No abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron death.More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1 mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1-mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In this study, we identified a dynein subunit as a component of the mutant SOD1-containing high molecular weight complexes using proteomic techniques. We further demonstrated interaction and colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the onset of symptoms in the ALS animal models and increased over the disease progression. Motor neurons with long axons are particularly susceptible to defects in axonal transport. Our results demonstrate a direct "gain-of-interaction" between mutant SOD1 and dynein, which may provide insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal transport or other dynein functions. The aberrant interaction is potentially critical to the formation of mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration in ALS.

The difficulty in accessing mammalian nephrons in vivo hinders the study of podocyte biology. The Drosophila nephrocyte shares remarkable similarities to the glomerular podocyte, but the lack of a functional readout for nephrocytes makes it challenging to study this model of the podocyte, which could potentially harness the power of Drosophila genetics. Here, we present a functional analysis of nephrocytes and establish an in vivo system to screen for renal genes. We found that nephrocytes efficiently take up secreted fluorescent protein, and therefore, we generated a transgenic line carrying secreted fluorescent protein and combined it with a nephrocyte-specific driver for targeted gene knockdown, allowing the identification of genes required for nephrocyte function. To validate this system, we examined the effects of knocking down sns and duf, the Drosophila homologs of nephrin and Neph1, respectively, in pericardial nephrocytes. Knockdown of sns or duf completely abolished the accumulation of the fluorescent protein in pericardial nephrocytes. Examining the ultrastructure revealed that the formation of the nephrocyte diaphragm and lacunar structure, which is essential for protein uptake, requires sns. Our preliminary genetic screen also identified Mec2, which encodes the homolog of mammalian Podocin. Taken together, these data suggest that the Drosophila pericardial nephrocyte is a useful in vivo model to help identify genes involved in podocyte biology and facilitate the discovery of renal disease genes.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together. Amyotrophic lateral sclerosis (ALS)3 is a progressive neurodegenerative disorder leading to the selective death of motor neurons (1, 2). The majority of cases are sporadic, but about 10% of all cases are familial (fALS). In ϳ20% of familial cases, a mutant allele of the copper-zinc superoxide dismutase (SOD1) enzyme has been identified (3-5). As of today, over 100 different mutations of SOD1 causing ALS have been reported, the majority of which are point mutations and act in a dominant fashion. Several ALS risk factors have been identified, but the etiology of the disease is largely unclear. A hallmark of the disease is the appearance of intracellular inclusions in degenerating motor neurons (6), both in the familial cases caused by mutations in SOD1 and in the more obscure sporadic cases (6 -11). The formation of such protein aggregates precedes neuronal death (12). The inclusions are typically SOD1 and ubiquitin immunoreactive in the mutant SOD1-mediated fALS cases (2, 7-9, 11-13). However, it is unclear how the SOD1 mutants form aggregates and whether other proteins play any role in the aggregation process or the aggregate-induced toxicity.p62 (also called sequestosome 1) was first identified as a phosphotyrosine-independent ligand for the Lck SH2 domain (14). It was later found to be a polyubiquitin-binding protein (15)(16)(17). The expression of p62 is up-regulated by several stress conditions, e.g. oxidative stress (18,19), proteasome inhibition (19 -21), or p...