Axons degenerate in the absence of mitochondria in C. elegans.

Many neurodegenerative disorders are associated with mitochondrial defects [1-3]. Mitochondria can play an active role in degeneration by releasing reactive oxygen species and apoptotic factors [4-7]. Alternatively, mitochondria can protect axons from stress and insults, for example by buffering calcium [8]. Recent studies manipulating mitochondria lend support to both of these models [9-13]. Here, we identify a C. elegans mutant, ric-7, in which mitochondria are unable to exit the neuron cell bodies, similar to the kinesin-1/unc-116 mutant. When axons lacking mitochondria are cut with a laser, they rapidly degenerate. Some neurons even spontaneously degenerate in ric-7 mutants. Degeneration can be suppressed by forcing mitochondria into the axons of the mutants. The protective effect of mitochondria is also observed in the wild-type: a majority of axon fragments containing a mitochondrion survive axotomy, whereas those lacking mitochondria degenerate. Thus, mitochondria are not required for axon degeneration and serve a protective role in C. elegans axons.

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans.

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. We identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror and LIN-18/Ryk. PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. We propose that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development.

The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions.

During development, all cells make the decision to live or die. Although the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C. elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C. elegans divisions that produce apoptotic cells [3]. We identified CNT-2, an Arf GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is essential for its function in these divisions. The N terminus of CNT-2, which contains a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2's function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions.

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless.

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin.

BACKGROUND: The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. RESULTS: We show here that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. CONCLUSIONS: mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance.

Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans.

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.

Multiple Wnts and frizzled receptors regulate anteriorly directed cell and growth cone migrations in Caenorhabditis elegans.

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.

Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones.

Protein folding in the mitochondria is assisted by nuclear-encoded compartment-specific chaperones but regulation of the expression of their encoding genes is poorly understood. We found that the mitochondrial matrix HSP70 and HSP60 chaperones, encoded by the Caenorhabditis elegans hsp-6 and hsp-60 genes, were selectively activated by perturbations that impair assembly of multi-subunit mitochondrial complexes or by RNAi of genes encoding mitochondrial chaperones or proteases, which lead to defective protein folding and processing in the organelle. hsp-6 and hsp-60 induction was specific to perturbed mitochondrial protein handling, as neither heat-shock nor endoplasmic reticulum stress nor manipulations that impair mitochondrial steps in intermediary metabolism or ATP synthesis activated the mitochondrial chaperone genes. These observations support the existence of a mitochondrial unfolded protein response that couples mitochondrial chaperone gene expression to changes in the protein handling environment in the organelle.

Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans.

Integrin receptors for extracellular matrix transmit mechanical and biochemical information through molecular connections to the actin cytoskeleton and to several intracellular signaling pathways. In Caenorhabditis elegans, integrins are essential for embryonic development, muscle cell adhesion and contraction, and migration of nerve cell axons and gonadal distal tip cells. To identify key components involved in distal tip cell migration, we are using an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. We have found that talin, a cytoskeletal-associated protein and focal adhesion component, is expressed in the distal tip cell and plays a central role in regulating its migration. Reduction of talin expression caused severe defects in gonad formation because of aberrant distal tip cell migration and also disrupted oocyte maturation and gonad sheath cell structure. Contractile muscle cells showed disorganization of the actin cytoskeleton leading to complete paralysis, a phenotype that was also observed with depletion of pat-2 and pat-3 integrins. These in vivo analyses show that talin is required not only for strong adhesion and cytoskeletal organization by contractile cells, but also for dynamic regulation of integrin signals during cell migration. In addition, induction of distal tip cell migration defects by bacterial RNAi in C. elegans provides an effective screen to identify genes involved in integrin signaling and function.

C. elegans ZAG-1, a Zn-finger-homeodomain protein, regulates axonal development and neuronal differentiation.

Neurons acquire distinct cell identities and implement differential gene programs to generate their appropriate neuronal attributes. On the basis of position, axonal structure and synaptic connectivity, the 302 neurons of the nematode Ceanorhabditis elegans are divided into 118 classes. The development and differentiation of many neurons require the gene zag-1, which encodes a deltaEF1/ZFH-1 Zn-finger-homeodomain protein. zag-1 mutations cause misexpression of neuron-specific genes, block formation of stereotypic axon branches, perturb neuronal migrations, and induce various axon-guidance, fasciculation and branching errors. A zag-1-GFP translational reporter is expressed transiently in most or all neurons during embryogenesis and in select neurons during the first larval stage. Analysis of the zag-1 promoter reveals that zag-1 is expressed in neurons and specific muscles, and that ZAG-1 directly represses its own expression. zag-1 activity also downregulates expression of genes involved in either the synthesis or reuptake of serotonin, dopamine and GABA. We propose that ZAG-1 acts as a transcriptional repressor to regulate multiple, discrete, neuron-specific aspects of terminal differentiation, including cell migration, axonal development and gene expression.

Genetic analysis of the myotubularin family of phosphatases in Caenorhabditis elegans.

