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In migrating NRK cells, aPKCs control the dynamics of turnover of paxillin-containing focal adhesions (FA) determining migration rate. Using a proteomic approach (two-dimensional fluorescence difference gel electrophoresis), dynein intermediate chain 2 (dynein IC2) was identified as a protein that is phosphorylated inducibly during cell migration in a PKC-regulated manner. By gene silencing and co-immunoprecipitation studies, we show that dynein IC2 regulates the speed of cell migration through its interaction with paxillin. This interaction is controlled by serine 84 phosphorylation, which lies on the aPKC pathway. The evidence presented thus links aPKC control of migration to the dynein control of FA turnover through paxillin.

Background: Intracellular transport of cargoes including organelles, vesicles, signalling molecules, protein complexes, and RNAs, is essential for normal function of eukaryotic cells. The cytoplasmic dynein complex is an important motor that moves cargos along microtubule tracks within the cell. In mammals this multiprotein complex includes dynein intermediate chains 1 and 2 which are encoded by two genes, Dync1i1 and Dync1i2. These proteins are involved in dynein cargo binding and dynein complexes with different intermediate chains bind to specific cargoes, although the mechanisms to achieve this are not known. The DYNC1I1 and DYNC1I2 proteins are translated from different splice isoforms, and specific forms of each protein are essential for the function of different dynein complexes in neurons., Methodology/principal Findings: Here we have undertaken a systematic survey of the dynein intermediate chain splice isoforms in mouse, basing our study on mRNA expression patterns in a range of tissues, and on bioinformatics analysis of mouse, rat and human genomic and cDNA sequences. We found a complex pattern of alternative splicing of both dynein intermediate chain genes, with maximum complexity in the embryonic and adult nervous system. We have found novel transcripts, including some with orthologues in human and rat, and a new promoter and alternative non-coding exon 1 for Dync1i2., Conclusions/significance: These data, including the cloned isoforms will be essential for understanding the role of intermediate chains in the cytoplasmic dynein complex, particularly their role in cargo binding within individual tissues including different brain regions.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute approximately 10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15-20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1(G93A). In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1(G93A) motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1(G93A) motor neurons.

Aims: The dynein-dynactin complex, mostly recognized for axonal retrograde transport in neurones, has an ever growing list of essential subcellular functions. Here, the distribution of complex subunits in human central nervous system (CNS) has been assessed using immunohistochemistry in order to test the hypothesis that this may be altered in neurodegenerative disease., Methods: Three dynactin and two dynein subunits were immunolocalized in the CNS of human post mortem sections from motor neurone disease, Alzheimer's disease and patients with no neurological disease., Results: Unexpectedly, coordinated distribution of complex subunits was not evident, even in normal tissues. Complex subunits were differentially localized in brain and spinal cord, and localization of certain subunits, but not others, occurred in pathological structures of motor neurone and Alzheimer's diseases., Conclusions: These results suggest that dynein-dynactin complex subunits may have specific subcellular roles, and primary events that disturb the function of individual components may result in disequilibrium of subunit pools, with the possibility that availability for normal cytoplasmic functions becomes impaired, with consequent organelle and axonal transport misfunction.

Cytoplasmic dynein 1 is a multi-subunit motor protein responsible for microtubule minus end-directed transport in axons. The cytoplasmic dynein intermediate chain subunit has a scaffold-like role in the dynein complex; it directly binds to four of the other five subunits, the heavy chain and the three light chains. The intermediate chain also binds the p150 subunit of dynactin, a protein that is essential for many dynein functions. We reexamined the generation of rat cytoplasmic dynein intermediate chain isoforms by the alternative splicing of the two genes that encode this subunit and identified an additional splicing site in intermediate chain gene 1. We reinvestigated the expression of the intermediate chain 1 isoforms in cultured cells and tissues. The Loa mouse, which is homozygote lethal, contains a missense mutation in the region of the cytoplasmic dynein heavy chain gene that binds the intermediate chain. Protein binding studies showed that all six intermediate chains were able to bind to the mutated heavy chain. GFP-tagged intermediate chains were constructed and PC12 cell lines with stable expression of the fusion proteins were established. Live cell imaging and comparative immunocytochemical analyses show that dynein is enriched in the actin rich region of growth cones., ((c) 2007 Wiley-Liss, Inc.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.

We have shown in a mouse model of motor neuron disease, the legs-at-odd-angles (Loa) mutant, and that mutations in the cytoplasmic dynein heavy chain gene (Dnchc1) cause motor neuron degeneration. Mice exhibiting the Loa phenotype suffer progressive loss of locomotor function and homozygous animals have neuronal inclusion bodies that are positive for SOD1, CDK5, neurofilament and ubiquitin proteins. As this phenotype models some aspects of human motor neuron degeneration disorders, we think there is a reasonable likelihood that dynein may be a causative gene or susceptibility factor in human motor neuron disease. Therefore we have screened exons of this gene in a set of human patients with familial forms of disparate motor neuron degeneration diseases, affecting both upper and lower motor neurons: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and hereditary spastic paraplegia. As part of this study, we have determined that DNCHC1 is a large gene of 78 exons spanning 86 kb genomic length. We have focused on the exons known to be mutated in Loa, and in a very similar mouse mutation, cramping 1 (Cra1); both mutations result in loss of anterior horn cells. The exons studied are highly conserved in a wide range of eukaryotes. We screened our patient samples by sequencing and although we detect single nucleotide polymorphisms, our results show these occur at the same frequency in our patient group as in control samples of unaffected individuals. Therefore we do not find any association between familial motor neuron disease and the genotypes presented here in the exons screened.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.