Capturing VCP: another molecular piece in the ALS jigsaw puzzle

TDP-43 mislocalization and aggregation are implicated in the pathogenesis of ALS and FTLD-U. Valosin containing protein (VCP) mutations also lead to TDP-43 deposition, resulting in Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). In this issue of Neuron, Johnson et?al. used whole-exome capture to identify VCP mutations in familial ALS. This extends the VCP phenotype to include motor neuron degeneration and provides another molecular tool to explore neurodegeneration disease mechanisms underlying the TDP-43 proteinopathies.     

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCP^R155H/+) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCP^R155H/+ mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (∼24–27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.     

The Homozygote VCPR155H/R155H Mouse Model Exhibits Accelerated Human VCP-Associated Disease Pathology

Valosin containing protein (VCP) mutations are the cause of hereditary inclusion body myopathy, Paget''s disease of bone, frontotemporal dementia (IBMPFD). VCP gene mutations have also been linked to 2% of isolated familial amyotrophic lateral sclerosis (ALS). VCP is at the intersection of disrupted ubiquitin proteasome and autophagy pathways, mechanisms responsible for the intracellular protein degradation and abnormal pathology seen in muscle, brain and spinal cord. We have developed the homozygous knock-in VCP mouse (VCP^R155H/R155H) model carrying the common R155H mutations, which develops many clinical features typical of the VCP-associated human diseases. Homozygote VCP^R155H/R155H mice typically survive less than 21 days, exhibit weakness and myopathic changes on EMG. MicroCT imaging of the bones reveal non-symmetrical radiolucencies of the proximal tibiae and bone, highly suggestive of PDB. The VCP^R155H/R155H mice manifest prominent muscle, heart, brain and spinal cord pathology, including striking mitochondrial abnormalities, in addition to disrupted autophagy and ubiquitin pathologies. The VCP^R155H/R155H homozygous mouse thus represents an accelerated model of VCP disease and can be utilized to elucidate the intricate molecular mechanisms involved in the pathogenesis of VCP-associated neurodegenerative diseases and for the development of novel therapeutic strategies.     

Proteomic analysis of a drosophila IBMPFD model reveals potential pathogenic mechanisms

IBMPFD, Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia, is a hereditary degenerative disorder due to single missense mutations in VCP (Valosin-Containing Protein). The mechanisms of how mutations of VCP lead to IBMPFD remain mysterious. Here we utilize two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry to study the IBMPFD disorder at the protein level. With this set-up, we are able to employ comparative proteomics to analyze IBMPFD disease using Drosophila melanogaster as our disease model organism. Head proteome of transgenic D. melanogaster expressing wild type VCP is compared, respectively, with the head proteome of transgenic mutant type VCPs that correspond to human IBMPFD disease alleles (TER94(A229E), TER94(R188Q), and TER94(R152H)). Of all the proteins identified, a significant fraction of proteins altered in TER94(A229E) and TER94(R188Q) mutants belong to the same functional categories, i.e. apoptosis and metabolism. Among these, Drosophila transferrin is observed to be significantly up-regulated in mutant flies expressing TER94(A229E). A knock-down experiment suggests that fly transferrin might be a potential modifier in IBMPFD disease. The molecular analysis of IBMPFD disease may benefit from the proteomics approach which combines the advantages of high throughput analysis and the focus on protein levels.     

Pathogenic VCP/TER94 alleles are dominant actives and contribute to neurodegeneration by altering cellular ATP level in a Drosophila IBMPFD model

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is caused by mutations in Valosin-containing protein (VCP), a hexameric AAA ATPase that participates in a variety of cellular processes such as protein degradation, organelle biogenesis, and cell-cycle regulation. To understand how VCP mutations cause IBMPFD, we have established a Drosophila model by overexpressing TER94 (the sole Drosophila VCP ortholog) carrying mutations analogous to those implicated in IBMPFD. Expression of these TER94 mutants in muscle and nervous systems causes tissue degeneration, recapitulating the pathogenic phenotypes in IBMPFD patients. TER94-induced neurodegenerative defects are enhanced by elevated expression of wild-type TER94, suggesting that the pathogenic alleles are dominant active mutations. This conclusion is further supported by the observation that TER94-induced neurodegenerative defects require the formation of hexamer complex, a prerequisite for a functional AAA ATPase. Surprisingly, while disruptions of the ubiquitin-proteasome system (UPS) and the ER-associated degradation (ERAD) have been implicated as causes for VCP-induced tissue degeneration, these processes are not significantly affected in our fly model. Instead, the neurodegenerative defect of TER94 mutants seems sensitive to the level of cellular ATP. We show that increasing cellular ATP by independent mechanisms could suppress the phenotypes of TER94 mutants. Conversely, decreasing cellular ATP would enhance the TER94 mutant phenotypes. Taken together, our analyses have defined the nature of IBMPFD-causing VCP mutations and made an unexpected link between cellular ATP level and IBMPFD pathogenesis.     

