Valosin containing protein mutations in fronto-temporal lobar degeneration: Clinical presentation, pathology and pathogenesis

Abstract

Inclusion body myopathy (IBM) associated with paget’s disease of the bone (PDB) and fronto-temporal dementia (FTD) or IBMPFD, is a rare multisystem degenerative disorder due to mutations in valosin containing protein (VCP). VCP is a ubiquitously expressed protein that facilitates the degradation of proteins via the ubiquitin proteasome and autophagy pathways. Affected brain and muscle tissue in IBMPFD have ubiquitinated and TAR DNA binding protein-43 (TDP-43) inclusions. In skeletal muscle, this pathology is consistent with IBM. While in the CNS, IBMPFD is a frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) subtype. Recent studies suggest that IBMPFD mutations in VCP disrupt its function in protein degradation. This review will explore the clinical phenotype and pathology of IBMPFD with an emphasis on central nervous system degeneration. In addition, we will discuss the current understanding regarding VCP’s function in terminally differentiated tissue and how disease associated mutations result in both myo- and neurodegeneration.

Introduction

IBMPFD is an autosomal dominantly inherited disorder with variable penetrance of three predominant phenotypic features (1). 90% of patients develop disabling weakness with a mean onset of 45 years of age. At this same age 51% of affected patients develop PDB. Finally 32% of patients develop FTD manifested by prominent language and behavior dysfunction with a mean onset of 54 years old (2). Other phenotypic features have been reported as well, including, dilated cardiomyopathy, hepatic steatosis, cataracts and sensory-motor axonal neuropathy (3–5). Patients do not need to express all three phenotypic components but can express only one or two in isolation. For example, while 90% of IBMPFD patients have muscle weakness, it is an isolated symptom in ~30% of patients. In contrast, 32% of patients clinically have FTD and 3% have FTD as an isolated phenotype (6). This heterogeneity is seen with the same mutation and even within isolated families bearing the same mutation. One study identified an arginine to histidine missense mutation at residue 159 in VCP (VCP-R159H) in three unrelated families (7). 6/6 affected patients in one R159H family exhibited FTD without associated PDB or IBM. Whereas in another family, only one of 5 affected family members had FTD and 5/5 had PDB and weakness. This was not due to variations in family members ages since family 1’s (all FTD) ages ranged from 67 to 86 and family 3’s (PDB and IBM) ages ranged from 60–76 or clinical reporting since all patients had neuropsychological assessment associated with a clinical evaluation. Because of this heterogeneity, any evaluation of supposed FTD should ask about a family history of PDB and muscular weakness, specifically IBM.

Clinical Description

FTD

Patients with IBMPFD develop a typical FTD but can also have some atypical features. One of the initial descriptions of an IBMPFD patient’s dementia highlights this (8). The patient was 44 years old and had the gradual onset of unexplained language difficulties. Although his speech was fluent, he had evidence of dysnomia, comprehension difficulties and paraphasic errors on exam. His episodic memory was preserved having 3 out of 3 recall but due to his language difficulties scored a 10/30 on the Mini-Mental State Examination. Other measures of psychometric performance were also impaired (for example he named only 3 animals in 60s (elderly control normal is 18)). The diagnosis of semantic dementia, a subtype of FTD, was made. Over the course of eight months self care deteriorated and re-examination found that his aphasia had progressed to the inability to repeat or follow one step commands. He died 9 months later. Notably, he remained ambulatory and strong until becoming bed bound from inanition.

Few studies have carefully evaluated the clinical features of IBMPFD dementia in large cohorts. One large clinical study found that four of 19 IBMPFD patients from 10 families with nine mutations had severe disabling dementia (9). Of the remaining 15 without overt dementia, 33% had impaired judgment, attention and reasoning as assessed by neuropsychological tests. This suggests that the clinical symptoms of FTD may be under appreciated in IBMPFD patients. Another study of 16 affected individuals from two families noted FTD in a much higher frequency, 81% of mutation carriers (7). Notably, 4 of these patients had the appearance of early psychotic features and carried the diagnosis of paranoid schizophrenia as early as 35 years of age.

