muscular dystrophy ( OPMD; Online Mendelian Inheritance in Man database no. 164300) Departments of Ophthalmology ( JDW, DAF, LKM), Neurology ( JDW), Neurosurgery ( JDW), and Neuroscience ( LKM), University of Minnesota Medical School, Minneapolis, Minnesota. Address correspondence to: Linda K. McLoon, PhD, Departments of Ophthalmology and Neuroscience, University of Minnesota, Room 374 LRB, 2001 6th Street SE, Minneapolis, MN 55455. Supported by grants EY11375 and EY13979 from the National Eye Institute, the Minnesota Lions and Lionesses, and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc. ( 1) to involve the craniofacial muscles, a distinct allotype that includes the extraocular muscles ( EOMs) ( 2,3). The craniofacial muscles have distinctive embryology ( 4); the unusual properties of the EOMs may predispose or protect them in neurogenic and myogenic diseases ( 5). The EOMs themselves are a heterogeneous subgroup within mamma­lian craniofacial muscles with distinct and unusual molecu­lar properties. We regard EOMs and certain craniofacial muscles as conceptually interchangeable, recognizing that each specific muscle may exhibit unique characteristics at the whole muscle and molecular levels. DIFFERENCES BETWEEN CRANIOFACIAL AND OTHER SKELETAL MUSCLES While the EOMs and craniofacial muscles are known to be quite similar to each other, they are distinctly different when compared with other skeletal muscles. Craniofacial muscles have more complex types of myofibers and smaller motor units than other skeletal muscles. The myosin heavy chain expression patterns of EOMs differ from those of other skeletal muscles ( 6). Adult EOMs express a variety of molecules, such as muscle myogenic and growth factors ( 7,8), which are normally downregulated in mature skeletal muscles ( 2, 9). Additionally, unlike adult skeletal muscles, EOMs undergo continuous addition of myonuclei ( 10- 12). We have not yet made a thorough study of the other cranio­facial muscles for evidence of continuous myonuclear ad­dition, but our preliminary data suggest that this process occurs in the laryngeal muscles as well. Our hypothesis for the selective involvement of cra­niofacial muscles in OPMD follows from our studies dem­onstrating that normal, uninjured, adult, mammalian EOMs undergo continuous addition and loss of myonuclei ( 10- 12). Thus, EOM myofibers cannot be considered postmi­totic cells in the usual sense. Previously, all adult skeletal muscles were considered to be postmitotic and did not un­dergo myonuclear addition unless they were injured or dis­eased. The evidence for continuous myofiber remodeling was visualized by the incorporation of bromodeoxyuridine 62 J Neuro- Ophthalmol, Vol. 24, No. 1, 2004 Viewpoint JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 into EOM satellite cells after intraperitoneal injection, fol­lowed by the incorporation of these labeled nuclei into existing myofibers ( 12). Subsequent studies used immuno-histologic markers of myogenic activity and cell cycling to extend our observations to monkeys and humans, and they have led us to believe that continuous myonuclear addition in EOMs may be a universal phenomenon in mammals ( 10). In addition, we have recently found evidence of myonuclear apoptosis, which is consistent with the concept of EOM myofiber remodeling. In these studies, individual myo­nuclear apoptosis and apparent satellite cell apoptosis were observed in adult rabbits, as visualized by positive terminal deoxynucleotidyltransferase- mediated nick end labeling ( TUNEL). Analysis of serial sections demonstrated that there was a distinct segmental localization of the activated caspase- 3 immunostaining within the cytoplasm of indi­vidual myofibers ( 13). Because the EOMs maintain a stable ratio of nuclear/ cytoplasmic volume, and a stable volume throughout adult life, a homeostatic process of myonuclear addition and loss would be required. Our experiments sup­port the model of EOM myonuclear homeostasis, which includes the random loss and addition of individual myonu-clei within existing multinucleated myofibers. Other inves­tigators have found evidence of apoptosis in 2.2% of normal human EOM myofibers ( 14). Their findings in normal hu­man EOMs support our observations. In summary, our studies have led us to conclude that EOM myofiber remod­eling occurs as a result of the insertion of replacement myo­cytes into mature EOM, concurrent with an ongoing process of localized nuclear apoptosis and cytoplasmic re­modeling affecting the myonuclear domains of the replace­ment myonuclei. Features of Oculopharyngeal Muscular Dystrophy OPMD ( 1) is an autosomal dominant disorder caused by a mutation in a single gene ( 15). It most frequently pre­sents in the fifth or sixth decade of life and causes ptosis, chronic progressive external ophthalmoplegia, dysphagia, and weakness of the limb- girdle muscles. Weakness and wasting of the face, neck, and distal limb muscles may also occur. OPMD can be fatal because of the selective involve­ment of the pharyngeal muscles. The molecular