The Apoptotic Engulfment Machinery Regulates Axonal Degeneration in C. elegans Neurons.
ABSTRACT: Axonal degeneration is a characteristic feature of neurodegenerative disease and nerve injury. Here, we characterize axonal degeneration in Caenorhabditis elegans neurons following laser-induced axotomy. We show that this process proceeds independently of the WLD(S) and Nmnat pathway and requires the axonal clearance machinery that includes the conserved transmembrane receptor CED-1/Draper, the adaptor protein CED-6, the guanine nucleotide exchange factor complex Crk/Mbc/dCed-12 (CED-2/CED-5/CED-12), and the small GTPase Rac1 (CED-10). We demonstrate that CED-1 and CED-6 function non-cell autonomously in the surrounding hypodermis, which we show acts as the engulfing tissue for the severed axon. Moreover, we establish a function in this process for CED-7, an ATP-binding cassette (ABC) transporter, and NRF-5, a lipid-binding protein, both associated with release of lipid-vesicles during apoptotic cell clearance. Thus, our results reveal the existence of a WLD(S)/Nmnat-independent axonal degeneration pathway, conservation of the axonal clearance machinery, and a function for CED-7 and NRF-5 in this process.
Project description:Glial cells efficiently recognize and clear cellular debris after nervous system injury to maintain brain homeostasis, but pathways governing glial responses to neural injury remain poorly defined. We identify the Drosophila melanogaster guanine nucleotide exchange factor complex Crk/Mbc/dCed-12 and the small GTPase Rac1 as modulators of glial clearance of axonal debris. We found that Crk/Mbc/dCed-12 and Rac1 functioned in a non-redundant fashion with the Draper transmembrane receptor pathway: loss of either pathway fully suppressed clearance of axonal debris. Draper signaling was required early during glial responses, promoting glial activation, which included increased Draper and dCed-6 expression and extension of glial membranes to degenerating axons. In contrast, the Crk/Mbc/dCed-12 complex functioned at later phases, promoting glial phagocytosis of axonal debris. Our work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris and termination of responses to injury are genetically separable events mediated by distinct signaling pathways.
Project description:Studies of naturally occurring mutant mice, wld(s), showing delayed Wallerian degeneration phenotype, suggest that axonal degeneration is an active process. We previously showed that increased nicotinamide adenine dinucleotide (NAD)-synthesizing activity by overexpression of nicotinamide mononucleotide adenylyltransferase (NMNAT) is the essential component of the Wld(s) protein, the expression of which is responsible for the delayed Wallerian degeneration phenotype in wld(s) mice. Indeed, NMNAT overexpression in cultured neurons provides robust protection to neurites, as well. To examine the effect of NMNAT overexpression in vivo and to analyze the mechanism that causes axonal protection, we generated transgenic mice (Tg) overexpressing NMNAT1 (nuclear isoform), NMNAT3 (mitochondrial isoform), or the Wld(s) protein bearing a W258A mutation, which disrupts NAD-synthesizing activity of the Wld(s) protein. Wallerian degeneration delay in NMNAT3-Tg was similar to that in wld(s) mice, whereas axonal protection in NMNAT1-Tg or Wld(s)(W258A)-Tg was not detectable. Detailed analysis of subcellular localization of the overexpressed proteins revealed that the axonal protection phenotype was correlated with localization of NMNAT enzymatic activity to mitochondrial matrix. Furthermore, we found that isolated mitochondria from mice showing axonal protection expressed unchanged levels of respiratory chain components, but were capable of increased ATP production. These results suggest that axonal protection by NMNAT expression in neurons is provided by modifying mitochondrial function. Alteration of mitochondrial function may constitute a novel tool for axonal protection, as well as a possible treatment of diseases involving axonopathy.
Project description:Axonal degeneration is a prominent feature of many neurological disorders that are associated with mitochondrial dysfunction, including Parkinson's disease, motor neuron disease, and inherited peripheral neuropathies. Studies of the Wld(s) mutant mouse, which undergoes delayed Wallerian degeneration in response to axonal injury, suggest that axonal degeneration is an active process. Wld(s) mice also have slower axonal degeneration and disease progression in numerous models of neurodegenerative disease. The Wld(s) mutation results in the production of a chimeric protein that contains the full-length coding sequence of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), which alone is sufficient for axonal protection in vitro. To test the effects of increased Nmnat expression on axonal degeneration induced by mitochondrial dysfunction, we examined dorsal root ganglion (DRG) neurons treated with rotenone. Rotenone induced profound axonal degeneration in DRG neurons; however, this degeneration was delayed by expression of Nmnat. Nmnat-mediated protection resulted in decreased axonal accumulation and sensitivity to reactive oxygen species (ROS) but did not affect the change in the rate of rotenone-induced loss in neuronal ATP. Nmnat also prevented axonal degeneration caused by exposure to exogenous oxidants and reduced the level of axonal ROS after treatment with vincristine, further supporting the idea that Nmnat promotes axonal protection by mitigating the effects of ROS.
Project description:Axonal degeneration is a key component of a variety of neurological diseases. Studies using wld(s) mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wld(s) protein is normally localized to the nucleus, and contains the N terminus of ubiquitination factor Ube4b fused to full-length Nmnat1, an NAD biosynthetic enzyme. While Nmnat enzymatic activity is necessary for Wld(s)-mediated axonal protection, several important questions remain including whether the Ube4b component of Wld(s) also plays a role, and in which cellular compartment (nucleus vs cytosol) the axonal protective effects of Nmnat activity are mediated. While Nmnat alone is clearly sufficient to delay axonal degeneration in cultured neurons, we sought to determine whether it was also sufficient to promote axonal protection in vivo. Using cytNmnat1, an engineered mutant of Nmnat1 localized only to the cytoplasm and axon, that provides more potent axonal protection than that afforded by Wld(s) or Nmnat1, we generated transgenic mice using the prion protein promoter (PrP). The sciatic nerve of these cytNmnat1 transgenic mice was transected, and microscopic analysis of the distal nerve segment 7 d later revealed no evidence of axonal loss or myelin debris, indicating that Nmnat alone, without any other Wld(s) sequences, is all that is required to delay axonal degeneration in vivo. These results highlight the importance of understanding the mechanism of Nmnat-mediated axonal protection for the development of new treatment strategies for neurological disorders.