The comprehensive evaluation of the neuroradiographic features in IBMPFD is also poorly described for large cohorts. Most affected patients have varying degrees of widening of the frontal and temporal sulci with ventricular enlargement similar to the gross neuropathologic changes described below. One study evaluated serial MRI changes in one IBMPFD patient with an R93C gene mutation (10). In this patient, an MRI at the age of 70, two years after the first clinical signs of dementia showed frontal atrophy. A repeat MRI 8 months later demonstrated an increase in frontal atrophy with an expansion of the ex vacuo dilatation at the frontal aspects of the lateral ventricles. A third MRI at the age of 74 revealed further progression of these features. At this same age, positron emission tomography using 2-[18F] fluoro-2-deoxy-D-glucose (FDG-PET) showed bilateral regions of hypometabolism in the striatum, thalamus and the frontal and temporal cortex of this patient. The symmetric frontotemporal hypometabolism signature was similarly seen on FDG-PET imaging of another patient with an R159H gene mutation (7). This patient had clinical symptoms of FTD for 6 months at the time imaging was performed.

Muscle Weakness

The pattern of skeletal muscle weakness is also quite variable (11). Most patients develop disabling weakness in the 4^th decade of life. This weakness progresses insidiously and patients are typically wheelchair bound 10–15 years after the initial onset of weakness. Patient demise occurs from complications of respiratory weakness ~10 years later. The myopathy in IBMPFD is characteristically limb-girdle in nature but may also have distal and scapulo-peroneal patterns as well. One characteristic but not essential feature is scapular winging. Supportive lab tests and other studies include serum creatine kinase which is often normal but can be modestly elevated in ~20% of patients. Electromyography reveals an “irritable myopathy” with brief small polyphasic action potentials, fibrillations, and positive sharp waves. Regardless of these findings, the mainstay of diagnosis is muscle biopsy (discussed below). As expected the variable muscle weakness patterns have led to the misdiagnosis of these patients as limb girdle muscular dystrophy, fascioscapular humeral dystrophy, distal myopathies (i.e. Welander and Miyoshi) and ALS.

PDB

PDB is the most common age associated degenerative bone disease. It is caused by an imbalance between the activity of osteoblasts and osteoclasts. IBMPFD patients develop typical PDB which clinically manifests as pain and pathologic fractures (1). Clinically significant lesions are patchy and exist in isolated vertebrae, pelvis or long bones of the arm and leg. Radiographic images of pageotoid lesions show areas of lucency consistent with lytic bone lesions. PDB is diagnosed by bone scan, plain film radiographs of affected bone or serum LDH. Of the three predominant phenotypes in IBMPFD, PDB is the only one that is treatable with bisphosphonates being the mainstay of therapy. This makes the diagnosis of PDB one of the most important in any IBMPFD patient.

Clinical Summary

IBMPFD is a heterogenous disorder and patients need not manifest all three phenotypic features. Clinicians evaluating a patient with suspected FTD, either sporadic or familial, must ask about other symptoms such as weakness and bone pain. In addition, a careful family history should be taken not just for a history of FTD and dementia but also for a history of PDB, IBM and other disorders of progressive weakness. These challenges make IBMPFD an under diagnosed and likely under recognized clinical syndrome.