Impaired protein aggregate handling and clearance underlie the pathogenesis of p97/VCP-associated disease

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.     

Early pathogenesis in the adult-onset neurodegenerative disease amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motor neurons starting in adulthood. Most of our knowledge about the pathophysiological mechanisms of ALS comes from transgenic mice models that emulate a subgroup of familial ALS cases (FALS), with mutations in the gene encoding superoxide dismutase (SOD1). In the more than 15 years since these mice were generated, a large number of abnormal cellular mechanisms underlying motor neuron degeneration have been identified, but to date this effort has led to few improvements in therapy, and no cure. Here, we consider that this surfeit of mechanisms is best interpreted by current insights that suggest a very early initiation of pathology in motor neurons, followed by a diversity of secondary cascades and compensatory mechanisms that mask symptoms for decades, until trauma and/or aging overloads their protective function. This view thus posits that adult‐onset ALS is the consequence of processes initiated during early development. In fact, motor neurons in neonatal mutant SOD mice display important alterations in their intrinsic electrical properties, synaptic inputs and morphology that are accompanied by subtle behavioral abnormalities. We consider evidence that human mutant SOD1 protein in neonatal hSOD1^G93A mice instigates motor neuron degeneration by increasing persistent sodium currents and excitability, in turn altering synaptic circuits that control excessive motor neuron firing and leads to excitotoxicity. We also discuss how therapies that are aimed at suppressing abnormal neuronal activity might effectively mitigate or prevent the onset of irreversible neuronal damage in adulthood. J. Cell. Biochem. 113: 3301–3312, 2012. © 2012 Wiley Periodicals, Inc.     

A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, we have generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These studies uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS.     

Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.     

Protein Arginine Methyltransferase 1 and 8 Interact with FUS to Modify Its Sub-Cellular Distribution and Toxicity In Vitro and In Vivo

Amyotrophic lateral sclerosis (ALS) is a late onset and progressive motor neuron disease. Mutations in the gene coding for fused in sarcoma/translocated in liposarcoma (FUS) are responsible for some cases of both familial and sporadic forms of ALS. The mechanism through which mutations of FUS result in motor neuron degeneration and loss is not known. FUS belongs to the family of TET proteins, which are regulated at the post-translational level by arginine methylation. Here, we investigated the impact of arginine methylation in the pathogenesis of FUS-related ALS. We found that wild type FUS (FUS-WT) specifically interacts with protein arginine methyltransferases 1 and 8 (PRMT1 and PRMT8) and undergoes asymmetric dimethylation in cultured cells. ALS-causing FUS mutants retained the ability to interact with both PRMT1 and PRMT8 and undergo asymmetric dimethylation similar to FUS-WT. Importantly, PRMT1 and PRMT8 localized to mutant FUS-positive inclusion bodies. Pharmacologic inhibition of PRMT1 and PRMT8 activity reduced both the nuclear and cytoplasmic accumulation of FUS-WT and ALS-associated FUS mutants in motor neuron-derived cells and in cells obtained from an ALS patient carrying the R518G mutation. Genetic ablation of the fly homologue of human PRMT1 (DART1) exacerbated the neurodegeneration induced by overexpression of FUS-WT and R521H FUS mutant in a Drosophila model of FUS-related ALS. These results support a role for arginine methylation in the pathogenesis of FUS-related ALS.     

