NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Heme Oxygenase 1 in Schwann Cells Regulates Peripheral Nerve Degeneration Against Oxidative Stress.

During Wallerian degeneration, Schwann cells lose their characteristic of myelinating axons and shift into the state of developmental promyelinating cells. This recharacterized Schwann cell guides newly regrowing axons to their destination and remyelinates reinnervated axons. This Schwann cell dynamics during Wallerian degeneration is associated with oxidative events. Heme oxygenases (HOs) are involved in the oxidative degradation of heme into biliverdin/bilirubin, ferrous iron, and carbon monoxide. Overproduction of ferrous iron by HOs increases reactive oxygen species, which have deleterious effects on living cells. Thus, the key molecule for understanding the exact mechanism of Wallerian degeneration in the peripheral nervous system is likely related to oxidative stress-mediated HOs in Schwann cells. In this study, we demonstrate that demyelinating Schwann cells during Wallerian degeneration highly express HO1, not HO2, and remyelinating Schwann cells during nerve regeneration decrease HO1 activation to levels similar to those in normal myelinating Schwann cells. In addition, HO1 activation during Wallerian degeneration regulates several critical phenotypes of recharacterized repair Schwann cells, such as demyelination, transdedifferentiation, and proliferation. Thus, these results suggest that oxidative stress in Schwann cells after peripheral nerve injury may be regulated by HO1 activation during Wallerian degeneration and oxidative-stress-related HO1 activation in Schwann cells may be helpful to study deeply molecular mechanism of Wallerian degeneration.

An Atypical SCF-like Ubiquitin Ligase Complex Promotes Wallerian Degeneration through Regulation of Axonal Nmnat2.

Axon degeneration is a tightly regulated, self-destructive program that is a critical feature of many neurodegenerative diseases, but the molecular mechanisms regulating this program remain poorly understood. Here, we identify S-phase kinase-associated protein 1A (Skp1a), a core component of a Skp/Cullin/F-box (SCF)-type E3 ubiquitin ligase complex, as a critical regulator of axon degeneration after injury in mammalian neurons. Depletion of Skp1a prolongs survival of injured axons in vitro and in the optic nerve in vivo. We demonstrate that Skp1a regulates the protein level of the nicotinamide adenine dinucleotide (NAD)+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) in axons. Loss of axonal Nmnat2 contributes to a local ATP deficit that triggers axon degeneration. Knockdown of Skp1a elevates basal levels of axonal Nmnat2, thereby delaying axon degeneration through prolonged maintenance of axonal ATP. Consistent with Skp1a functioning through regulation of Nmnat2, Skp1a knockdown fails to protect axons from Nmnat2 knockdown. These results illuminate the molecular mechanism underlying Skp1a-dependent axonal destruction.

DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.

Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.

A caspase cascade regulating developmental axon degeneration.

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.

Mutations in a P-type ATPase gene cause axonal degeneration.

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

WldS prevents axon degeneration through increased mitochondrial flux and enhanced mitochondrial Ca2+ buffering.

Wld(S) (slow Wallerian degeneration) is a remarkable protein that can suppress Wallerian degeneration of axons and synapses, but how it exerts this effect remains unclear. Here, using Drosophila and mouse models, we identify mitochondria as a key site of action for Wld(S) neuroprotective function. Targeting the NAD(+) biosynthetic enzyme Nmnat to mitochondria was sufficient to fully phenocopy Wld(S), and Wld(S) was specifically localized to mitochondria in synaptic preparations from mouse brain. Axotomy of live wild-type axons induced a dramatic spike in axoplasmic Ca(2+) and termination of mitochondrial movement-Wld(S) potently suppressed both of these events. Surprisingly, Wld(S) also promoted increased basal mitochondrial motility in axons before injury, and genetically suppressing mitochondrial motility in vivo dramatically reduced the protective effect of Wld(S). Intriguingly, purified mitochondria from Wld(S) mice exhibited enhanced Ca(2+) buffering capacity. We propose that the enhanced Ca(2+) buffering capacity of Wld(S+) mitochondria leads to increased mitochondrial motility, suppression of axotomy-induced Ca(2+) elevation in axons, and thereby suppression of Wallerian degeneration.

A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity.

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.

Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe.

We have previously shown that driving PI3K levels up or down leads to increases or reductions in the number of synapses, respectively. Using these tools to assay their behavioral effects in Drosophila melanogaster, we showed that a loss of synapses in two sets of local interneurons, GH298 and krasavietz, leads to olfaction changes toward attraction or repulsion, while the simultaneous manipulation of both sets of neurons restored normal olfactory indexes. We show here that olfactory central adaptation also requires the equilibrated changes in both sets of local interneurons. The same genetic manipulations directed to projection (GH146) or mushroom body (201Y, MB247) neurons did not affect adaptation. Also, we show that the equilibrium is a requirement for the glomerulus-specific size changes which are a morphological signature of central adaptation. Since the two sets of local neurons are mostly, although not exclusively, inhibitory (GH298) and excitatory (krasavietz), we interpret that the normal phenomena of sensory perception, measured as an olfactory index, and central adaptation rely on an inhibition/excitation ratio.

