Katrina J. Llewellyn

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 (VCPR155H/+) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCPR155H/+ 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.

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

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.

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, Pagets 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 (VCPR155H/R155H) model carrying the common R155H mutations, which develops many clinical features typical of the VCP-associated human diseases. Homozygote VCPR155H/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 VCPR155H/R155H mice manifest prominent muscle, heart, brain and spinal cord pathology, including striking mitochondrial abnormalities, in addition to disrupted autophagy and ubiquitin pathologies. The VCPR155H/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.

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.

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 Pagets 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 VCPR155H/+ 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 VCPR155H/+ 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 VCPR155H/+ mice did not have any detrimental value to the function of muscle, and may offer effective therapeutic options for patients with VCP-associated diseases.

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.

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, Pagets 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.

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-1β, 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-1β(+)F4/80(+)Ly6C(+) inflammatory macrophages that infiltrate the muscles of VCPR155H/+ mice; (iv) NLRP3 inflammasome activation and accumulation IL-1β(+)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-1β(+) macrophage infiltrates in the muscle, and significantly ameliorated muscle strength. Together, these results suggest that (i) NLRP3 inflammasome and local IL-1β(+)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.

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 Pagets 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.