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1.  Immunoreactivity of valosin-containing protein in sporadic amyotrophic lateral sclerosis and in a case of its novel mutant 
Background
Mutations in the valosin-containing protein (VCP) gene were first found to cause inclusion- body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD). Mutations in the VCP gene were later reported to occur in familial amyotrophic lateral sclerosis (ALS). But the role of VCP in the neurodegenerative processes that occur in ALS remains unknown. The purpose of the present study was to elucidate the role of VCP in the neurodegeneration seen in sporadic and VCP mutant ALS.
Results
Immunohistochemistry demonstrated that the frequency of distinct VCP-positive nuclei of spinal motor neurons of patients with sporadic ALS (SALS) and the ALS with VCP novel mutation (ALS-VCP, M158V) was increased, compared with that of the control cases. No VCP-positive inclusion bodies were observed in SALS patients, a ALS-VCP patient or in control subjects. Neuropathologic examination of the ALS-VCP case showed loss of motor neurons, the presence of Bunina bodies, and degeneration of the corticospinal tracts. Bunina bodies detected in this case were confirmed to show immunohistochemical and ultrastructural features similar to those previously described. Furthermore, neuronal intracytoplasmic inclusions immunopositive for TAR DNA-binding protein 43 kDa (TDP-43), phosphorylated TDP-43, ubiquitin (Ub), p62, and optineurin were identified in the spinal and medullary motoneurons, but not in the neocortex. Gene analysis of this ALS-VCP patient confirmed the de novo mutation of M158V, which was not found in control cases; and bioinformatics using several in silico analyses showed possible damage to the structure of VCP. Immunocytochemical study of cultured cells showed increased cytoplasmic translocation of TDP-43 in cells transfected with several mutant VCP including our patient’s compared with wild-type VCP.
Conclusion
These findings support the idea that VCP is associated with the pathomechanism of SALS and familial ALS with a VCP mutation, presumably acting through a dominant-negative mechanism.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0172-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s40478-014-0172-0
PMCID: PMC4297454  PMID: 25492614
Amyotrophic lateral sclerosis; Paget disease of bone; Valosin-containing protein (VCP); Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD); TAR DNA-binding protein 43 kDa (TDP-43); Golgi apparatus fragmentation
2.  Optineurin immunoreactivity in neuronal and glial intranuclear inclusions in adult-onset neuronal intranuclear inclusion disease 
Optineurin (OPTN) is a multifunctional protein involved in cellular morphogenesis, vesicle trafficking, maintenance of the Golgi complex, and transcription activation through its interactions with the Rab8, myosin 6 (MYO6), huntingtin. Recently, OPTN immunoreactivity has been reported in intranuclear inclusions in patients with neuronal intranuclear inclusions disease (NIID). Other studies have shown that the RNA-binding protein, fused in sarcoma (FUS), is a component of intranuclear inclusions in NIID. We aimed to investigate the relationship between OPTN, its binding protein MYO6 and FUS in this study. In control subjects, OPTN (C-terminal) (OPTN-C) and MYO6 immunoreactivity was mainly demonstrated in the cytoplasm of neurons. In NIID patients, both neuronal intranuclear inclusions (NII) and glial intranuclear inclusions (GII) were immunopositive for MYO6 as well as OPTN-C. However, the intensity of OPTN-C immunostaining of the neuronal cytoplasm with and without NII was less than that of the control subjects. Double immunofluorescence staining for OPTN-C, ubiquitin (Ub), p62 and FUS revealed co-localization of these proteins within NII. Moreover, Ub positive inclusions were co-localized with MYO6. The percentage of co-localization of Ub with OPTN-C, FUS or MYO6 in NII was 100%, 52% and 92%, respectively. Ultrastructurally, the inclusions consisted of thin and thick filaments. Both filaments were immunopositive for Ub and OPTN-C. These findings suggest that OPTN plays a central role in the disease pathogenesis, and that OPTN may be a major component of NII.
PMCID: PMC4162590  PMID: 25232514
Optineurin; Myosin-6; neuronal intranuclear inclusion disease; FUS; intranuclear inclusion
3.  FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations 
Brain  2011;134(9):2595-2609.
Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing’s sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing’s sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing’s sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing’s sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.
doi:10.1093/brain/awr201
PMCID: PMC3170539  PMID: 21856723
FUS; TAF15; EWS; amyotrophic lateral sclerosis; frontotemporal dementia

Results 1-3 (3)