IBMPFD pathology

CNS

Prior to the description of IBMPFD as a clinical entity and the identification of mutations in VCP as the cause, many patients were miscategorized as frontotemporal dementia lacking distinctive histopathology. However since 2004, descriptions of genetically defined IBMPFD patient brains classify it as an FTLD-U subtype (subtype 4) (12, 13). The initial descriptions of the brain pathology from two patients with IBMPFD, prior to the identification of causal mutations in VCP, found evidence of atrophy with decreased cortical thickness and ventricular dilatation (8). Subsequent studies have evaluated CNS tissue from genetically defined IBMPFD patients. The largest of these studies evaluated 8 affected patients from 5 different families harboring three unique mutations in VCP (R155H, R155C and N387H) (13). Of these patients only 6 had clinically diagnosed FTD. These studies identified variable evidence of cerebral atrophy ranging from focal involvement of the frontal or medial temporal lobes to severe diffuse atrophy and ventriculomegaly. Subcortical nuclei, cerebellum and brainstem were unaffected. Consistent with their dementia, microscopy showed neuron loss and gliosis in affected brain regions most prominently in the upper cortical layers. No atrophy was apparent in the two patients that did not clinically manifest FTD. Immunohistopathology for β-amyloid and tau demonstrated a low density of senile plaques and neurofibrillary tangles in a small subset of patients but this did not meet neuropathologic criteria for Alzheimer’s disease or other defined tauopathy. Similarly α-synuclein immunostaining identified rare cortical lewy bodies in one patient. These findings are in sharp contrast to ubiquitin immunohistochemistry. Antibodies to ubiquitin immunostain prominent neuronal intranuclear inclusions (NIIs), dystrophic neurites and neuronal cytoplasmic inclusions in all patients regardless of dementia status. The marked abundance of NIIs, led to the designation of IBMPFD as a novel subtype of FTLD-U (subtype 4). Figure 1 shows ubiquitin immunostaining of FTLD-U subtypes 1–4. The ubiquitinated inclusions did not co-label with antibodies to tau, α-synuclein, neurofilament, β-amyloid, α-internexin or polyglutamine expansions. Notably, they did not co-label with VCP which was identified in rare NIIs. In contrast, UBIs prominently labeled with antibodies to TDP-43 (14). TDP-43 inclusions were found in the same brain regions as the ubiquitin pathology and were predominantly intranuclear. This was accompanied by a decrease in the normal nuclear staining of TDP-43 in many non-inclusion bearing neurons of affected cortex. This neuropathologic description is similar to that of two unrelated IBMPFD families with an R159H mutation in VCP (7). These studies describe IBMPFD neuropathology as an FTLD-U with prominent ubiquitin and TDP-43 positive NIIs. Further characterization of IBMPFD patient brains using differential solubility to separate aggregating proteins from non-aggregating proteins failed to see changes in VCP protein levels or solubility (13). In contrast TDP-43 as well as pathologic fragments and hyperphosphorylated forms of TDP-43 do become enriched in the urea insoluble fractions of IBMPFD CNS lysates (14). Interestingly, one paper that failed to detect ubiquitinated inclusions in three patients with an R155C gene mutation in VCP did find an increase in soluble high molecular weight ubiquitinated proteins suggesting that both soluble and insoluble ubiqutinated species accumulate in IBMPFD patient tissue (3).

Figure 1.

Frontotemporal lobar degeneration-U, subtypes 1–4. a–d Type 1 is characterized by long and tortuous dystrophic neurites (DNs) in lamiae II/III with relatively few neuronal cytoplasmic inclusions (NCIs) and no neuronal intranuclear inclusion (NII). b Type 2 has numerous NCIs, relatively few DNs, and no NII is present. c Type 3 has numerous NCIs and DNs and an occasional NII in lamina II. d Type 4 pathology in a case of FTD with VCP mutation is characterized by numerous NII and DN, but few NCI. TDP-43 immunohistochemistry. Scale bar 10 micrometers (a–d). [Reproduced with permission from American Journal of Pathology 207;171:227–240].

Muscle

Previous reports of muscle biopsies in IBMPFD patients found that only 39% of patients had been correctly diagnosed as IBM (6). While all patients had evidence of an active myopathy (i.e. variation in fiber size, increased endomysial connective tissue) most are classified as a non-specific myopathy. In fact our own study of 11 patients with genetically defined IBMPFD, found evidence of IBM pathology in only 8/11 patients (11). This may be due to the patchy nature of IBMPFD, technical issues regarding muscle selection for biopsy or that IBM pathology is not necessary for weakness. The skeletal muscle pathology in IBMPFD and other hereditary IBMs is distinctive. In addition to a myopathy, affected fibers have vacuoles rimmed by and containing proteinaceous debris (Figure 2A). Protein inclusions visualized by congo red staining can also be found within fibers. Although these inclusions likely contain multiple proteins the most commonly defined in IBMPFD are ubiquitin, p62/sequestosome, desmin, and TDP-43 (15–17) (Figure 2B and 2D). Ultrastructural analysis of IBMPFD patient muscle identifies tubulofilamentous inclusions, membranous debris and myofibrillar disorganization. Interestingly, many fibers had intranuclear inclusions. Degenerating myonuclei appeared to extrude 15–21nm tubular filaments into the sarcoplasm (18). While TDP-43 immunostaining is normally nuclear, TDP-43 inclusions were present in the sarcoplasm of normal, atrophic and vacuolated IBMPFD myofibers (17) (Figure 2D). In fibers containing TDP-43 sarcoplasmic inclusions, there was clearing of the normal TDP-43 from myonuclei. Most sarcoplasmic TDP-43 inclusions co-labeled with ubiquitin similar to that seen in IBMPFD brain tissue.