basis for OPMD is guanine- cytosine-guanine ( GCG) trinucleotide/ tandem repeat expansions in exon 1 of the polyadenylate binding protein, nuclear 1, PABPN1 ( also termedPABPZ) gene. ( 1) This mutation has arisen independently in many populations and has a world­wide distribution. The abnormal gene is located on chromo­some 14ql 1.2- ql3. A GCG repeat at the 5' end of the exon encoding the N terminus of the protein is expanded from the normal number of 6 to a range between 8 and 13 repeats. The gene expansion of this trinucleotide repeat causes ex­pansion of the normal 10- alanine stretch to 12- 17 alanines in the resultant PABPN1. Homozygous patients with auto­somal dominant OPMD ( GCG) 8- 13 have the onset of clini­cal manifestations at least a decade earlier than do heterozy-gotes, and have approximately twice as many intranuclear filament inclusions ( 9.4%) on electron microscopic exami­nation of muscle biopsies ( 16). The ( GCG) 7 allele is present in about 2% of the population. For ( GCG) 7, homozygotes have an autosomal recessive form of OPMD. There is no exact correlation between the number of repeats and the phenotypic expression of the disease ( 17). Polymorphisms affecting the PABPN1 gene have also been described. These may be clinically neutral or affect the severity of the disease ( 18). PABPN1 is a nuclear protein of 33 kDa whose func­tion is to stimulate the progressive addition of poly( A) to the 3' tail of newly transcribed mRNA. PABPN1 is a con­served protein in eukaryotic cells and has been implicated in the regulation of mRNA stability and translation critical for mRNA processing ( 19- 24). PABPN1 is largely con­fined within the nucleus ( 25). However, perhaps important in the pathogenesis of OPMD is the fact that mammalian cells display a cell cycle- dependent cytoplasmic polyade-nylation, suggesting that translational control by polyade-nylation might be a general feature of mitosis ( 26,27). OPMD is characterized by the accumulation of mutant PABPN1 ( mPABPNl) oligomers that form unique intra­nuclear tubulofilamentous inclusions with an outer diam­eter of 8.5 nm in skeletal muscle fibers ( 28). The conditions favoring aggregation of mPABNl have been partially de­fined ( 29). The Prior Hypothesis Based on the properties of mPABPNl, several toxic gain- of- function mechanisms have been previously pro­posed for this protein's role in OPMD ( 30) ( Fig. 1). The cumulative effect of these mechanisms is to create a " nuclear trap," sequestering proteins needed by the cyto­plasm. Oligomers of mPABPNl- alal7 may accumulate and form aggregates or insoluble fibrils that are resistant to deg­radation by the ubiquitin- proteasome pathway and thus be­come cytotoxic ( 28,30,31). Inclusions of mPABPNl may interfere with mRNA traffic through the nuclear pores by blocking nucleocytoplasmic transport. mPABPNl may se­quester specific proteins, such as heterogeneous nuclear ri-bonucleoproteins, and ski- interacting proteins that control processes essential for myogenesis and myocyte survi­val ( 32- 34). Several unresolved questions about the pathogenesis of OPMD have been posed: ( 1) Is the nuclear localization of the OPMD inclusions required for disease progression? ( 2) Does the mRNA binding capacity of the mPABNl trap 63 JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Wirtschafter et al. mPABPNl Pathogenic " gain of function"? Pathogenic " loss of function"? Insoluble protein inclusions with ? sequestration of: • mRNA • hnKNP Al • linRNP A/ B Disabled mRNA Disabled polyadenylation Normal and disabled mRNA plus myogenic proteins that may be quantitatively deficient, trapped, or defectively exported from nucleus Interference with transcription of extraocular and craniofacial muscle specific and/ or other myogenic genes Disabled cell cycling and differentiation O • < o v> 3 Fig. 1. Proposed molecular and cellular level pathogenic mechanisms of oculopharyngeal muscular dystrophy ( OPMD). Pathogenic ( toxic) gain- of- function is represented by insoluble nuclear inclusions of aggregated mutant pro­teins sequestering mRNA and heterogeneous nuclear ribo-nucleoproteins. Pathogenic loss- of- function is represented by the presence of disabled mRNA and polyadenylation. Disabled cell cycling and differentiation may result from gain- of- function or loss- of- function effects. Modified from Fan and Rouleau ( 30). mRNA and interfere with its export from the nucleus? ( 3) Why do mutations in the ubiquitous protein PABNl induce intranuclear inclusions mainly in muscle cells? ( 4) Why is OPMD an adult- onset disorder? To this list of important unresolved questions, we would add a fifth: Why are EOMs selectively targeted for presentation of the disease phenotype? hypothesis relies on the cumulative pathogenic loss- of-function of the mRNA, cell cycling, and differentiation re­quired to sustain the processes of continuous remodeling of the muscles most affected in OPMD. In proposing this model, we recognize the possibility of multiple pathogenic mechanisms, any of which could be related to continuous