Yo Sasaki

A dual leucine kinase–dependent axon self-destruction program promotes Wallerian degeneration

Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. We found that dual leucine kinase (DLK) promoted degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promoted degeneration locally in axons as they committed to degenerate. This pathway also promoted degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program.

Stimulation of Nicotinamide Adenine Dinucleotide Biosynthetic Pathways Delays Axonal Degeneration after Axotomy

Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

Nicotinamide Mononucleotide Adenylyl Transferase-Mediated Axonal Protection Requires Enzymatic Activity But Not Increased Levels of Neuronal Nicotinamide Adenine Dinucleotide

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 (Wlds) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wlds 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 Wlds-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.

Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain

Extracellular nucleotides, including ATP, have been demonstrated to transmit important physiological signals in the brain through either G‐protein‐coupled P2Y receptors or P2X receptors, which are ligand‐gated ion channels. In this study, we performed a detailed analysis of the expression of the Gi/o‐coupled receptor P2Y12 in the brain. Northern blot analysis demonstrated that P2Y12 is expressed predominantly in the brain, and to a lesser extent in the spleen. The cellular localization of P2Y12 was investigated by in situ hybridization, and P2Y12 mRNA was detected in small cells distributed throughout the brain, including the hippocampus. Expression of P2Y12 was also observed in naive and axotomized facial nuclei, and the number of P2Y12‐expressing cells increased following facial nerve axotomy. Selective expression of P2Y12 mRNA in microglia was confirmed by double‐label in situ hybridization and immunohistochemistry with antibodies against NeuN and Iba1 as an immunohistochemical marker for neurons and microglia, respectively. Hardly any P2Y12 mRNA was detected in macrophages obtained from the spleen and abdominal cavity, which share many surface molecules with microglia.

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration

Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimers disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.

Microglia/macrophage‐specific protein Iba1 binds to fimbrin and enhances its actin‐bundling activity

Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage‐specific calcium‐binding protein. Iba1 has the actin‐bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1‐related intracellular signalling pathway in greater detail, we employed a yeast two‐hybrid screen to isolate an Iba1‐interacting molecule and identified another actin‐bundling protein, L‐fimbrin. In response to stimulation, L‐fimbrin accumulated and co‐localized with Iba1 in membrane ruffles induced by M‐CSF‐stimulation and phagocytic cups formed by IgG‐opsonized beads in microglial cell line MG5. L‐fimbrin was shown to associate with Iba1 in cell lysate of COS‐7 expressing L‐fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L‐fimbrin was demonstrated by immunoprecipitation, glutathione S‐transferase pull‐down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin‐bundling activity of L‐fimbrin. These results indicate that Iba1 forms complexes with L‐fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co‐operates with L‐fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.

Transgenic Mice Expressing the Nmnat1 Protein Manifest Robust Delay in Axonal Degeneration In Vivo

Axonal degeneration is a key component of a variety of neurological diseases. Studies using wlds mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wlds 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 Wlds-mediated axonal protection, several important questions remain including whether the Ube4b component of Wlds 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 Wlds 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 Wlds 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.

Aberrant Schwann Cell Lipid Metabolism Linked to Mitochondrial Deficits Leads to Axon Degeneration and Neuropathy

Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells [SCs]) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response (ISR) through the actions of heme-regulated inhibitor (HRI) kinase, and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines (ACs), an intermediate of fatty acid β-oxidation. Importantly, we show that ACs are released from SCs and induce axonal degeneration. A maladaptive ISR as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies.

SARM1 activation triggers axon degeneration locally via NAD+ destruction

SARM1-driven axon degeneration Axons, the long protrusions of nerve cells, are programmed to self-destruct under certain conditions that occur during development, stress, or disease states. Gerdts et al. outline a biochemical mechanism that controls such axon degeneration. The authors designed versions of SARM1 (sterile alpha and TIR motif—constraining 1) that could be activated or inhibited in cells. Their experiments showed that the activation of SARM1 was necessary and sufficient to cause axon destruction in cultured mouse neurons. SARM1-mediated destruction was associated with depletion of the metabolic cofactor NAD+ from cells. Science, this issue p. 453 A critical role is elucidated for destruction of a metabolic cofactor in neuronal degeneration. Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif–containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD+) after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll–interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD+, whereas SARM1-induced axon destruction could be counteracted by increased NAD+ synthesis. SARM1-induced depletion of NAD+ may explain the potent axon protection in Wallerian degeneration slow (Wlds) mutant mice.

Sarm1-Mediated Axon Degeneration Requires Both SAM and TIR Interactions

Axon degeneration is an evolutionarily conserved pathway that eliminates damaged or unneeded axons. Manipulation of this poorly understood pathway may allow treatment of a wide range of neurological disorders. In an RNAi-based screen performed in cultured mouse DRG neurons, we observed strong suppression of injury-induced axon degeneration upon knockdown of Sarm1 [SARM (sterile α-motif-containing and armadillo-motif containing protein)]. We find that a SARM-dependent degeneration program is engaged by disparate neuronal insults: SARM ablation blocks axon degeneration induced by axotomy or vincristine treatment, while SARM acts in parallel with a soma-derived caspase-dependent pathway following trophic withdrawal. SARM is a multidomain protein that associates with neuronal mitochondria. Deletion of the N-terminal mitochondrial localization sequence disrupts SARM mitochondrial localization in neurons but does not alter its ability to promote axon degeneration. In contrast, mutation of either the SAM (sterile α motif) or TIR (Toll-interleukin-1 receptor) domains abolishes the ability of SARM to promote axonal degeneration, while a SARM mutant containing only these domains elicits axon degeneration and nonapoptotic neuronal death even in the absence of injury. Protein–protein interaction studies demonstrate that the SAM domains are necessary and sufficient to mediate SARM–SARM binding. SARM mutants lacking a TIR domain bind full-length SARM and exhibit strong dominant-negative activity. These results indicate that SARM plays an integral role in the dismantling of injured axons and support a model in which SAM-mediated multimerization is necessary for TIR-dependent engagement of a downstream destruction pathway. These findings suggest that inhibitors of SAM and TIR interactions represent therapeutic candidates for blocking pathological axon loss and neuronal cell death.