Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles in Alzheimer's disease (AD) and related tauopathies. It is not understood, however, why the neurofibrillary tangle-containing neurons seen in the AD brains do not die of apoptosis but rather degeneration even though they are constantly awash in a proapoptotic environment. Here, we show that cells overexpressing tau exhibit marked resistance to apoptosis induced by various apoptotic stimuli, which also causes correlated tau hyperphosphorylation and glycogen synthase kinase 3 (GSK-3) activation. GSK-3 overexpression did not potentiate apoptotic stimulus-induced cell apoptosis in the presence of high levels of tau. The resistance of neuronal cells bearing hyperphosphorylated tau to apoptosis was also evident by the inverse staining pattern of PHF-1-positive tau and activated caspase-3 or fragmented nuclei in cells and the brains of rats or tau-transgenic mice. Tau hyperphosphorylation was accompanied by decreases in ␤-catenin phosphorylation and increases in nuclear translocation of ␤-catenin. Reduced levels of ␤-catenin antagonized the antiapoptotic effect of tau, whereas overexpressing ␤-catenin conferred resistance to apoptosis. These results reveal an antiapoptotic function of tau hyperphosphorylation, which likely inhibits competitively phosphorylation of ␤-catenin by GSK-3␤ and hence facilitates the function of ␤-catenin. Our findings suggest that tau phosphorylation may lead the neurons to escape from an acute apoptotic death, implying the essence of neurodegeneration seen in the AD brains and related tauopathies.Alzheimer's disease ͉ tau hyperphosphorylation ͉ glycogen synthase kinase-3 C hronic neurodegeneration characterized by accumulation of hyperphosphorylated tau and formation of neurofibrillary tangles (NFTs) is a hallmark lesion in Alzheimer's disease (AD) and related tauopathies (1-4). Although the mechanism underlying neurodegeneration remains elusive, the idea that neurons undergo apoptosis in the course of neurodegeneration is supported by studies showing that AD-related toxic stimuli, such as ␤-amyloid, cause cell death as manifested by up-regulation of apoptotic markers (5, 6). However, apoptosis accounts for only a minor proportion of neurons lost in AD brains (7); most NFT-bearing neurons undergo chronic degeneration (8-13) rather than apoptosis, even though they are constantly exposed to apoptotic stimuli, suggesting that mechanism(s) exist enabling neurons to escape apoptosis.Studies on postmortem AD brains have demonstrated that abnormally hyperphosphorylated tau is the major protein subunit of NFT (1-4), which suggests that hyperphosphorylation of tau may play a role in leading the neuronal cells to desert apoptosis. Tau is a microtubule-associated protein. The major function of tau is to promote microtubule assembly and maintain the stability of the microtubules. The roles of tau hyperphosphorylation and accumulation in the development of neurofibrillary degeneration seen in the AD brains (1-4) and related t...

Neurofibrillary tangles (NFTs) consisting of the hyperphosphorylated microtubule-associated protein tau are a defining pathological characteristic of Alzheimer's disease (AD). Hyperphosphorylation of tau is hypothesized to impair the microtubule stabilizing function of tau, leading to the formation of paired helical filaments and neuronal death. Glycogen synthase kinase-3 (GSK-3) has been shown to be one of several kinases that mediate tau hyperphosphorylation in vitro. However, molecular mechanisms underlying overactivation of GSK-3 and its potential linkage to AD-like pathologies in vivo remain unclear. Here, we demonstrate that injection of wortmannin (a specific inhibitor of phosphoinositol-3 kinase) or GF-109203X (a specific inhibitor of protein kinase C) into the left ventricle of rat brains leads to overactivation of GSK-3, hyperphosphorylation of tau at Ser 396/404/199/202 and, most significantly, impaired spatial memory. The effects of wortmannin and GF-109203X are additive. Significantly, specific inhibition of GSK-3 activity by LiCl prevents hyperphosphorylation of tau, and spatial memory impairment resulting from PI3K and PKC inhibition. These results indicate that in vivo inhibition of phosphoinositol-3 kinase and protein kinase C results in overactivation of GSK-3 and tau hyperphosphorylation and support a direct role of GSK-3 in the formation of AD-like cognitive deficits.

Microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and other tauopathies and is believed to lead to neurodegeneration in this family of diseases. Here we show that infusion of forskolin, a specific cAMP-dependent protein kinase A (PKA) activator, into the lateral ventricle of brain in adult rats induced activation of PKA by severalfold and concurrently enhanced the phosphorylation of tau at Ser-214, Ser-198, Ser-199, and or Ser-202 (Tau-1 site) and Ser-396 and or Ser-404 (PHF-1 site), which are among the major abnormally hyperphosphorylated sites seen in AD. PKA activation positively correlated to the extent of tau phosphorylation at these sites. Infusion of forskolin together with PKA inhibitor or glycogen synthase kinase-3 (GSK-3) inhibitor revealed that the phosphorylation of tau at Ser-214 was catalyzed by PKA and that the phosphorylation at both the Tau-1 and the PHF-1 sites is induced by basal level of GSK-3, because forskolin activated PKA and not GSK-3 and inhibition of the latter inhibited the phosphorylation at Tau-1 and PHF-1 sites. Inhibition of cdc2, cdk5, or MAPK had no significant effect on the forskolin-induced hyperphosphorylation of tau. Forskolin inhibited spatial memory in a dose-dependent manner in the absence but not in the presence of R p -adenosine 3 ,5 -cyclic monophosphorothioate triethyl ammonium salt, a PKA inhibitor. These results demonstrate for the first time that phosphorylation of tau by PKA primes it for phosphorylation by GSK-3 at the Tau-1 and the PHF-1 sites and that an associated loss in spatial memory is inhibited by inhibition of the hyperphosphorylation of tau. These data provide a novel mechanism of the hyperphosphorylation of tau and identify both PKA and GSK-3 as promising therapeutic targets for AD and other tauopathies.