myofiber remodeling, and that loss- of- function and gain-of- function pathogenic mechanisms may be additive rather than mutually exclusive. Moreover, mPABPNl and disabled mRNA may also react with muscle- specific proteins that are unique to EOMs or uniquely upregulated in adult EOMs, thus providing an explanation for the selectivity of this disease to EOMs. Gene array studies show a number of genes that are uniquely upregulated or downregulated in EOMs when compared with leg muscles ( 2,36). We propose that non-craniofacial skeletal muscles are less affected in OPMD be­cause if they remodel, it is at a low rate, mostly as a result of exercise, injury, toxic insult, or other disease processes. This hypothesis offers an explanation for the selective in­volvement of the craniofacial muscles and the adult- onset of symptoms, and is compatible with the observed histopa-thologyofOPMD. Rejected Hypotheses We rejected two other models based on continuous myofiber remodeling to explain the clinical pattern of OPMD because of lack of evidentiary support. The first rejected hypothesis was somatic mosaicism, in which the repeated division of the muscle satellite cells could result in an increasing number of GCG repeats. Somatic mosa­icism is defined as the presence of a different number of trinucleotide repeats in different cells or different tissues of Our Hypothesis The underlying theme of our hypothesis is that ongo­ing remodeling of the EOMs and other craniofacial muscle myofibers is associated with cell signaling or biochemical processes that are relatively upregulated in the affected muscles as compared with other uninjured and healthy adult skeletal muscles ( 2). This upregulation causes the affected muscles to be selectively vulnerable to the effects of the mutant gene ( Fig. 2). We hypothesize that, in OPMD, the ongoing production of mPABPNl in muscles undergoing continuous remodeling results in the generation of disabled mRNA, which causes deficient polyadenylation that cumu­latively fails to support the continuous remodeling process. Defective polyadenylation may interfere with the cell cy­cling and differentiation required for continuous EOM remodeling ( 35). Over time, deficiencies in this process lead to myonuclear loss and, finally, to myofiber loss. Our Other Skeletal Muscles Extraocular and Craniofacial Muscles Post- milotic myofibers • Maintenance levels of mRNA and protein synthesis and degradation • Lesser susceptibility to failure • No cell cycling 1 Continuously remodeling myol'ibers- • Upregulation of mRNA • High rate vi protein synthesis and degradation • Increased susceptibility lo failure of synthesis and proteolysis • Low rate of cell cycling T OPMD disables cell cycling and differentiation X Milder pathology and symptoms in myofibers of other skeletal muscles Cumulative death of ocular and pharyngeal myofibers with clinical weakness by fifth decade Fig. 2. Our hypothesis of continuous remodeling of adult extraocular muscles as an explanation for selective cranio­facial vulnerability in OPMD. The hypothesis relies on the cumulative pathogenic loss- of- function of the disabled mRNA, and polyadenylation on the processes of cell cycling and differentiation required to sustain the continuous re­modeling of the muscles most affected in OPMD. 64 © 2004 Lippincott Williams & Wilkins Viewpoint JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 an individual patient. It occurs frequently in some " long" dynamic trinucleotide repeat disorders, such as X- linked bulbospinal muscular atrophy ( Kennedy disease), but not in other trinucleotide repeat disorders, including Huntington's disease ( HD) ( 37,38). Dynamic instability of the number of repeats has been studied in the HD mutant mouse, where the normal number of cytosine- adenine- guanine repeats is 9 to 35, and where the pathogenic number of repeats may be over 200 ( 39). However, the presence of a varying number of repeats in pathologic tissue obtained from different re­gions of a single affected human HD brain could be the result, rather than the cause, of the pathology. Somatic mo­saicism has not been found in OPMD, which is considered to be a " short" trinucleotide repeat disorder ( 17). A second rejected hypothesis was that of a toxic gain-of- function based on the cumulative retention of non-degraded mPABPNl in the myofibers following each cycle of myofiber remodeling. OPMD intranuclear inclusions have been proposed as " poly( A) RNA death traps" that in­terfere with RNA export and thus cause muscle cell death ( 34). It is possible that toxic aggregates of self- associated complexes of beta- pleated- sheet complexes of the mutant protein mPABPNl- alal7 accumulate because they are not readily degraded by proteasomes ( 29,40). Such accumula­tions have been described as possibly pathogenic in a num­ber of neurologic disorders, including HD and spinocer­ebellar ataxia ( 41,42). However, histopathologic studies do not show a clear correlation between aggregates of mPABPNl retained in the nucleus and OPMD muscle pa­thology ( 