Myotubularins (MTMs) constitute a large family of lipid phosphatases that specifically dephosphorylate phosphatidylinositol (3)P. MTM1 and MTM2 are mutated in X-linked myotubular myopathy and Charcot-Marie-Tooth disease (type 4B), respectively, although the mechanisms whereby MTM dysfunction leads to these diseases is unknown. To gain insight into MTM function, we undertook the study of MTMs in the nematode Caenorhabditis elegans, which possesses representative homologues of the four major subgroups of MTMs identified in mammals. As in mammals, we found that C. elegans MTMs mediate distinct functions. let-512 (vps34) encodes the C. elegans homologue of the yeast and mammalian homologue of the phosphatidylinositol 3-kinase Vps34. We found that reduction of mtm-6 (F53A2.8) function by RNA inhibition rescued the larval lethality of let-512 (vps34) mutants and that the reduction of mtm-1 (Y110A7A.5) activity by RNA inhibition rescued the endocytosis defect of let-512 animals. Together, these observations provide genetic evidence that MTMs negatively regulate phosphatidylinositol (3)P levels. Analysis of MTM expression patterns using transcriptional green fluorescence protein reporters demonstrated that these two MTMs exhibit mostly non-overlapping expression patterns and that MTM-green fluorescence protein fusion proteins are localized to different subcellular locations. These observations suggest that some of the different functions of MTMs might, in part, be a consequence of unique expression and localization patterns. However, our finding that at least three C. elegans MTMs play essential roles in coelomocyte endocytosis, a process that also requires VPS34, indicates that MTMs do not simply turn off VPS34 but unexpectedly also function as positive regulators of biological processes.

A survival pathway for Caenorhabditis elegans with a blocked unfolded protein response.

The unfolded protein response (UPR) counteracts stress caused by unprocessed ER client proteins. A genome-wide survey showed impaired induction of many UPR target genes in xbp-1 mutant Caenorhabditis elegans that are unable to signal in the highly conserved IRE1-dependent UPR pathway. However a family of genes, abu (activated in blocked UPR), was induced to higher levels in ER-stressed xbp-1 mutant animals than in ER-stressed wild-type animals. RNA-mediated interference (RNAi) inactivation of a representative abu family member, abu-1 (AC3.3), activated the ER stress marker hsp-4::gfp in otherwise normal animals and killed 50% of ER-stressed ire-1 and xbp-1 mutant animals. Abu-1(RNAi) also enhanced the effect of inactivation of sel-1, an ER-associated protein degradation gene. The nine abu genes encode highly related type I transmembrane proteins whose lumenal domains have sequence similarity to a mammalian cell surface scavenger receptor of endothelial cells that binds chemically modified extracellular proteins and directs their lysosomal degradation. Our findings that ABU-1 is an intracellular protein located within the endomembrane system that is induced by ER stress in xbp-1 mutant animals suggest that ABU proteins may interact with abnormal ER client proteins and this function may be particularly important in animals with an impaired UPR.

PAG-3, a Zn-finger transcription factor, determines neuroblast fate in C. elegans.

During Caenorhabditis elegans development, the patterns of cell divisions, cell fates and programmed cell deaths are reproducible from animal to animal. In a search for mutants with abnormal patterns of programmed cell deaths in the ventral nerve cord, we identified mutations in the gene pag-3, which encodes a zinc-finger transcription factor similar to the mammalian Gfi-1 and Drosophila Senseless proteins. In pag-3 mutants, specific neuroblasts express the pattern of divisions normally associated with their mother cells, producing with each reiteration an abnormal anterior daughter neuroblast and an extra posterior daughter cell that either terminally differentiates or undergoes programmed cell death, which accounts for the extra cell corpses seen in pag-3 mutants. In addition, some neurons do not adopt their normal fates in pag-3 mutants. The phenotype of pag-3 mutants and the expression pattern of the PAG-3 protein suggest that in some lineages pag-3 couples the determination of neuroblast cell fate to subsequent neuronal differentiation. We propose that pag-3 counterparts in other organisms determine blast cell identity and for this reason may lead to cell lineage defects and cell proliferation when mutated.

IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.

The unfolded protein response (UPR), caused by stress, matches the folding capacity of endoplasmic reticulum (ER) to the load of client proteins in the organelle. In yeast, processing of HAC1 mRNA by activated Ire1 leads to synthesis of the transcription factor Hac1 and activation of the UPR. The responses to activated IRE1 in metazoans are less well understood. Here we demonstrate that mutations in either ire-1 or the transcription-factor-encoding xbp-1 gene abolished the UPR in Caenorhabditis elegans. Mammalian XBP-1 is essential for immunoglobulin secretion and development of plasma cells, and high levels of XBP-1 messenger RNA are found in specialized secretory cells. Activation of the UPR causes IRE1-dependent splicing of a small intron from the XBP-1 mRNA both in C. elegans and mice. The protein encoded by the processed murine XBP-1 mRNA accumulated during the UPR, whereas the protein encoded by unprocessed mRNA did not. Purified mouse IRE1 accurately cleaved XBP-1 mRNA in vitro, indicating that XBP-1 mRNA is a direct target of IRE1 endonucleolytic activity. Our findings suggest that physiological ER load regulates a developmental decision in higher eukaryotes.