ABSTRACT
The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor ß-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished ß-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.
Subject(s)
Forkhead Transcription Factors/metabolism , Huntington Disease/etiology , Neurons/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Wnt/metabolism , Aged , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Line , Female , Humans , Huntington Disease/metabolism , Male , Mice , Mice, Transgenic , Middle Aged , Oligonucleotide Array Sequence Analysis , Presenilin-1/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Serotonin Plasma Membrane Transport Proteins/genetics , Serotonin Plasma Membrane Transport Proteins/metabolism , Wnt Signaling PathwayABSTRACT
Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Histone Deacetylases/genetics , Longevity/physiology , Sirtuins/genetics , Aging/genetics , Aging/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caloric Restriction , Crosses, Genetic , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Gene Expression , Histone Deacetylases/metabolism , Longevity/genetics , Male , RNA, Messenger/analysis , RNA, Messenger/genetics , Sirtuins/metabolismABSTRACT
One of the current challenges of neurodegenerative disease research is to determine whether signaling pathways that are essential to cellular homeostasis might contribute to neuronal survival and modulate the pathogenic process in human disease. In Caenorhabditis elegans, sir-2.1/SIRT1 overexpression protects neurons from the early phases of expanded polyglutamine (polyQ) toxicity, and this protection requires the longevity-promoting factor daf-16/FOXO. Here, we show that this neuroprotective effect also requires the DAF-16/FOXO partner bar-1/ß-catenin and putative DAF-16-regulated gene ucp-4, the sole mitochondrial uncoupling protein (UCP) in nematodes. These results fit with a previously proposed mechanism in which the ß-catenin FOXO and SIRT1 proteins may together regulate gene expression and cell survival. Knockdown of ß-catenin enhanced the vulnerability to cell death of mutant-huntingtin striatal cells derived from the HdhQ111 knock-in mice. In addition, this effect was compensated by SIRT1 overexpression and accompanied by the modulation of neuronal UCP expression levels, further highlighting a cross-talk between ß-catenin and SIRT1 in the modulation of mutant polyQ cytoxicity. Taken together, these results suggest that integration of ß-catenin, sirtuin and FOXO signaling protects from the early phases of mutant huntingtin toxicity.
Subject(s)
Caenorhabditis elegans Proteins/biosynthesis , Caenorhabditis elegans Proteins/physiology , Cytoskeletal Proteins/biosynthesis , Nerve Tissue Proteins/toxicity , Signal Transduction/physiology , Sirtuins/physiology , Transcription Factors/biosynthesis , beta Catenin/biosynthesis , Animals , Animals, Genetically Modified , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Cell Survival/drug effects , Cell Survival/physiology , Cytoskeletal Proteins/genetics , Forkhead Transcription Factors , Huntingtin Protein , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Sirtuins/genetics , Transcription Factors/genetics , beta Catenin/geneticsABSTRACT
Most animals exhibit mirror-symmetric body plans, yet the molecular constituents from which they are formed are often chiral. In planarian flatworms, centrioles are arranged in a bilaterally symmetric pattern across the ventral epidermis. Here, we found that this pattern is generated by a network of centrioles with prominent chiral asymmetric properties. We identify centriole components required for establishing asymmetric connections between centrioles and balancing their effects to align centrioles along polarity fields. SMED-ODF2, SMED-VFL1, and SMED-VFL3 affect the assembly of centriole appendages that tether cytoskeletal connectors to position the centrioles. We further show that the medio-lateral polarization of centrioles relies on mechanisms that are partly distinct on the left and right sides of the planarian body. Our findings shed light on how bilaterally symmetrical patterns can emerge from chiral cellular organizations.
Subject(s)
Body Patterning/physiology , Cell Polarity/physiology , Planarians/metabolism , Animals , Cell Cycle Proteins/metabolism , Centrioles/physiology , Cilia/physiology , Cytoskeleton , Epidermal Cells , Epidermis , MicrotubulesABSTRACT
In the past few years, the freshwater planarian Schmidtea mediterranea has emerged as a powerful model system to study the assembly and function of cilia. S. mediterranea is a free-living flatworm that uses the beating of cilia on its ventral epidermis for locomotion. The ventral epidermis is composed of a single layer of multiciliated cells highly similar to the multiciliated cells that line the airway, the brain ventricles, and the oviducts in humans. The genome of S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference (RNAi) are available. Locomotion defects induced by perturbing the expression of ciliary genes can be often detected by simple visual screening, and more subtle defects can be detected by measuring locomotion speed. Cilia are present in large numbers and are directly accessible, which facilitates analyses by immunofluorescence and electron microscopy. Here we describe a set of methods for maintaining planarians in the lab. These include gene knockout by RNAi, cilia visualization by immunofluorescence, transmission electron microscopy, and live imaging.