The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity.

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.

CLIPR-59: a protein essential for neuromuscular junction stability during mouse late embryonic development.

CLIPR-59 is a new member of the cytoplasmic linker proteins (CLIP) family mainly localized to the trans-Golgi network. We show here that Clipr-59 expression in mice is restricted to specific pools of neurons, in particular motoneurons (MNs), and progressively increases from embryonic day 12.5 (E12.5) until the first postnatal days. We generated a Clipr-59 knockout mouse model that presents perinatal lethality due to respiratory defects. Physiological experiments revealed that this altered innervation prevents the normal nerve-elicited contraction of the mutant diaphragm that is reduced both in amplitude and fatigue-resistance at E18.5, despite unaffected functional muscular contractility. Innervation of the mutant diaphragm is not altered until E15.5, but is then partially lost in the most distal parts of the muscle. Ultrastructural observations of neuromuscular junctions (NMJs) in the distal region of the diaphragm reveal a normal organization, but a lower density of nerve terminals capped by terminal Schwann cells in E18.5 mutant when compared with control embryos. Similar defects in NMJ stability, with a hierarchy of severity along the caudo-rostral axis, are also observed in other muscles innervated by facial and spinal MNs in Clipr-59 mutant mice. Clipr-59 deficiency therefore affects axon maintenance but not axon guidance toward muscle targets. Thus, CLIPR-59 is involved in the stabilization of specific motor axons at the NMJ during mouse late embryogenesis and its role is crucial for mouse perinatal development.

Integration of ß-catenin, sirtuin, and FOXO signaling protects from mutant huntingtin toxicity.

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.

Meclizine is neuroprotective in models of Huntington's disease.

Defects in cellular energy metabolism represent an early feature in a variety of human neurodegenerative diseases. Recent studies have shown that targeting energy metabolism can protect against neuronal cell death in such diseases. Here, we show that meclizine, a clinically used drug that we have recently shown to silence oxidative metabolism, suppresses apoptotic cell death in a murine cellular model of polyglutamine (polyQ) toxicity. We further show that this protective effect extends to neuronal dystrophy and cell death in Caenorhabditis elegans and Drosophila melanogaster models of polyQ toxicity. Meclizine's mechanism of action is not attributable to its anti-histaminergic or anti-muscarinic activity, but rather, strongly correlates with its ability to suppress mitochondrial respiration. Since meclizine is an approved drug that crosses the blood-brain barrier, it may hold therapeutic potential in the treatment of polyQ toxicity disorders, such as Huntington's disease.

The initiation factor eIF3-f is a major target for atrogin1/MAFbx function in skeletal muscle atrophy.

In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3-f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3-f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3-f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3-f expression induces atrophy in myotubes. Finally, eIF3-f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3-f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3-f seems to be an attractive therapeutic target.

Trio controls the mature organization of neuronal clusters in the hindbrain.

During the embryonic development of the hindbrain, movements of neuronal clusters allow the formation of mature "pools", in particular for inferior olivary (ION) and facial motor (fMN) nuclei. The cellular mechanisms of neuron clustering remain uncharacterized. We report that the absence of the Rho-guanine exchange factor Trio, which can activate both RhoG and Rac1 in vivo, prevents the proper formation of ION and fMN subnuclei. Rac1, but not RhoG, appears to be a downstream actor in Trio-induced lamellation. In addition, we report that Cadherin-11 is expressed by a subset of neurons through the overall period of ION and fMN parcellations, and defects observed in trio mutant mice are located specifically in Cadherin-11-expressing regions. Moreover, endogenous Cadherin-11 is found in a complex with Trio when lamellation occurs. Altogether, those results establish a link between Trio activity, the subsequent Rac1 activation, and neuronal clusters organization, as well as a possible recruitment of the Cadherin-11 adhesive receptor to form a complex with Trio.

Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 in S. cerevisiae.

Orderly progression through the eukaryotic cell cycle is a complex process involving both regulation of cyclin dependent kinase activity and control of specific substrate-Cdk interactions. In Saccharomyces cerevisiae, the mitotic cyclin Clb2 has a central role in regulating the onset of anaphase and in maintaining the cellular shape of the bud by inhibiting growth polarization induced in G1. However, how Clb2 and the partially redundant cyclin Clb1 confer specificity to Cdk1 in these processes still remains unclear. Here, we show that Clb2 mutants impaired in nuclear import or export are differentially affected for subsets of Clb2 functions while remaining fully functional for others. Our data support a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation. By contrast, the nuclear form of the cyclin is required for timely initiation of anaphase. Clb2 localization influences its stage-specific degradation as well. We report that Clb2 trapped in the cytoplasm is stabilized during anaphase but not at the time of mitotic exit. Altogether, our results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function.

The pro-apoptotic activity of a vertebrate Bar-like homeobox gene plays a key role in patterning the Xenopus neural plate by limiting the number of chordin- and shh-expressing cells.

Targeted disruption of effectors molecules of the apoptotic pathway have demonstrated the occurrence and magnitude of early programmed cell death (EPCD), a form of apoptosis that affects proliferating and newly differentiated cells in vertebrates, and most dramatically cells of the central nervous system (CNS). Little is known about the molecular pathways controlling apoptosis at these early developmental stages, as the roles of EPCD during patterning of the developing nervous system. We describe a new function, in Xenopus neurodevelopment, for a highly conserved homeodomain protein Barhl2. Barhl2 promotes apoptosis in the Xenopus neuroectoderm and mesoderm, acting as a transcriptional repressor, through a mechanism that cannot be attributed to an unspecific cellular stress response. We show that the pro-apoptotic activity of Barhl2 is essential during normal neural plate formation as it limits the number of chordin- and Xshh-expressing cells in the prospective notochord and floorplate, which act as organizing centers. Our findings show that Barhl2 is part of a pathway regulating EPCD. They also provide evidence that apoptosis plays an important role in regulating the size of organizing centers.

RLIP, an effector of the Ral GTPases, is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis.

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. We have looked for actors that associate with RLIP during mitosis. We show here that RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and that this interaction is crucial for the mitotic phosphorylation of Epsin that, once phosphorylated, is no longer competent for endocytosis. We show also that this latter phosphorylation is dependent on Ral signaling. We propose that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off.

Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit.

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.