Figure 2.

Histochemical and immunohistochemical stains of IBMPFD patient muscle biopsies. (A) Congo red staining demonstrating fibers with vacuoles and basophilic debris. (B) Immunostaining with an anti-ubiquitin antibody (FK2) demonstrates large inclusions that are myonuclear and subsarcolemmal. (C) A vacuolated fiber with VCP inclusions. (D) TDP-43 immunostaining sarcoplasmic inclusions. Closed arrows highlight vacuoles and open arrows denote inclusions. [Portions are reproduced with permission from Neuromuscular Disorders. 19(5): 308-15].

VCP is normally found around and within myonuclei (1, 19). It is also seen beneath the sarcolemma and diffusely throughout the sarcoplasm. Rare VCP positive sarcoplasmic inclusions are identified in IBMPFD muscle tissue (1) (Figure 2C). VCP inclusions can co-localize with TDP-43 and ubiquitin but this is not a common feature (17). This pattern of pathology is similar to that seen in sporadic inclusion body myositis (sIBM) with the exception of muscle inflammation which occurs in sIBM. The solubility of VCP and the muscle specific intermediate filament, desmin, were unchanged in IBMPFD patient muscle (16). This was in contrast to TDP-43 protein levels which were increased and had evidence of an additional more slowly migrating TDP-43 isoform (17). This may be the “hyperphosphorylated” form that is seen in FTLD-U patients. .

Bone

In contrast to the descriptions of CNS and muscle pathology, there are limited descriptions of the pathology seen in the pagetoid lesions of IBMPFD patients. This is likely because the diagnosis of PDB is principally made by radiography. However in one family harboring an R155P mutation in VCP, 4 family members were affected with PDB and had a bone biopsy (6). The pagetoid lesions were typical for PDB with disruption of the normal pattern of cortical and cancellous bone, proliferation of fibrous connective tissue in the medullary spaces, hypertrophic trabeculi with mosaic patterns, and focal new bone formation. Ultrastructural analysis of IBMPFD patient osteoclasts identified intranuclear inclusions containing straight, tubular structures of 15nm diameter which are commonly found in sporadic PDB.

Pathologic Summary

Although disease mutations in VCP cause pathology in disparate tissues (i.e. brain, muscle and bone) there are clear common pathologic features. 1) progressive tissue degeneration; 2) accumulation of ubiquitinated protein species; 3) prominent nuclear pathology; and 4) TDP-43 inclusions. How these components relate to IBMPFD pathogenesis is only beginning to be unraveled. Regardless it suggests that the pathogenic mechanism in these three tissues may be similar in IBMPFD.

Valosin Containing Protein (VCP)

VCP function

IBMPFD is due to mutations in the AAA+ (ATPase associated with a variety of cellular activities) protein VCP (also termed p97 and cdc48) (1). As implied by its protein genre name (AAA+ protein), VCP is instrumental in coordinating multiple distinct cellular events. These include cell division, organelle biogenesis, nuclear envelope formation and protein degradation via the UPS (20). However it is not VCP alone which mediates these processes. Instead, VCP performs these functions by binding to specific cofactor/s that utilize VCP as the “motor” for each task (20). For example, VCP in association with the co-factors UFD1 and Npl4 facilitates the delivery of ubiquitinated substrates to the 20S proteasome . Whereas golgi biogenesis requires VCP’s association with p47. To date VCP has been found to associate with >50 different proteins. This extensive interactome allows VCP to participate in these diverse cellular processes (21).

