Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related Amyotrophic lateral sclerosis (ALS)2 is an adult onset, typically fatal, neurodegenerative disorder of poorly understood etiology that destroys motor neurons (1). Dominant mutations in superoxide dismutase 1 were the first established cause of ALS (2, 3); however, ϳ90% of cases occur sporadically with no clear genetic link, and there is no effective treatment for the condition (3).An important breakthrough toward understanding ALS etiology was made by Neumann et al. (4), who showed that TDP-43 is a major constituent of cytoplasmic ubiquitin-positive inclusions that accumulate in the degenerating motor neurons of ALS patients and individuals with ubiquitin-positive fronto-temporal lobar degeneration. TDP-43 is an essential nuclear RNA-binding protein that participates in transcriptional repression, exon splicing inhibition, and mRNA stabilization (5-8). More recently, dominant mutations in the TARDBP gene encoding TDP-43 were found to cause a subset of inherited ubiquitinpositive fronto-temporal lobar degeneration and ALS cases (9 -11), which strongly supports a direct role for TDP-43 aggregation in ALS pathogenesis.The mechanisms whereby mutations in TDP-43 cause neurodegeneration are not known, but models invoking toxic gain of function and loss of critical nuclear function are equally plausible. Virtually all ALS-associated mutations in TDP-43 occur in an unstructured Gly-rich domain that binds to heterogeneous ribonucleoprotein A/B complexes (12-14). ALS-associated mutants of TDP-43 are hyperphosphorylated, ubiquitylated, aggregationprone, and cleaved into 25-and 35-kDa C-terminal fragments that exhibit cytotoxicity in cellulo (15-18). TDP-43 is degraded by proteasome and autophagy-dependent pathways, which may be mediated in part through its association with the ubiquitin-binding protein .Recent studies have shown that expression of mutant or wild-type TDP-43 is neurotoxic to zebrafish, Drosophila, and mice (22)(23)(24)(25)(26). In several of these studies, TDP-43-induced neurodegeneration occurred in the absence of detectable cytosolic TDP-43 aggregation (22,23,25). Combined with the finding that TDP-43 loss of function induces motor neuron deficits in Drosophila (27), it is plausible that too much or too little nuclear TDP-43 disrupts RNA processing events critical for motor neuron function.Remarkably, mutations in a structurally related RNA-binding protein, FUS/TLS, also cause dominantly inherited ALS (28,29). Like TDP-43, FUS/TLS forms cytosolic aggregates in degenerating neurons of ALS patients, and FUS/TLS mutants exhibit increased cytosolic localization in transfected cells (28,29). The mechanisms linking FUS/TLS mutation to motor neuron degeneration are unclear; however, FUS/TLS localizes to dendritic spines following mGluR activation, where it may function in the localized protein translation important for neuron functio...

Amyotrophic lateral sclerosis (ALS)5 is a progressive, typically fatal, neurodegenerative disease that targets motor neurons (1). The cumulative lifetime risk for ALS is ϳ1 in 1000, which is comparable with the occurrence rate of multiple sclerosis (1). However, only about 3,000 cases of ALS are observed at any given time in the United States, because of the low fiveyear median survival rate (20%) for this disease. There are currently no effective treatments to halt the course of ALS.Approximately 10% of ALS cases, termed familial ALS (fALS), have a clear genetic link. Dominant mutations in the Cu,Zn-superoxide dismutase 1 (SOD1) are responsible for ϳ20% of fALS (2). Well over 100 different disease-associated mutations in SOD1 have been identified in humans (3). The mutations occur throughout the SOD1 open reading frame and do not alter the catalytic properties of the SOD1 enzyme per se (4). Instead, mutant SOD1 proteins are thought to acquire an abnormal fold that confers a toxic gain-of-function activity (4). Multiple cellular processes, including mitochondrial function, axon transport, and glutamate transporter function are adversely affected by toxic SOD1 mutants (5-10). Remarkably, mouse models of SOD1-induced ALS have shown that it is not a neuron-autonomous disease; expression of toxic SOD1 mutants in non-neuronal glial cells is sufficient to induce neuron pathology, whereas expression of toxic SOD1 mutants in motor neurons is insufficient for full expression of the disease phenotype (11,12). The SOD1 mouse model has been used to test candidate ALS therapeutics, including gene therapies directed toward silencing toxic SOD1 alleles in fALS (13)(14)(15).Sporadic ALS (sALS), representing 90 -95% of all ALS cases, occurs in the absence of a family history of disease, but follows a clinical course that is similar to SOD1 ALS. A hallmark of sALS is the presence of ubiquitin (Ub)-positive cytosolic aggregates in degenerating motor neurons (16). Recently, Neumann et al. (17) demonstrated that the 43-kDa human immunodeficiency virus transactivating region DNA-binding domain protein (TDP-43) is a major protein component of ubiquitin-positive aggregates in patients with sALS or ubiquitin-positive fronto-temporal lobular dementia. First identified as a binding factor of the long-terminal repeat region of the human immunodeficiency virus genome, TDP-43 is a nuclear RNA-binding protein that regulates splicing of the cystic fibrosis transmembrane conductance regulator mRNA (18 -20). Inactivation of TDP-43 through RNA interference caused enhanced transcription of cyclin-dependent kinase 6, hyperphosphorylation of the

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1␦ (CK1 ␦) and casein kinase 1⑀ (CK1⑀) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662 3 Ala mutation that abrogated PER2 phosphorylation significantly reduced its halflife, whereas a phosphomimetic Ser-662 3 Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.

Small-molecule inhibitors of protein kinases have contributed immensely to our understanding of biological signaling pathways and have been exploited therapeutically for the treatment of cancers and other disease states. The pyridinyl imidazole compounds SB 203580 and SB 202190 were identified as ATP competitive antagonists of the p38 stress-activated protein kinases and have been widely used to elucidate p38-dependent cellular processes. Here, we identify SB 203580 and SB 202190 as potent inhibitors of stress-induced CREB phosphorylation on Serine 111 (Ser-111) in intact cells. Unexpectedly, we found that the inhibitory activity of SB 203580 and SB 202190 on CREB phosphorylation was independent of p38, but instead correlated with inhibition of casein kinase 1 (CK1) in vitro. The inhibition of CK1-mediated CREB phosphorylation by concentrations of pyridinyl imidazoles commonly employed to suppress p38, suggests that in some cases conclusions of p38-dependence derived solely from the use of these inhibitors may be invalid.

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.