Project description:Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. We previously showed that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, we screened candidate suppressor genes and found that the Wallerian degeneration slow (Wld(S)) protein blocked JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1) portion of Wld(S) is required, we found that its nicotinamide adenine dinucleotide (NAD(+)) enzyme activity and the Wld(S) N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. We suggest that Wld(S)-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions.
Project description:Axonal degeneration is a hallmark of many neurological disorders. Studies in animal models of neurodegenerative diseases indicate that axonal degeneration is an early event in the disease process, and delaying this process can lead to decreased progression of the disease and survival extension. Overexpression of the Wallerian degeneration slow (Wld(s)) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wld(s) protein consists of the N-terminal portion of the ubiquitination factor Ube4b fused to the nicotinamide adenine dinucleotide (NAD(+)) biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). We previously showed that the Nmnat1 portion of this fusion protein was the critical moiety for Wld(s)-mediated axonal protection. Here, we describe the development of an automated quantitative assay for assessing axonal degeneration. This method successfully showed that Nmnat1 enzymatic activity is important for axonal protection as mutants with reduced enzymatic activity lacked axon protective activity. We also found that Nmnat enzymes with diverse sequences and structures from various species, including Drosophila melanogaster, Saccharomyces cerevisiae, and archaebacterium Methanocaldococcus jannaschii, which encodes a protein with no homology to eukaryotic Nmnat enzymes, all mediate robust axonal protection after axotomy. Besides the importance of Nmnat enzymatic activity, we did not observe changes in the steady-state NAD(+) level, and we found that inhibition of nicotinamide phosphoribosyltransferase (Nampt), which synthesizes substrate for Nmnat in mammalian cells, did not affect the protective activity of Nmnat1. These results provide the possibility of a role for new Nmnat enzymatic activity in axonal protection in addition to NAD(+) synthesis.
Project description:Axonal degeneration is an early and important component of many neurological disorders. Overexpression of nicotinamide mononucleotide adenylyltransferase (Nmnat), a component of the slow Wallerian degeneration (Wld(s)) protein, protects axons from a variety of insults. We found that transduction of Nmnat protein into severed axons via virus-like particles prevented axonal degeneration. The post-injury efficacy of Nmnat indicates that its protective effects occur locally within the axon and provides an opportunity to develop novel agents to treat axonal damage.
Project description:Deficits in axonal transport are thought to contribute to the pathology of many neurodegenerative diseases. Expressing the slow Wallerian degeneration protein (Wld(S)) or related nicotinamide mononucleotide adenyltransferases (NmNATs) protects axons against damage from a broad range of insults, but the ability of these proteins to protect against inhibition of axonal transport has received little attention. We set out to determine whether these proteins can protect the axons of cultured hippocampal neurons from damage due to hydrogen peroxide or oxygen-glucose deprivation (OGD) and, in particular, whether they can reduce the damage that these agents cause to the axonal transport machinery. Exposure to these insults inhibited the axonal transport of both mitochondria and of the vesicles that carry axonal membrane proteins; this inhibition occurred hours before the first signs of axonal degeneration. Expressing a cytoplasmically targeted version of NmNAT1 (cytNmNAT1) protected the axons against both insults. It also reduced the inhibition of transport when cells were exposed to hydrogen peroxide and enhanced the recovery of transport following both insults. The protective effects of cytNmNAT1 depend on mitochondrial transport. When mitochondrial transport was inhibited, cytNmNAT1 was unable to protect axons against either insult. The protective effects of mitochondrially targeted NmNAT also were blocked by inhibiting mitochondrial transport. These results establish that NmNAT robustly protects the axonal transport system following exposure to OGD and reactive oxygen species and may offer similar protection in other disease models. Understanding how NmNAT protects the axonal transport system may lead to new strategies for neuroprotection in neurodegenerative diseases.
Project description:Axons damaged by acute injury, toxic insults, or during neurodegenerative diseases undergo Wallerian or Wallerian-like degeneration, which is an active and orderly cellular process, but the underlying mechanisms are poorly understood. Drosophila has been proven to be a successful system for modeling human neurodegenerative diseases. In this study, we established a novel in vivo model of axon injury using the adult fly wing. The wing nerve highlighted by fluorescent protein markers can be directly visualized in living animals and be precisely severed by a simple wing cut, making it highly suitable for large-scale screening. Using this model, we confirmed an axonal protective function of Wld(S) and nicotinamide mononucleotide adenylyltransferase (Nmnat). We further revealed that knockdown of endogenous Nmnat triggered spontaneous, dying-back axon degeneration in vivo. Intriguingly, axonal mitochondria were rapidly depleted upon axotomy or downregulation of Nmnat. The injury-induced mitochondrial loss was dramatically suppressed by upregulation of Nmnat, which also protected severed axons from degeneration. However, when mitochondria were genetically eliminated from axons, upregulation of Nmnat was no longer effective to suppress axon degeneration. Together, these findings demonstrate an essential role of endogenous Nmnat in maintaining axonal integrity that may rely on and function by stabilizing mitochondria.
Project description:The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.