Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies

VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo.

Lysosomes are classically viewed as vesicular structures to which cargos are delivered for degradation. Here, we identify a network of dynamic, tubular lysosomes that extends throughout Drosophila muscle, in vivo. Live imaging reveals that autophagosomes merge with tubular lysosomes and that lysosomal membranes undergo extension, retraction, fusion and fission. The dynamics and integrity of this tubular lysosomal network requires VCP, an AAA-ATPase that, when mutated, causes degenerative diseases of muscle, bone and neurons. We show that human VCP rescues the defects caused by loss of Drosophila VCP and overexpression of disease relevant VCP transgenes dismantles tubular lysosomes, linking tubular lysosome dysfunction to human VCP-related diseases. Finally, disruption of tubular lysosomes correlates with impaired autophagosome-lysosome fusion, increased cytoplasmic poly-ubiquitin aggregates, lipofuscin material, damaged mitochondria and impaired muscle function. We propose that VCP sustains sarcoplasmic proteostasis, in part, by controlling the integrity of a dynamic tubular lysosomal network

Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells.

DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ~25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R(2)=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation

Multisystem proteinopathy due to a homozygous p.Arg159His VCP mutation : a tale of the unexpected

ObjectiveTo assess the clinical, radiologic, myopathologic, and proteomic findings in a patient manifesting a multisystem proteinopathy due to a homozygous valosin-containing protein gene (VCP) mutation previously reported to be pathogenic in the heterozygous state.MethodsWe studied a 36-year-old male index patient and his father, both presenting with progressive limb-girdle weakness. Muscle involvement was assessed by MRI and muscle biopsies. We performed whole-exome sequencing and Sanger sequencing for segregation analysis of the identified p.Arg159His VCP mutation. To dissect biological disease signatures, we applied state-of-the-art quantitative proteomics on muscle tissue of the index case, his father, 3 additional patients with VCP-related myopathy, and 3 control individuals.ResultsThe index patient, homozygous for the known p.Arg159His mutation in VCP, manifested a typical VCP-related myopathy phenotype, although with a markedly high creatine kinase value and a relatively early disease onset, and Paget disease of bone. The father exhibited a myopathy phenotype and discrete parkinsonism, and multiple deceased family members on the maternal side of the pedigree displayed a dementia, parkinsonism, or myopathy phenotype. Bioinformatic analysis of quantitative proteomic data revealed the degenerative nature of the disease, with evidence suggesting selective failure of muscle regeneration and stress granule dyshomeostasis.ConclusionWe report a patient showing a multisystem proteinopathy due to a homozygous VCP mutation. The patient manifests a severe phenotype, yet fundamental disease characteristics are preserved. Proteomic findings provide further insights into VCP-related pathomechanisms

Rare manifestation of a c.290 C\u3eT, p.Gly97Glu VCP mutation

Introduction. The valosin-containing protein (VCP) regulates several distinct cellular processes. Consistent with this, VCP mutations manifest variable clinical phenotypes among and within families and are a diagnostic challenge. Methods. A 60-year-old man who played ice hockey into his 50’s was evaluated by electrodiagnostics, muscle biopsy, and molecular genetics. Results. With long-standing pes cavus and toe walking, our patient developed progressive weakness, cramps, memory loss, and paresthesias at age 52. An axonal sensorimotor neuropathy was found upon repeated testing at age 58. Neuropathic histopathology was present in the quadriceps, and exome sequencing revealed the VCP mutation c.290 C>T, p.Gly97Glu. Conclusions. Our patient reflects the clinical heterogeneity of VCP mutations, as his neurological localization is a spectrum between a lower motor neuron disorder and a hereditary axonal peripheral neuropathy such as CMT2. Our case demonstrates a rare manifestation of the c.290 C>T, pGly97Glu VCP mutation

Uptake of an OHS code of practice by construction firms : barriers and enablers in an Australian industry

The Australian construction industry, reflecting a global trend, is moving towards the implementation of a voluntary code of practice (hereafter VCP) for occupational health and safety. The evidence suggests that highlyvisible clients and project management firms, in addition to their subcontractors, look set to embrace such a code. However, smaller firms not operating in high-profile contracting regimes may prove reticent to adopt a VCP. This paper incorporates qualitative data from a high-profile research project commissioned by Engineers Australia and supported by the Australian Contractors’ Association, Property Council of Australia, Royal Australian Institute of Architects, Association of Consulting Engineers Australia, Australian Procurement and Construction Council, Master Builders Australia and the Australian CRC for Construction Innovation. The paper aims to understand the factors that facilitate or prevent the uptake of the VCP by smaller firms, together with pathways to the adoption of a VCP by industry

Vector cross product in n-dimensional vector space

The definition of vector cross product (VCP) introduced by Eckmann only exists in thethree- and the seven- dimensional vector space. In this paper, according to the orthogonal completeness, magnitude of basis vector cross product and all kinds of combinations of basis vector $\hat{e}_i$, the generalized definition of VCP in the odd n-dimensional vector space is given by introducing a cross term $X_{AB}$. In addition, the definition is validated by reducing the generalization definition to the fundamental three- and seven-dimensional vector space.Comment: 9 pages, 1 figure, comments are welcom

Cell Surface Expression of the Vaccinia Virus Complement Control Protein is Mediated by Interaction with the Viral A56 Protein and Protects Infected Cells from Complement Attack

