Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation. Amyotrophic lateral sclerosis (ALS)1 is a neurodegenerative disease characterized by the loss of motor neurons in the cerebral cortex, brain stem, and spinal cord (1, 2). Although most patients with ALS are sporadic cases, 5-10% of ALS patients show a familial trait, and in 10 -20% of these patients this trait is associated with mutations in the SOD1 gene (3). Although many scenarios for understanding ALS pathogenesis have been proposed (4 -6), to date the exact mechanisms causing the disease are still unknown. In considering the pathogenesis of ALS, it is important that the inclusion bodies, which consist of aggregated, ubiquitylated proteins surrounded by disorganized filaments, are frequently seen in the surviving motor neurons in both sporadic and familial ALS (7-12). Interestingly, SOD1 is reported to be the major component of the neuronal hyaline inclusion (NHI) of familial ALS associated with SOD1 mutation (13). In addition, the NHI in the motor neurons of familial ALS is commonly stained with anti-ubiquitin (Ub)-antibody, but it remains uncertain whether SOD1 itself is ubiquitylated with respect to the inclusion body formation. To clarify the mechanisms underlying the inclusion body formation and consequent motor neuron degeneration in ALS, it is important to know how SOD1 and/or other protein component(s) are ubiquitylated.Dorfin is a gene product we cloned from the anterior horn tissues of the human spinal cord (14). Dorfin contains two RING finger motifs and an in-between RING finger (IBR) domain at the N terminus (14). The RING finger/IBR motif was identified in several protein sequences through a data base search (15). It was reported that HHARI (human homologue of ariadne) and H7-AP1 (UbcH7-associated protein), which are both RING finger/IBR motif-containing proteins, interact with the ubiquitin-conjugating enzyme (E2) UbcH7 through the RING finger/IBR motif and that a distinct subclass of RING finger/IBR motif-containing proteins represents a new family of proteins that specificall...

Amyotrophic lateral sclerosis is a devastating, progressive neurodegenerative disease that affects upper and lower motor neurons. Although several genes are identified as the cause of familial cases, the pathogeneses of sporadic forms, which account for 90% of amyotrophic lateral sclerosis, have not been elucidated. Transactive response DNA-binding protein 43 a nuclear protein regulating RNA processing, redistributes to the cytoplasm and forms aggregates, which are the histopathological hallmark of sporadic amyotrophic lateral sclerosis, in affected motor neurons, suggesting that loss-of-function of transactive response DNA-binding protein 43 is one of the causes of the neurodegeneration. To test this hypothesis, we assessed the effects of knockout of transactive response DNA-binding protein 43 in mouse postnatal motor neurons using Cre/loxp system. These mice developed progressive weight loss and motor impairment around the age of 60 weeks, and exhibited degeneration of large motor axon, grouped atrophy of the skeletal muscle, and denervation in the neuromuscular junction. The spinal motor neurons lacking transactive response DNA-binding protein 43 were not affected for 1 year, but exhibited atrophy at the age of 100 weeks; whereas, extraocular motor neurons, that are essentially resistant in amyotrophic lateral sclerosis, remained preserved even at the age of 100 weeks. Additionally, ultra structural analysis revealed autolysosomes and autophagosomes in the cell bodies and axons of motor neurons of the 100-week-old knockout mice. In summary, the mice in which transactive response DNA-binding protein 43 was knocked-out specifically in postnatal motor neurons exhibited an age-dependent progressive motor dysfunction accompanied by neuropathological alterations, which are common to sporadic amyotrophic lateral sclerosis. These findings suggest that transactive response DNA-binding protein 43 plays an essential role in the long term maintenance of motor neurons and that loss-of-function of this protein seems to contribute to the pathogenesis of amyotrophic lateral sclerosis.

The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases.The 43-kDa TAR DNA-binding protein 2 has recently been identified as a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinpositive inclusions (1, 2). Subsequently several point mutations located in the glycine-rich domain of TDP-43 have been identified as the disease-causing mutations of familial and sporadic ALS (3-7). TDP-43 has been shown to be a fundamental component of ubiquitin-positive neuronal cytoplasmic and intranuclear inclusions as well as that of neuronal dystrophic neurites in the affected neurons or glial cells in these neurodegenerative diseases. TDP-43 is known to regulate gene transcription, exon splicing, and exon inclusion through interactions with RNA, heterogeneous nuclear ribonucleoproteins, and nuclear bodies (8 -12). Recently it has been reported that TDP-43 stabilizes human low molecular weight neurofilament mRNA through direct interaction with the 3Ј-untranslated region (13) and that it regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression (14). However, the physiological function of TDP-43 in the central nervous system has not been fully elucidated, and it remains unclear how this protein is implicated in the pathogenesis of neurodegeneration.The Rho family of GTPases are members of the Ras superfamily and are known for regulating actin cytoskeletal dynamics (15-18). RhoA, Rac1, and Cdc42, the most studied proteins of this family, also modulate functions such as cell movement, motility, transcription, cell growth, and cell survival (18). In neurons, RhoA, Rac1, and Cdc42 have been shown to regulate neurite outgrowth (19 -21).Although TDP-43 ...

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains. In this study, we report that Dorfin, a RING finger-type ubiquityl ligase for mutant superoxide dismutase-1, was localized with ubiquitin in LBs. Recently, synphilin-1 was identified to associate with ␣-synuclein and to be a major component of LBs. We found that overexpression of synphilin-1 in cultured cells led to the formation of large juxtanuclear inclusions, but showed no cytotoxicity. Dorfin colocalized in these large inclusions with ubiquitin and proteasomal components. In contrast to full-length synphilin-1, overexpression of the central portion of synphilin-1, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding domain, predominantly led to the formation of small punctate aggregates scattered throughout the cytoplasm and showed cytotoxic effects. Dorfin and ubiquitin did not localize in these small aggregates. Overexpression of the N or C terminus of synphilin-1 did not lead to the formation of any aggregates. Dorfin physically bound and ubiquitylated synphilin-1 through its central portion, but did not ubiquitylate wild-type or mutant ␣-synuclein. These results suggest that the central domain of synphilin-1 has an important role in the formation of aggregates and cytotoxicity and that Dorfin may be involved in the pathogenic process of PD and LB formation by ubiquitylation of synphilin-1.

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosincontaining protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400 -600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP K524A , a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.