p97/VCP at the intersection of the autophagy and the ubiquitin proteasome system

A feature of aged onset degenerative disease is ubiquitinated protein inclusions. Similar inclusions are found in different tissues ranging from the central nervous, cardiovascular, musculoskeletal and gastrointestinal systems; whether, the same pathomechanism is responsible for the similar pathology in these disparate tissues is not known. To address this question, we explored the pathogenesis of a multi-system degenerative disorder, IBMPFD or inclusion body myopathy (IBM), paget's disease of the bone (PDB) and fronto-temporal dementia (FTD) of which ubiquitinated inclusions are a key pathological feature in muscle, brain and bone tissue. IBMPFD is caused by mutations in the ubiquitin proteasome system (UPS) chaperone p97/VCP. Previous reports suggest dysfunctional UPS in IBMPFD, however, we find that autophagic protein degradation and autophagosome maturation are diminished in IBMPFD mutant-expressing mice, patients and cell models. Moreover, a loss of p97/VCP function recapitulates the same effects, suggesting that p97/VCP is essential for autophagy. Thus, the degenerative phenotype in IBMPFD and its phenotypic components (IBM, PDB and FTD) may be disorders of impaired autophagy. p97/VCP is likely important in regulating both UPS- and autophagy-mediated protein degradation. This places p97/VCP in a key regulatory position at the intersection of these two proteolytic pathways.     

Functional role of post-translational modifications of Sp1 in tumorigenesis

Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.     

The Human G93A-Superoxide Dismutase-1 Mutation,Mitochondrial Glutathione and Apoptotic Cell Death

Mutations in Cu/Zn superoxide dismutase are a cause of motor neuron death in about 20% of cases of familial amyotrophic lateral sclerosis (ALS). Although the molecular mechanism of which these mutations induce motor neuron cell death is to a large extent unknown, there is significant evidence that effects on mitochondrial function and development of oxidative stress make a major contribution to the selective death of motor neurons in this disease. In this overview article we review the current understanding of mutant SOD1-mediated motor neuron degeneration in ALS with focus on oxidative damage and mitochondrial dysfunction. We also present novel information on the role of mitochondrial glutathione for the survival of NSC-34 cells stably transfected with the human SOD1^G93A mutation, putting forward the hypothesis that this antioxidant pool provides a potentially useful target for therapeutic intervention. Special issue article in Honor of Dr. Graham Johnston.     

Targeted Exon Capture and Sequencing in Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.     

Neuromuscular effects of G93A-SOD1 expression in zebrafish

ABSTRACT: BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis. RESULTS: We demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease. CONCLUSIONS: This sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.     

From neurodevelopment to neurodegeneration: the interaction of neurofibromin and valosin-containing protein/p97 in regulation of dendritic spine formation

Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget''s disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients'' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.     

Highlight Commentary on "Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis"

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterized by rapid degeneration of and loss of function in the motor cortex, brain stem, and spinal cord, particularly the anterior horn cells. Since the pioneering work of Brown and colleagues, more than 100 mutations in Cu,Zn superoxide dismutase (SOD1) have been described (P. Pasinelli, R. H. Brown, Nat. Rev. Neurosci.7, 710-723, 2006). There are toxic gain-of-function alterations in SOD1, because the enzymatic activity of this protein is not different in ALS from that of controls. The paper by Butterfield and colleagues reporting the use of redox proteomics to identify oxidatively modified proteins in the spinal cord in the G93A-SOD1 mouse model of familial amyotrophic lateral sclerosis was identified by the SCOPUS science literature information system to be one of the top 20 downloaded papers for 2005-2006 in Free Radical Biology and Medicine. Here my thoughts on the importance and impact of this paper are reported.     

Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, adult-onset motor neuron disease that arises as a dominantly inherited trait in approximately 10% of ALS cases. Mutations in one gene, cytosolic Cu/Zn superoxide dismutase (SOD1), account for approximately 25% of familial ALS (FALS) cases. We have performed a genetic linkage screen in 16 pedigrees with FALS with no evidence for mutations in the SOD1 gene and have identified novel ALS loci on chromosomes 16 and 20. The analysis of these genes will delineate pathways implicated as determinants of motor-neuron viability and provide insights into possible therapies for ALS.     

Genetics of amyotrophic lateral sclerosis: an update

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder involving both upper motor neurons (UMN) and lower motor neurons (LMN). Enormous research has been done in the past few decades in unveiling the genetics of ALS, successfully identifying at least fifteen candidate genes associated with familial and sporadic ALS. Numerous studies attempting to define the pathogenesis of ALS have identified several plausible determinants and molecular pathways leading to motor neuron degeneration, which include oxidative stress, glutamate excitotoxicity, apoptosis, abnormal neurofilament function, protein misfolding and subsequent aggregation, impairment of RNA processing, defects in axonal transport, changes in endosomal trafficking, increased inflammation, and mitochondrial dysfunction. This review is to update the recent discoveries in genetics of ALS, which may provide insight information to help us better understanding of the disease neuropathogenesis.     

Recent advances in p97/VCP/Cdc48 cellular functions

p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases.