Ceramide contributes to pathogenesis and may be targeted for therapy in VCP inclusion body myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

Myogenic differentiation of VCP disease-induced pluripotent stem cells: A novel platform for drug discovery.

Valosin Containing Protein (VCP) disease is an autosomal dominant multisystem proteinopathy caused by mutations in the VCP gene, and is primarily associated with progressive muscle weakness, including atrophy of the pelvic and shoulder girdle muscles. Currently, no treatments are available and cardiac and respiratory failures can lead to mortality at an early age. VCP is an AAA ATPase multifunction complex protein and mutations in the VCP gene resulting in disrupted autophagic clearance. Due to the rarity of the disease, the myopathic nature of the disorder, ethical and practical considerations, VCP disease muscle biopsies are difficult to obtain. Thus, disease-specific human induced pluripotent stem cells (hiPSCs) now provide a valuable resource for the research owing to their renewable and pluripotent nature. In the present study, we report the differentiation and characterization of a VCP disease-specific hiPSCs into precursors expressing myogenic markers including desmin, myogenic factor 5 (MYF5), myosin and heavy chain 2 (MYH2). VCP disease phenotype is characterized by high expression of TAR DNA Binding Protein-43 (TDP-43), ubiquitin (Ub), Light Chain 3-I/II protein (LC3-I/II), and p62/SQSTM1 (p62) protein indicating disruption of the autophagy cascade. Treatment of hiPSC precursors with autophagy stimulators Rapamycin, Perifosine, or AT101 showed reduction in VCP pathology markers TDP-43, LC3-I/II and p62/SQSTM1. Conversely, autophagy inhibitors chloroquine had no beneficial effect, and Spautin-1 or MHY1485 had modest effects. Our results illustrate that hiPSC technology provide a useful platform for a rapid drug discovery and hence constitutes a bridge between clinical and bench research in VCP and related diseases.

Activation of the NLRP3 Inflammasome Is Associated with Valosin-Containing Protein Myopathy.

Aberrant activation of the NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome, triggers a pathogenic inflammatory response in many inherited neurodegenerative disorders. Inflammation has recently been associated with valosin-containing protein (VCP)-associated diseases, caused by missense mutations in the VCP gene. This prompted us to investigate whether NLRP3 inflammasome plays a role in VCP-associated diseases, which classically affects the muscles, bones, and brain. In this report, we demonstrate (i) an elevated activation of the NLRP3 inflammasome in VCP myoblasts, derived from induced pluripotent stem cells (iPSCs) of VCP patients, which was significantly decreased following in vitro treatment with the MCC950, a potent and specific inhibitor of NLRP3 inflammasome; (ii) a significant increase in the expression of NLRP3, caspase 1, IL-1ß, and IL-18 in the quadriceps muscles of VCPR155H/+ heterozygote mice, an experimental mouse model that has many clinical features of human VCP-associated myopathy; (iii) a significant increase of number of IL-1ß(+)F4/80(+)Ly6C(+) inflammatory macrophages that infiltrate the muscles of VCPR155H/+ mice; (iv) NLRP3 inflammasome activation and accumulation IL-1ß(+)F4/80(+)Ly6C(+) macrophages positively correlated with high expression of TDP-43 and p62/SQSTM1 markers of VCP pathology in damaged muscle; and (v) treatment of VCPR155H/+ mice with MCC950 inhibitor suppressed activation of NLRP3 inflammasome, reduced the F4/80(+)Ly6C(+)IL-1ß(+) macrophage infiltrates in the muscle, and significantly ameliorated muscle strength. Together, these results suggest that (i) NLRP3 inflammasome and local IL-1ß(+)F4/80(+)Ly6C(+) inflammatory macrophages contribute to pathogenesis of VCP-associated myopathy and (ii) identified MCC950 specific inhibitor of the NLRP3 inflammasome with promising therapeutic potential for the treatment of VCP-associated myopathy.

A Fine Balance of Dietary Lipids Improves Pathology of a Murine Model of VCP-Associated Multisystem Proteinopathy.

The discovery of effective therapies and of disease mechanisms underlying valosin containing protein (VCP)-associated myopathies and neurodegenerative disorders remains elusive. VCP disease, caused by mutations in the VCP gene, are a clinically and genetically heterogeneous group of disorders with manifestations varying from hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia (IBMPFD), and amyotrophic lateral sclerosis (ALS). In the present study, we examined the effects of higher dietary lipid percentages on VCPR155H/R155H, VCPR155H/+ and Wild Type (WT) mice from birth until 15 months of age by immunohistochemical and biochemical assays. Findings illustrated improvement in the muscle strength, histology, and autophagy signaling pathway in the heterozygote mice when fed 9% lipid-enriched diets (LED). However, increasing the LED by 12%, 30%, and 48% showed no improvement in homozygote and heterozygote survival, muscle pathology, lipid accumulation or the autophagy cascade. These findings suggest that a balanced lipid supplementation may have a therapeutic strategy for patients with VCP-associated multisystem proteinopathies.

Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics.

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).

Administration of CoQ10 analogue ameliorates dysfunction of the mitochondrial respiratory chain in a mouse model of Angelman syndrome.

Genetic defects in the UBE3A gene, which encodes for the imprinted E6-AP ubiquitin E3 ligase (UBE3A), is responsible for the occurrence of Angelman syndrome (AS), a neurodegenerative disorder which arises in 1 out of every 12,000-20,000 births. Classical symptoms of AS include delayed development, impaired speech, and epileptic seizures with characteristic electroencephalography (EEG) readings. We have previously reported impaired mitochondrial structure and reduced complex III in the hippocampus and cerebellum in the Ube3a(m-/p+) mice. CoQ10 supplementation restores the electron flow to the mitochondrial respiratory chain (MRC) to ultimately increase mitochondrial antioxidant capacity. A number of recent studies with CoQ10 analogues seem promising in providing therapeutic benefit to patients with a variety of disorders. CoQ10 therapy has been reported to be safe and relatively well-tolerated at doses as high as 3000mg/day in patients with disorders of CoQ10 biosynthesis and MRC disorders. Herein, we report administration of idebenone, a potent CoQ10 analogue, to the Ube3a(m-/p+) mouse model corrects motor coordination and anxiety levels, and also improves the expression of complexes III and IV in hippocampus CA1 and CA2 neurons and cerebellum in these Ube3a(m-/p+) mice. However, treatment with idebenone illustrated no beneficial effects in the reduction of oxidative stress. To our knowledge, this is the first study to suggest an improvement in mitochondrial respiratory chain dysfunction via bioenergetics modulation with a CoQ10 analogue. These findings may further elucidate possible cellular and molecular mechanism(s) and ultimately a clinical therapeutic approach/benefit for patients with Angelman syndrome.

Global gene expression profiling in R155H knock-in murine model of VCP disease.

Dominant mutations in the valosin-containing protein (VCP) gene cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, which is characterized by progressive muscle weakness, dysfunction in bone remodeling, and frontotemporal dementia. More recently, VCP has been linked to 2% of familial amyotrophic lateral sclerosis cases. VCP plays a significant role in a plethora of cellular functions including membrane fusion, transcription activation, nuclear envelope reconstruction, postmitotic organelle reassembly, and cell cycle control. To elucidate the pathological mechanisms underlying the VCP disease progression, we have previously generated a VCP(R155H/+) mouse model with the R155H mutation. Histological analyses of mutant muscle showed vacuolization of myofibrils, centrally located nuclei, and disorganized muscle fibers. Global expression profiling of VCP(R155H/+) mice using gene annotations by DAVID identified key dysregulated signaling pathways including genes involved in the physiological system development and function, diseases and disorders, and molecular and cellular functions. There were a total of 212 significantly dysregulated genes, several of which are involved in the regulation of proteasomal function and NF-κB signaling cascade. Findings of the gene expression study were validated by using quantitative reverse transcriptase polymerase chain reaction analyses to test genes involved in various signaling cascades. This investigation reveals the importance of the VCP(R155H/+) mouse model in the understanding of cellular and molecular mechanisms causing VCP-associated neurodegenerative diseases and in the discovery of novel therapeutic advancements and strategies for patients suffering with these debilitating disorders.

Targeted excision of VCP R155H mutation by Cre-LoxP technology as a promising therapeutic strategy for valosin-containing protein disease.

Inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia is attributed to mutations in the valosin-containing protein (VCP) gene, mapped to chromosomal region 9p13.3-12. Affected individuals exhibit scapular winging and die from progressive muscle weakness and cardiac and respiratory failure in their 40s to 50s. Mutations in the VCP gene have also been associated with amyotrophic lateral sclerosis in 10-15% of individuals with hereditary inclusion body myopathy and 2-3% of isolated familial amyotrophic lateral sclerosis. Currently, there are no effective treatments for VCP-related myopathy or dementia. To determine the effects of targeted excision of the most common R155H mutation in VCP disease, we generated the Cre-ER™-VCPR155H/+ tamoxifen-inducible model. We administered tamoxifen (0.12 mg/g body weight) or corn oil (vehicle) to the pregnant dams by oral gavage and monitored survival and muscle strength measurements of the pups until 18 months of age. We confirmed efficient removal of exons 4 and 5 and recombination of the mutant/floxed VCP copies by Q-PCR analyses. The activity and specificity of Cre recombinase was confirmed by immunostaining. Herein, we report that Cre-ER™-VCPR155H/+ mice demonstrated improved muscle strength and quadriceps fibers architecture, autophagy signaling pathway, reduced brain neuropathology, decreased apoptosis, and less severe Paget-like bone changes. The Cre-ER™-VCPR155H/+ mouse model provides proof of principle by demonstrating that removal of the mutated exons could be beneficial to patients with VCP-related neurodegenerative diseases, and serves as an excellent platform in understanding the underlying pathophysiological mechanism(s) in the hopes of a promising therapeutic approach.

