A novel calcium-binding protein is associated with tau proteins in tauopathy.

Tauopathies are a group of neurological disorders characterized by the presence of intraneuronal hyperphosphorylated and filamentous tau. Mutations in the tau gene have been found in kindred with tauopathy. The expression of the human tau mutant in transgenic mice induced neurodegeneration, indicating that tau plays a central pathological role. However, the molecular mechanism leading to tau-mediated neurodegeneration is poorly understood. To gain insights into the role that tau plays in neurodegeneration, human tau proteins were immunoprecipitated from brain lysates of the tauopathy mouse model JNPL3, which develops neurodegeneration in age-dependent manner. In the present work, a novel EF-hand domain-containing protein was found associated with tau proteins in brain lysate of 12-month-old JNPL3 mice. The association between tau proteins and the novel identified protein appears to be induced by the neurodegeneration process as these two proteins were not found associated in young JNPL3 mice. Consistently, the novel protein co-purified with the pathological sarkosyl insoluble tau in terminally ill JNPL3 mice. Calcium-binding assays demonstrated that this protein binds calcium effectively. Finally, the association between tau and the novel calcium-binding protein is conserved in human and enriched in Alzheimer's disease brain. Taken together, the identification of a novel calcium-binding protein associated with tau protein in terminally ill tauopathy mouse model and its confirmation in human brain lysate suggests that this association may play an important physiological and/or pathological role.

Disruption of the dynamic sub-cellular localization of the Xenopus tumorhead protein causes embryonic lethality at the early gastrula transition.

The Xenopus laevis tumorhead (TH) protein, a positive regulator of cell proliferation during embryogenesis, shuttles from the cell periphery into the nucleus during embryogenesis. In these studies, we performed a detailed analysis of TH's subcellular localization pattern to characterize its dynamic behavior. We found that TH exhibits distinct patterns of localization in different germ layers. At the blastula stage, TH is present in the apical cell periphery of prospective mesodermal and ectodermal cells. At the gastrula stage, TH is distributed throughout the entire cytoplasm of prospective mesodermal and ectodermal cells, whereas it shows nuclear localization in presumptive endodermal cells. TH moves into the nucleus of mesodermal and ectodermal cells during the neurula and early tailbud stages. To understand if TH is regulated by changes in its subcellular localization, we used a TH mutant containing signals for farnesylation and palmitoylation to tether the protein to the plasma membrane. Ubiquitous overexpression of this mutant causes embryonic lethality at the early gastrula transition. Further examination using TUNEL assays indicated that wild-type TH overexpression induces apoptosis during gastrulation, and that this effect is exacerbated by the overexpression of the membrane-bound TH mutant. Taken together, our results suggest that changes in the sub-cellular localization of the TH protein are important for its function because blocking the nuclear translocation of overexpressed TH increases apoptosis and causes embryos to die. Our data also suggest that TH plays a role outside the nucleus when it is present at the cell periphery.

Identification of TH-B, a new oligomeric variant of the Xenopus morphogenetic factor, tumorhead.

The Xenopus laevis gene tumorhead (TH) is a regulator of cell proliferation of the ectodermal germ layer during embryonic development. TH overexpression results in increased cell proliferation within the developing ectoderm, causing an expansion of the neural plate. Conversely, loss of TH function results in inhibition of proliferation of ectodermal cells. Embryos with altered levels of TH protein are unable to express neural differentiation markers, indicating that the effect of TH in proliferation is linked with differentiation in the nervous system. To date, the molecular mechanism by which TH affects cell proliferation during embryogenesis is unknown. We have utilized the yeast two-hybrid system to identify protein partners of TH that could lead us to define the mechanism or pathway through which TH functions. Using this assay we have identified a new variant of TH designated TH-B, as a potential protein partner of the original TH, now referred to as TH-A. The sequence for TH-B was found to be 85% identical at the amino acid level to the TH-A sequence. Further characterization of the TH-B variant using RT-PCR indicates that it is expressed ubiquitously throughout development from early cleavage stages until at least the tadpole stage. TH-B association with TH-A was confirmed in co-immnoprecipitation studies in Xenopus, indicating that the two variants may function as an oligomer in vivo. These studies reveal the presence of an isoform of TH that may possess novel functional capabilities.

Tumorhead distribution to cytoplasmic membrane of neural plate cells is positively regulated by Xenopus p21-activated kinase 1 (X-PAK1).

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.

The Xenopus laevis morphogenetic factor, tumorhead, causes defects in polarized growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe.

Tumorhead (TH) is a maternally expressed gene product that regulates neural tube morphogenesis in the amphibian, Xenopus laevis. Here we describe the effects of TH expression in the rod-shaped fission yeast, Schizosaccharomyces pombe. Expression of TH in S. pombe resulted in severe morphological defects, including ovoid, bottle-shaped, and enlarged cells. Multi-septated cells were also observed in TH expressing cultures, indicating that TH is inhibitory to a process required for the completion of cytokinesis. TH expression caused significant actin and microtubule cytoskeletal defects, including depolarization of the cortical F-actin cytoskeleton and increased microtubule formation. Immunostaining experiments showed that TH is localized to the cell cortex, cell tips, and septum in S. pombe cells. Localization of TH to the cell cortex was dependent on the S. pombe PAK homolog, Shk1. Moreover, TH expression was inhibitory to the growth of a mutant defective in Shk1 function, suggesting that TH may interact with a component(s) of a PAK-mediated morphogenetic regulatory pathway in S. pombe. Taken together, our findings demonstrate that S. pombe may be a useful model organism for identifying potential TH interacting factors.