Of VCP’s promiscuous functions, mediating protein degradation is the best studied. VCP can associate with polyubiquitinated proteins, 26S proteasome subunits, E3 ubiquitin ligases and deubiquitinating enzymes (20). Initial studies found that depletion of p97/VCP from cellular extracts or via RNAi silencing led to the accumulation of polyubiquitinated proteins (22). Subsequently, it has been demonstrated that the degradation of ubiquitinated cytosolic protein reporters is decreased under similar siRNA knockdown conditions (23). VCP also mediates the degradation of improperly folded proteins from the membrane and lumen of the endoplasmic reticulum or ERAD (24). Co-expression of the prototypical ERAD substrate, ΔF508-CFTR with dominant-negative p97/VCP or following RNAi silencing of VCP increases the amount of ubiquitinated ΔF508-CFTR protein in cells (23, 25). These cells also have an expansion of ER membrane which becomes swollen and vacuolated consistent with the accumulation of misfolded and undegraded proteins that are stuck in the ER lumen (25). Whether VCP mediates the degradation of all ERAD or ubiquitinated substrates is not known. However it seems more likely that VCP is responsible for the degradation of specific proteins or substrates. Several VCP specific degradation substrates have been proposed; all with varying degrees of evidence to support them. These include IκB, HIF1α, auroraB kinase and UNC-45b (26–29). For example, UNC-45b is a skeletal muscle specific myosin chaperone (29). Too much or too little UNC-45b in c. elegans leads to myofibrillar disorganization and non-motile worms. Janiesch and colleagues identified UNC-45B in a complex with CHN1, UFD2 and VCP (29). Subsequently it was confirmed that mammalian UNC-45B is degraded by the proteasome in a VCP dependent manner. Loss of VCP in worms led to the accumulation of ubiquitinated UNC-45 and a decrease in functioning myosin heavy chain B.

VCP also participates in degradation of protein aggregates. Prior to its identification as the causal protein in IBMPFD, VCP was identified as a polyglutamine interacting protein (30). Specifically, VCP was found to interact preferentially with expanded ataxin-3 in cells. This interaction was between the N-terminal half of VCP and the polyglutamine stretch. Subsequently VCP was identified as a component of huntingtin positive inclusions in Huntington’s disease, Lewy bodies in Parkinson’s disease and UBIs in amyotrophic lateral sclerosis (ALS) (30, 31). VCP is also essential for protein inclusion or aggresome formation (31). Aggresomes are actively generated, microtubule dependent, cellular structures containing protein aggregates and the molecular machinery necessary for their degradation (32). VCP co-localizes to aggresomes and siRNA knockdown or dominant negative VCP expression abrogates aggresome formation. Interestingly, VCP also associates with another aggresome essential protein, histone deacetylase 6 (HDAC6) (33). Although classified as a histone deacetylase, HDAC6 resides in the cytosol. HDAC6 associates with ubiquitinated proteins and facilitates their delivery to the aggresome (34).

IBMPFD pathogenesis

VCP is a type II AAA+ protein. It forms a homohexamer consisting of two tandemly encoded AAA+ ATPase domains (D1 and D2) that are stacked upon each other in a double ring-like structure (20). Each monomer has an N-domain, two AAA+ domains and a C-terminal region. It is speculated that the N-domain and C-terminal regions are necessary for substrate and co-factor binding. Of the two AAA+ ATPase domains, hydrolysis of ATP by the D2 domain is most essential for protein activity while ATP binding at the D1 domain is more essential for hexamer formation and protein stability (35). Movement at the interface between the N and D1 domains is greatest during ATP hydrolysis suggesting that this is the region of substrate and cofactor association/dissociation (36, 37). IBMPFD mutations in VCP reside at the N-terminal half of the protein within the N domain, linker 1 or D1 ATPase domain (Figure 3A). Mutations at residue 155 affect greater than half of IBMPFD patients. The crystal structure of VCP allows positioning of the mutant residues within the context of a VCP hexamer. Interestingly, all 13 IBMPFD mutated residues in VCP lie at the N-D1 domain interface (Figure 3B and 3C). IBMPFD mutations in VCP do not appear to disrupt gross hexamer formation as assessed by gel filtration and electron microscopy of mutant protein (38, 39). Surprisingly, IBMPFD mutations in VCP lead to an increase in basal ATPase activity in vitro (38). Whether this correlates with an increase in VCP functionality in vivo is not established.

Figure 3.

A) Linear diagram of VCP with the location of 19 identified missense mutations at 13 different residues (in red). B–C) Renderings of the crystallographic structure a VCP hexamer (each individual monomer is a unique color). B illustrates hexameric VCP with the positions of the D1 domain (top barrel) and D2 domain (bottom barrel) with the N and C domains protruding to the sides. The red oval denotes where all 13 mutant residues reside. C is a view from the top looking down of the D1 and N domains. The red residues are mutant residues at the interface between the N and D1 domains.