The vaccinia virus (VACV) complement control protein (VCP) is the major protein secreted from VACV-infected cells. It has been reported that VCP binds to the surfaces of uninfected cells by interacting with heparan sulfate proteoglycans (HSPGs). In this study, we show that VCP is also expressed on the surfaces of infected cells and demonstrate that surface localization occurs independently of HSPGs. Since VCP does not contain a transmembrane domain, we hypothesized that VCP interacts with a membrane protein that localizes to the infected-cell surface. We show that the VACV A56 membrane protein is necessary for the cell surface expression of VCP and demonstrate that VCP and A56 interact in VACV-infected cells. Since the surface expression of VCP was abrogated by reducing agents, we examined the contribution of an unpaired cysteine residue on VCP to VCP surface expression and VCP\u27s interaction with A56. To do this, we mutated the unpaired cysteine in VCP and generated a recombinant virus expressing the altered form of VCP. Following the infection of cells with the mutant virus, VCP was neither expressed on the cell surface nor able to interact with A56. Importantly, the cell surface expression of VCP was found to protect infected cells from complement-mediated lysis. Our findings suggest a new function for VCP that may be important for poxvirus pathogenesis and impact immune responses to VACV-based vaccines

VALOSIN CONTAINING PROTEIN IN AMYOTROPHIC LATERAL SCLEROSIS: NEW INSIGHT IN PATHOLOGICAL MECHANISMS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of upper and lower motor neurons (MNs). ALS can be classified in familiar forms (fALS), occurring in 10% of the cases, and sporadic form (sALS), occurring in the remaining 90%. Many pathways have been found to concur with MNs death, among which has a great importance alteration in proteostasis. Indeed, all ALS forms are characterized by the presence of protein aggregates and vacuoles in the brain tissue of effected patients. These are signs of an altered protein quality control (PQC) system. Protein aggregates can be either triggered by the mutations of genes that express unstable proteins that misfold and aggregate, or by mutations of genes that express proteins involved in the degradation pathways as ubiquitin-proteasome system (UPS) and autophagy. One of these genes associated to ALS is VCP that encodes for Valosin Containing Protein (VCP), a protein involved in many steps of the PQC system. One of VCP roles is to disassemble protein aggregates and concur to their degradation. Thus, in the first part of my thesis, I focused on VCP contribute in the removal of protein aggregates positive to ALS-associated SOD1-mutant (SOD1 G93A). The aim of this part was to define if the modulation of VCP expression could improve altered proteostasis, ameliorating the pathological condition. Moreover, I also studied two VCP-mutants associated to ALS (VCP R155H and VCP R191Q) comparing to VCP WT, their contribution in this pathway. To study VCP role I overexpressed VCP WT and VCP-mutants in NSC34 cells, an immortalized motor neuron cell line, that transiently expressed SOD1 G934A. My work demonstrated that VCP WT overexpression enhanced SOD1-mutant clearance through the UPS. Surprisingly also VCP-mutants increased SOD1-mutants clearance. VCP-mutants contribute in SOD1-mutants clearance differed from VCP WT as it resulted dependent from the autophagic pathway. In the second part of my thesis, I focused on VCP-mutants to better understand their pathological mechanisms. In particular, I studied their effects on lysosomal damage and their impact on the autophagic flux. To define VCP-mutants pathological contribute in an ALS-model I overexpressed VCP R155H and VCP R191Q in NSC34 cells. Firstly, I determined that VCP-mutants aggregated in this model. Moreover, I found that VCP-mutants expression was associated to lysosome alteration in content, size, morphology, and membrane breakage. Lysosomal damaged induced by VCP-mutants led to the specific activation of TFE3, a transcription factor regulator of autophagy and lysosome biogenesis. In fact, by studying autophagic markers, I determined that the presence of VCP-mutants was associated to the activation of the autophagic flux. Finally, in this part, I found that VCP-mutants prevented damaged lysosome clearance when it was induced by chemical compounds, as trehalose, or by SOD1-mutant overexpression. Conversely, VCP WT overexpression enhanced lysosomal damage clearance in both of these conditions. Overall with my work, I determined new pathological mechanisms of VCP-mutants ALS-associated. Moreover, I found that VCP modulation could ameliorate ALS-pathological conditions through different pathways

Voluntary Cleanups and Redevelopment Potential: Lessons from Baltimore, Maryland

Policy has increasingly shifted towards economic incentives and liability attenuation for promoting cleanup and redevelopment of contaminated sites, but little is known about the effectiveness of such policies. An example of such legislation is State Voluntary Cleanup Programs (VCPs), which were established in the US in the 1990s and to date have been implemented in almost every state. We examine Baltimore properties that participated in the Maryland VCP from its inception in 1997 to the end of 2006. Specifically, we examine what type of properties tend to participate in these programs, how these properties compare to other eligible but non-participating sites, and what is the redevelopment potential of VCP properties and implications towards open space conversion. We find that most applicants (66%) actually requested a “No Further Action Determination” directly, rather than proposing cleanup. VCP properties tend to be industrial, located in industrial areas, and away from residential neighborhoods. In more recent years larger industrial properties have increasingly enrolled in the program. The majority of sites are reused as industrial or commercial. In contrast to Alberini (2007), this suggests that pressure for residential development does not drive VCP participation. Based on differences in zoning requirements, the VCP may reduce demand for potentially contaminating activities on pristine land by as much as 1,238 to 6,444 acres, in Baltimore alone.Brownfields, Contaminated Sites, Voluntary Cleanup Programs, Incentives