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The formation of neurofibrillary tangles, mainly composed of hyperphosphorylated tau protein, is a hallmark in the brain of human tauopathies, including Alzheimer's disease (AD). Although neurons bearing neurofibrillary tangles are constantly exposed to various apoptotic stimuli, they do not appear to preferentially die by apoptosis. The underlying mechanism for such resistance to apoptosis remains elusive. Previously, we studied the role of tau phosphorylation in apoptosis and found that tau hyperphosphorylation by glycogen synthase kinase-3 (GSK-3) rendered cells more resistant to apoptosis. In this study, we show that the overexpression of tau without any exogenous activation of kinases also confers increased resistance to apoptosis in both N2a cells and in a tau transgenic mouse model. Mechanistically, the overexpression of tau was associated with a reduced p53 level, decreased release of cytochrome C from mitochondria, and inhibition of caspases-9/-3. Additionally, a decreased phosphorylation and increased nuclear translocation of beta-catenin were also detected in N2a/tau cells, and knockdown of beta-catenin eliminated the anti-apoptotic effect of tau. Furthermore, tau was spontaneously hyperphosphorylated upon overexpression and by staurosporine treatment. The phosphorylation level of p53 decreased upon tau overexpression, and a more profound reduction of the phosphorylated p53 was detected when the cells were treated with lithium and roscovitine, inhibitors of GSK-3 and cyclin-dependent kinase-5 (Cdk-5). These results suggest that the overexpression of tau, which may be hyperphosphorylated by endogenous GSK-3 and Cdk-5, is anti-apoptotic by mechanisms involving modulation of multiple anti-apoptotic factors, including beta-catenin and p53-mitochondria-caspase-mediated apoptotic pathways.

Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment.

Fish often encounters exposures to acute environmental hypoxia either spatially or temporally. Gill organ plays important roles in response to hypoxic stress in fish. Few studies focus on the molecular regulation mechanisms of gills under hypoxic stress. In this study, we investigated the transcriptomic response to 12-h acute hypoxia in gill of a hypoxia tolerant fish, Nile tilapia Oreochromis niloticus through RNA sequencing (RNA-Seq). We sequenced messenger RNA from three control samples and three hypoxia-treated samples. Bioinformatics analysis identified 239 differentially expressed genes (DEG) and 34 genes (DUES) that had significant differential alternative isoform regulation events in at least one exonic region in gill in response to acute hypoxia. The spatiotemporal expression analysis in five tissues (heart, liver, brain, gill, and spleen) sampled at three time points (6, 12, and 24 h) under hypoxia treatment confirmed the significant association of differential exon usages in two DUES genes (TLDC2 and SSX2IPA) with hypoxia conditions. Further functional analysis suggested several energy and immune response-related pathways, e.g., metabolic pathway and antigen processing and presentation, contained the most abundant DEG genes. We found that some GO biological processes for DEG genes were significantly enriched under hypoxic stress, such as glycolysis, metabolic process, generation of precursor metabolites and energy, and cholesterol metabolic process. Our findings suggest abundant differential gene expression changes and alternative isoform regulation events in genes involved in the hypoxia response in gill. Our results provide a basis for exploring the gene regulation mechanism under hypoxic stress in fish.

Exposure to hypoxia induces both acute and chronic stress responses, which plays an important role in health of cultured organisms including growth, reproduction, immunity, and other energy demanding activities. Application of advanced genomic technologies allows rapid identification of hypoxia trait-associated genes and precise selection of superior brood stocks with high tolerance in tilapia. By applying QTL-seq and double-digest restriction-site associated DNA sequencing (ddRAD-seq) techniques, we identified four genome-wide significant quantitative trait loci (QTLs) for hypoxia tolerance and many suggestive QTLs in Nile tilapia. These QTLs explained 6.6-14.7% of the phenotypic variance. Further analysis revealed that single nucleotide polymorphisms (SNPs) in exons of both GPR132 and ABCG4 genes located in genome-wide QTL intervals were significantly associated with hypoxia-tolerant traits. Expression analysis of both genes suggested that they were strong candidate genes involved into hypoxia tolerance in tilapia. Our findings suggest that both QTL-seq and ddRAD-seq techniques can be effectively utilized in QTL mapping of hypoxia traits in fish. Our data supply a basis for further marker-assisted selection of super lines with a high level of tolerance against low oxygen stress in the tilapia.