The initial studies evaluating IBMPFD mutant expression found that ubiquitinated proteins were increased in cell culture models similar to that in patient tissue. This accumulation of ubiquitinated proteins was comparable to that seen with proteasome inhibition or expression of a dominant negative VCP (25, 33, 39). Expression of IBMPFD mutations in transgenic mice under the control of a muscle specific or ubiquitously expressing chicken β-actin promoter recapitulates the features seen in IBMPFD patients with progressive weakness, prominent ubiquitinated inclusions, vacuolation and TDP-43 aggregates in skeletal muscle beginning at ~6 months of age (15, 40). Transgenic mice expressing one of two IBMPFD mutations, R155H or A232E under the control of a chicken β-actin promoter also developed evidence of pagetoid lesions and neuropathologic changes consistent with IBMPFD but at later ages than muscle weakness (13 and 14 months respectively) (41). Specifically, these mice developed cytosolically localized ubiquitin and TDP-43 positive inclusions. This was accompanied by clearing of TDP-43 from the nuclei of cortical and spinal cord neurons. Interestingly, there were no intranuclear ubiquitin or TDP-43 positive inclusions, a proposed hallmark of VCP-associated FTLD-U. These mice also developed behavioural changes consistent with frontal lobe dysfunction. In particular, elevated zero maze testing, a measure of anxiety was persistently abnormal in IBMPFD transgenic mice as early as 16 weeks of age well before the onset of weakness or neuropathologic changes. Much later at 14 months of age these mice also developed declarative memory changes using a novel object recognition task. This behavioral task has been reported to be abnormal in other mouse models of FTD (42).

The buildup of ubiquitinated proteins that occurs in patients, transgenic mice and cell culture suggests a defect in protein degradation pathways either via the UPS or autophagy. It is well established that inhibition of the proteasome leads to the accumulation of ubiquitinated proteins. Recently it has also been shown that cells deficient in key autophagic proteins such as ATG5 or ATG7 accumulate ubiquitinated protein inclusions (43, 44). Subsequent studies explored the degradation of putative proteasome substrates in IBMPFD mutant expressing cells but with mixed results. UNC-45B is degraded by the proteasome in a VCP dependent manner. Expression of IBMPFD mutant VCP stabilizes UNC-45B in cells (29). This has been demonstrated in IBMPFD transgenic mouse (41) and patient skeletal muscle (unpublished data) suggesting that the degradation of UNC-45B is truly impaired in IBMPFD muscle. These data point toward an impairment in UPS mediated protein degradation as potential pathogenic mechanism in IBMPFD. However, in contrast to UNC-45B, the degradation of ubiquitin fusion domain substrates such as Ub-G76V-GFP (45) or tetraubiquitinated-luciferase (unpublished data) and the activity of isolated 20S proteasome (46) do not seem to be affected in IBMPFD mutant expressing cells unlike that seen with siRNA knockdown of VCP or dominant negative VCP expression (23). We originally reported that IBMPFD mutants led to the accumulation of the prototypical ERAD substrate ΔF508CFTR as insoluble inclusions that co-localized with VCP in cultured myoblasts (39). A recent study failed to identify a defect in the ERAD degradation of CD3δ following IBMPFD mutant expression (45). This raises the question of whether IBMFPD mutants mishandle specific ERAD or UPS substrates (aggregated vs. misfolded) or that perhaps another degradation system is involved since aggregated ERAD substrates may also be degraded via autophagy (47, 48).

IBMPFD mutations in VCP do affect the degradation of protein aggregates (33). IBMPFD mutant expression impairs aggresome formation following proteasome inhibition or of an aggregate prone polyglutamine expressing protein. This was accompanied by an increase in insoluble protein aggregates and a reduction in their clearance from the cell (33). This is similar to that seen with loss of VCP or with dominant negative VCP expression (49, 50). Interestingly, aggresome formation was improved in IBMPFD mutant expressing cells following expression of the VCP cofactor, histone deacetylase 6 (HDAC6) (33).