Cytokine profiling in patients with VCP-associated disease.

Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia [IBMPFD] syndrome) is caused by mutations in the gene encoding VCP classically affecting the muscle, bone and brain. Although the genetic cause has been identified, details regarding the pathogenesis of IBMPFD have not been fully determined. Muscle wasting observed in VCP disease is suggestive of cytokine imbalance. We hypothesized that dysfunctional protein homeostasis caused by VCP mutations leads to cytokine imbalances thereby contributing to the muscle wasting phenotype. Circulating levels of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF a) and epidermal growth factor (EGF) were measured in plasma of patients with VCP disease or controls. TNF a and EGF were significantly altered in VCP disease as compared to control. TNF a was up-regulated, consistent with a cachexia phenotype and EGF levels were increased. No significant differences were observed in IL-4 and IL-6. Cytokine imbalances may be associated with VCP disease and may play a contributory role in VCP myopathy. Further understanding of how VCP dysfunction leads to aberrant protein homeostasis and subsequent cytokine imbalances may also aid in the understanding of other proteinopathies and in the development of novel treatments.

Lipid-enriched diet rescues lethality and slows down progression in a murine model of VCP-associated disease.

Valosin-containing protein (VCP)-associated disease caused by mutations in the VCP gene includes combinations of a phenotypically heterogeneous group of disorders such as hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Currently, there are no effective treatments for VCP myopathy or dementia. VCP mouse models carrying the common R155H mutation include several of the features typical of the human disease. In our previous investigation, VCP(R155H/R155H) homozygous mice exhibited progressive weakness and accelerated pathology prior to their early demise. Herein, we report that feeding pregnant VCP(R155H/+) heterozygous dams with a lipid-enriched diet (LED) results in the reversal of the lethal phenotype in VCP(R155H/R155H) homozygous offspring. We examined the effects of this diet on homozygous and wild-type mice from birth until 9 months of age. The LED regimen improved survival, motor activity, muscle pathology and the autophagy cascade. A targeted lipidomic analysis of skeletal muscle and liver revealed elevations in tissue levels of non-esterified palmitic acid and ceramide (d18:1/16:0), two lipotoxic substances, in the homozygous mice. The ability to reverse lethality, increase survival, and ameliorate myopathy and lipids deficits in the VCP(R155H/R155H) homozygous animals suggests that lipid supplementation may be a promising therapeutic strategy for patients with VCP-associated neurodegenerative diseases.

Exercise training reverses skeletal muscle atrophy in an experimental model of VCP disease.

BACKGROUND: The therapeutic effects of exercise resistance and endurance training in the alleviation of muscle hypertrophy/atrophy should be considered in the management of patients with advanced neuromuscular diseases. Patients with progressive neuromuscular diseases often experience muscle weakness, which negatively impact independence and quality of life levels. Mutations in the valosin containing protein (VCP) gene lead to Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) and more recently affect 2% of amyotrophic lateral sclerosis (ALS)-diagnosed cases. METHODS/PRINCIPLE FINDINGS: The present investigation was undertaken to examine the effects of uphill and downhill exercise training on muscle histopathology and the autophagy cascade in an experimental VCP mouse model carrying the R155H mutation. Progressive uphill exercise in VCP(R155H/+) mice revealed significant improvement in muscle strength and performance by grip strength and Rotarod analyses when compared to the sedentary mice. In contrast, mice exercised to run downhill did not show any significant improvement. Histologically, the uphill exercised VCP(R155H/+) mice displayed an improvement in muscle atrophy, and decreased expression levels of ubiquitin, P62/SQSTM1, LC3I/II, and TDP-43 autophagy markers, suggesting an alleviation of disease-induced myopathy phenotypes. There was also an improvement in the Paget-like phenotype. CONCLUSIONS: Collectively, our data highlights that uphill exercise training in VCP(R155H/+) mice did not have any detrimental value to the function of muscle, and may offer effective therapeutic options for patients with VCP-associated diseases.

A progressive translational mouse model of human valosin-containing protein disease: the VCP(R155H/+) mouse.

INTRODUCTION: Mutations in the valosin-containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis. METHODS: The VCP(R155H/+) knock-in mouse model was assessed for muscle strength and immunohistochemical, Western blot, apoptosis, autophagy, and microPET/CT imaging analyses. RESULTS: VCP(R155H/+) mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons. CONCLUSIONS: VCP(R155H/+) knock-in mice represent an excellent preclinical model for understanding VCP-associated disease mechanisms and future treatments.

The homozygote VCP(R¹55H/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.

A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos.

We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.