Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.

Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-ß.

Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.

Homeostatic generation of reactive oxygen species protects the zebrafish liver from steatosis.

UNLABELLED: Nonalcoholic fatty liver disease is the most common liver disease in both adults and children. The earliest stage of this disease is hepatic steatosis, in which triglycerides are deposited as cytoplasmic lipid droplets in hepatocytes. Through a forward genetic approach in zebrafish, we found that guanosine monophosphate (GMP) synthetase mutant larvae develop hepatic steatosis. We further demonstrate that activity of the small GTPase Rac1 and Rac1-mediated production of reactive oxygen species (ROS) are down-regulated in GMP synthetase mutant larvae. Inhibition of Rac1 activity or ROS production in wild-type larvae by small molecule inhibitors was sufficient to induce hepatic steatosis. More conclusively, treating larvae with hydrogen peroxide, a diffusible ROS that has been implicated as a signaling molecule, alleviated hepatic steatosis in both GMP synthetase mutant and Rac1 inhibitor-treated larvae, indicating that homeostatic production of ROS is required to prevent hepatic steatosis. We further found that ROS positively regulate the expression of the triglyceride hydrolase gene, which is responsible for the mobilization of stored triglycerides in hepatocytes. Consistently, inhibition of triglyceride hydrolase activity in wild-type larvae by a small molecule inhibitor was sufficient to induce hepatic steatosis. CONCLUSION: De novo GMP synthesis influences the activation of the small GTPase Rac1, which controls hepatic lipid dynamics through ROS-mediated regulation of triglyceride hydrolase expression in hepatocytes.

Mutation of zebrafish Snapc4 is associated with loss of the intrahepatic biliary network.

Biliary epithelial cells line the intrahepatic biliary network, a complex three-dimensional network of conduits. The loss of differentiated biliary epithelial cells is the primary cause of many congenital liver diseases. We identified a zebrafish snapc4 (small nuclear RNA-activating complex polypeptide 4) mutant in which biliary epithelial cells initially differentiate but subsequently disappear. In these snapc4 mutant larvae, biliary epithelial cells undergo apoptosis, leading to degeneration of the intrahepatic biliary network. Consequently, in snapc4 mutant larvae, biliary transport of ingested fluorescent lipids to the gallbladder is blocked. Snapc4 is the largest subunit of a protein complex that regulates small nuclear RNA (snRNA) transcription. The snapc4(s445) mutation causes a truncation of the C-terminus, thereby deleting the domain responsible for a specific interaction with Snapc2, a vertebrate specific subunit of the SNAP complex. This mutation leads to a hypomorphic phenotype, as only a subset of snRNA transcripts are quantitatively altered in snapc4(s445) mutant larvae. snapc2 knockdown also disrupts the intrahepatic biliary network in a similar fashion as in snapc4(s445) mutant larvae. These data indicate that the physical interaction between Snapc2 and Snapc4 is important for the expression of a subset of snRNAs and biliary epithelial cell survival in zebrafish.

Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development.

Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

Significant association of the neurexin-1 gene (NRXN1) with nicotine dependence in European- and African-American smokers.

The neurexin-1 gene (NRXN1) has been shown to play a fundamental role in synaptogenesis and synaptic maintenance, as well as Ca(2+) channel and NMDA receptor recruitment. A recent study reported that NRXN1 is associated with nicotine dependence (ND); this, together with the intriguing physiological functions of the gene, motivated us to investigate the involvement of NRXN1 with ND in independent samples. In this study, we analyzed 21 single nucleotide polymorphisms (SNPs) within NRXN1 for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerström test for ND (FTND). Individual SNP and haplotype association tests were carried out in a sample consisting of 2037 individuals from 602 nuclear families of African-American (AA) or European-American (EA) origin. Individual SNP analysis revealed significant associations of rs2193225 with SQ, HSI and FTND (P = 0.00014-0.0010) in the EA sample and with SQ (P = 0.0019) in the pooled sample under the dominant model and rs6721498 with SQ, HSI and FTND in the AA (P = 0.000090-0.0000086) and pooled (P = 0.0010-0.00099) samples under the additive model, following correction for multiple testing. Haplotype analysis revealed six major haplotypes in the AA sample (minimum P-value = 0.000079), one major haplotype in the EA sample (P = 0.0062) and five major haplotypes in the pooled sample (minimum P-value = 0.00083), which showed significant association with all three ND measures; all of these contained one specific allele from one of the two aforementioned SNPs. Based on our findings that NRXN1 has significant association with ND in two independent samples, recent findings that NRXN1 plays an important role in synaptic development, and the previous report of association, we conclude that this gene represents a strong candidate for involvement in the etiology of ND.

Significant association of glutamate receptor, ionotropic N-methyl-D-aspartate 3A (GRIN3A), with nicotine dependence in European- and African-American smokers.

The glutamate receptor gene, ionotropic N-methyl-D-aspartate 3A (GRIN3A), is one of the seven that code for subunits of N-methyl-D-aspartate receptors, which play an essential role at many synapses in the brain, regulating ion flow across membranes in response to glutamate signaling. In this study, we analyzed 25 single nucleotide polymorphisms (SNPs) within GRIN3A for association with nicotine dependence (ND), which was assessed by smoking quantity, heaviness of smoking index, and the Fagerström test for ND. Both individual SNP and haplotype association tests were performed in African-American (AA) and European-American (EA) samples as well as in the pooled sample consisting of 2,037 individuals from 602 nuclear families. Individual SNP analysis revealed significant associations of 5, 5, and 4 SNPs with at least one ND measure in the pooled, EA, and AA samples, respectively. Of them, SNPs rs17189632 and rs10121600 in the pooled sample and rs11788456 in the EA sample remained significant after correction for multiple testing. On the basis of the blocks determined with Haploview, we performed haplotype-based association analysis and found 2, 4, and 1 haplotype(s) that are significantly associated with at least one ND measure in the pooled, EA, and AA samples, respectively. Some of them remained significant after correction for multiple testing. We concluded that GRIN3A represents a strong candidate for involvement in the etiology of ND and warrants further investigation in independent samples.

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function.

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Alzheimer disease: a tale of two prions.

Alzheimer disease (AD) has traditionally been thought to involve the misfolding and aggregation of two different factors that contribute in parallel to pathogenesis: amyloid-ß (Aß) peptides, which represent proteolytic fragments of the transmembrane amyloid precursor protein, and tau, which normally functions as a neuronally enriched, microtubule-associated protein that predominantly accumulates in axons. Recent evidence has challenged this model, however, by revealing numerous functional interactions between Aß and tau in the context of pathogenic mechanisms for AD. Moreover, the propagation of toxic, misfolded Aß and tau bears a striking resemblance to the propagation of toxic, misfolded forms of the canonical prion protein, PrP, and misfolded Aß has been shown to induce tau misfolding in vitro through direct, intermolecular interaction. In this review we discuss evidence for the prion-like properties of both Aß and tau individually, as well as the intriguing possibility that misfolded Aß acts as a template for tau misfolding in vivo.