These data raised the possibility that IBMPFD mutations in VCP may affect autophagic protein degradation as opposed to or in addition to UPS mediated degradation. Several lines of evidence support this line of reasoning. 1) Loss of autophagy in tissues results in UBIs and accumulation of protein aggregates (43, 44), 2) many other vacuolar myopathies are due to mutations in proteins associated with the autophagosomal-lysosomal system (51), 3) mutations in the critical autophagy adaptor protein p62 cause PDB (52), and 4) a pathologically similar FTLD-U is due to mutations in CHMP2B also known to be involved in autophagy (53–55). Consistent with this hypothesis, a recent study found that IBMPFD skeletal muscle from patients and transgenic animals accumulated p62 and LC3 positive structures that in some cases localize to RVs (15). In addition, p62 and the autophagosome associated LC3II isoform accumulated in IBMPFD muscle suggesting a defect in autophagic protein degradation (15). Subsequent cell culture experiments evaluating both IBMPFD mutants and loss of function models of VCP found that autophagosomes were formed but failed to mature into autolysosomes and degrade autophagosomal contents (15). This was accompanied by impaired protein aggregate degradation in IBMPFD mutant mouse muscle (15). A subsequent study extended these initial findings and found that the immature autophagosomes in IBMPFD mutant expressing cells were predominantly ubiquitin positive (45). Moreover, VCP’s role in autophagosome maturation was more pronounced under basal and proteasome inhibition induced autophagy as opposed to starvation induced autophagy. The VCP co-factor, HDAC6, was also found to mediate the maturation of autophagosome under basal conditions (56). These data implicate autophagic dysregulation in IBMPFD. They highlight a novel role for VCP in the autophagic degradation of protein inclusions and the basal clearance of ubiquitinated proteins by autophagy. How VCP participates in autophagosome maturation is not known. VCP may be involved in autophagosome trafficking, autophagosome-lysosome fusion or the selective loading of ubiqutinated cargo into an autophagosome. Future studies are needed to address these questions.

VCP and TDP-43

The identification of TDP-43 inclusions in IBMPFD patient muscle and brain suggested that VCP may be involved in TDP-43 protein trafficking or degradation. Current models of TDP-43 pathogenesis suggest that its redistribution from the nucleus to the cytoplasm correlates with toxicity (57). Whether this is an active response resulting in pathogenesis or a passive consequence of another pathogenic mechanism is unclear. In an effort to model this in cell culture, the localization of endogenous TDP-43 was evaluated in cells expressing IBMPFD mutant VCP (46). Endogenous TDP-43 was found to co-localize with cytosolic IBMPFD mutant VCP suggesting it had redistributed from the nucleus to the cytoplasm. Subsequent studies found that endogenous VCP, VCP-WT and IBMPFD mutant VCPs co-immunoprecipitated with TDP-43 in cells and patient tissue (46). However it should be noted that VCP does not co-localize with TDP-43 inclusions in IBMPFD patient muscle or CNS tissue (14, 17). Moreover, VCP and TDP-43 fail to co-localize in transgenic mouse tissue (15, 41). Therefore the relevance of these experimental results is unclear. Another study found that exogenously expressed fluorescently tagged TDP-43 accumulated in the cytoplasm of IBMPFD mutant expressing cells similar to the previous study (15). However, this was similar to what was seen when similar cells were treated with inhibitors of lysosomal degradation such as bafilomycinA and chloroquine. TDP-43 has also been shown to leave the nucleus and accumulate in the cytosol of cell treated with the application of proteasome inhibitors (58). This suggests that TDP-43’s accumulation in the cytosol, in some instances, can occur as a secondary response to proteotoxic stress.

Conclusions

IBMPFD is a unique clinical entity that predominantly affects three different tissues (muscle, brain and bone). Although the pathogenesis of IBMPFD is unknown, cellular degeneration in association with ubiquitinated and TDP-43 inclusions is a common pathologic feature in these disparate tissues. IBMPFD is caused by missense mutations in the ubiquitously expressed protein, VCP. How mutations in VCP lead to a degenerative phenotype is unclear but may relate to changes in protein degradation pathways such as the UPS and autophagy. It is conceivable that muscle, brain and bone are particularly sensitive to changes in proteostatic stress conferred by alterations in protein degradation. Alternatively, IBMPFD mutant VCP may specifically mishandle the degradation of specific co-factors or substrates that are uniquely expressed in muscle, brain and bone. Insight into the pathogenesis of IBMPFD will translate into treatments for many age associated degenerative disorders including sIBM, PDB and FTD.