43). Further Considerations The understanding of the pathogenesis and clinical presentation of genetic disorders is not always immediately evident after the identification of the mutant genes and their protein products. Structural changes that initiate the accu­mulation of mutant protein detected by microscopy are of­ten identified earlier than pathogenic biochemical mecha­nisms. Such protein aggregates have been assumed to ex­plain the selective targeting or sparing of specific tissues. In both HD and OPMD, the presence of abnormal accumula­tions was initially interpreted as supporting the concept that such accumulations cause a pathogenic, toxic gain-of- function. Currently, HD pathogenesis is being reevaluated and loss- of- function mechanisms are being proposed ( 41,42, 44- 46). Toxic gain- of- function and loss- of- function patho­genic mechanisms may ultimately prove to be complimen­tary even though they may be initially presented as mutu­ally exclusive. Intranuclear inclusions notwithstanding, OPMD lacks structurally identifiable abnormalities, such as aggregates of misfolded undegraded proteins, that are quantitatively correlated with the expression of the dis­ease at the organismal level ( 43). This finding tends to shift the balance of competing hypotheses toward loss- of-function hypotheses. Thus, it is reasonable to propose that mPABNl may fail to support the mRNA production required to maintain continuous EOM myofiber remodeling. Patients with OPMD have one normal copy and one mutant copy of the PABPN1 gene and therefore produce normal, wild- type PABPN1 and mutant PABPN1. The major known function of PABPN1 is to influence the production of normal mRNA ( 18,20,24). The continuous EOM remodeling process is re­flected at the RNA level in the expression profiles of the EOMs as compared with skeletal muscle from the leg. Genes related to muscle growth, development, and regen­eration are upregulated in EOMs ( 2). The production of mRNA, some proportion of which consists of disabled mRNA, could lead to cumulative loss of affected myofi­bers. Also, because polyadenylation is important in the regulation of the cell cycle, abnormalities in this process could interfere with the process of EOM myonuclear addi­tion and integration in the affected muscles. Failure of nor­mal withdrawal from the cell cycle and impaired differen­tiation have been shown to be pathogenic factors in myo­tonic dystrophy ( 47). mPABPNl may also react with unique EOM and cra­niofacial muscle- specific proteins that are not expressed in other skeletal muscles. As noted, gene array studies show that there are a number of genes uniquely upregulated or downregulated in EOM when compared with leg muscles. It is important to note that gene array data from whole EOMs include a mixture of activated muscle satellite cells and myoblasts, in addition to resident connective tis­sues, nerves, neuromuscular junctions, and blood vessels ( 2). Therefore, these reports reflect contributions from a mixture of tissues and make interpretation of the results more ambiguous. Our hypothesis bridges the gap between a specific ge­netic defect partially understood at the molecular and cellular level ( Fig. 1), and the clinical presentation at the organismal level ( Fig. 2), where there is presently no under­standing as to the selective susceptibility of the EOMs and certain craniopharyngeal muscles. The greatest problem with our hypothesis is its failure to explain the low- grade involvement of the proximal limb muscles if it is assumed that normal appendicular skeletal muscles do not undergo continuous myonuclear replacement. We speculate that proximal limb muscle may undergo non- exercise- induced myonuclear replacement at a undetectable level or at spe­cific regions of the muscles not yet studied, such as the tendonous insertions. We might also invoke some exercise/ injury- induced myonuclear addition to explain the 65 JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Wirtschafter et al. lesser involvement of the proximal appendicular muscles compared with greater abnormalities of the craniofacial muscles. Further studies of normal adult mammalian EOMs, craniofacial muscles, and noncraniofacial skeletal muscles will be needed to determine the relative concentra­tion of PABPNl- specific mRNA, the rate of mRNA turn­over, and the ratios of mPABPNl and wild- type PABPN1 present in the sarcoplasm compared to other proteins such as cyclophylin, which has been demonstrated to be stable during aging. It would also be useful to compare the activity of the major pathways for protein degradation, such as the ubiquitin- proteasome pathway, in skeletal muscles from various regions of the body. One would predict higher en­dogenous activity in muscles with higher protein turnover. It has been speculated that the accumulation of mPABPNl-alal7 in older individuals explains the delayed clinical on­set, given that older cells have a diminished capability to induce the production of heat shock proteins ( 31). 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