A conditioning lesion protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade.

Axons are vulnerable components of neuronal circuitry, and neurons are equipped with mechanisms for responding to axonal injury. A highly studied example of this is the conditioning lesion, in which neurons that have been previously injured have an increased ability to initiate new axonal growth (Hoffman, 2010). Here we investigate the effect of a conditioning lesion on axonal degeneration, which occurs in the distal stump after injury, and also occurs in neuropathies and neurodegenerative disorders (Coleman, 2005). We found that Drosophila motoneuron axons that had been previously injured had an increased resiliency to degeneration. This requires the function of a conserved axonal kinase, Wallenda (Wnd)/DLK, and a downstream transcription factor. Because axonal injury leads to acute activation of Wnd (Xiong et al., 2010), and overexpression studies indicate that increased Wnd function is sufficient to promote protection from degeneration, we propose that Wnd regulates an adaptive response to injury that allows neurons to cope with axonal stress.

c-Jun N-terminal kinase induces axonal degeneration and limits motor recovery after spinal cord injury in mice.

c-Jun N-terminal kinase (JNK) mediates neuronal death in response to stress and injury in the CNS and peripheral nervous system. Here, we show that JNK also regulates retrograde axonal degeneration (axonal dieback) after spinal cord injury (SCI) in mice. Activated phospho-JNK was highly expressed in damaged corticospinal tract (CST) axons after thoracic SCI by hemisection. Local administration of SP600125, a JNK inhibitor, prevented accumulation of amyloid-ß precursor protein and retraction of the severed CST axons as well as preserved the axonal arbors rostral to the injury site. The treatment with SP600125 also improved functional recovery of the hindlimbs, assessed by Basso mouse scale open-field scores and the grid-walking test. In Jnk1(-/-) and Jnk3(-/-) mice, we observed prevention of axonal degeneration and enhancement of motor recovery after SCI. These results indicate that both JNK1 and JNK3 induce axonal degeneration and limit motor recovery after SCI. Thus, a JNK inhibitor may be a suitable therapeutic agent for SCI.

Wallerian degeneration, wld(s), and nmnat.

Traditionally, researchers have believed that axons are highly dependent on their cell bodies for long-term survival. However, recent studies point to the existence of axon-autonomous mechanism(s) that regulate rapid axon degeneration after axotomy. Here, we review the cellular and molecular events that underlie this process, termed Wallerian degeneration. We describe the biphasic nature of axon degeneration after axotomy and our current understanding of how Wld(S)--an extraordinary protein formed by fusing a Ube4b sequence to Nmnat1--acts to protect severed axons. Interestingly, the neuroprotective effects of Wld(S) span all species tested, which suggests that there is an ancient, Wld(S)-sensitive axon destruction program. Recent studies with Wld(S) also reveal that Wallerian degeneration is genetically related to several dying back axonopathies, thus arguing that Wallerian degeneration can serve as a useful model to understand, and potentially treat, axon degeneration in diverse traumatic or disease contexts.

Aldose reductase deficiency improves Wallerian degeneration and nerve regeneration in diabetic thy1-YFP mice.

This study examined the role of aldose reductase (AR) in diabetes-associated impaired nerve regeneration using thy1-YFP (YFP) mice. Sciatic nerves of nondiabetic and streptozotocin-induced diabetic AR(+/+)YFP and AR(-/-)YFP mice were transected after 4 weeks of diabetes. Wallerian degeneration and nerve regeneration were evaluated at 1 and 2 weeks postaxotomy by fluorescence microscopy. Motor nerve conduction velocity recovery and regenerating nerve morphometric parameters were determined at 10 and 20 weeks, respectively. There was no difference in the extent of Wallerian degeneration, size of regenerating stump, motor nerve conduction velocity recovery, or caliber of regenerating fibers between nondiabetic AR(+/+)YFP and AR(-/-)YFP mice. In diabetic AR(+/+)YFP mice, Wallerian degeneration was delayed, associated with slower macrophage invasion and abnormal vascularization. Those mice had smaller regenerating stumps, slower motor nerve conduction velocity, and smaller regenerating fibers compared with nondiabetic mice. These features of impaired nerve regeneration were largely attenuated in diabetic AR(-/-)YFP mice. Retarded macrophage invasion and vascularization associated with Wallerian degeneration were normalized in diabetic AR(-/-)YFP mice. These results indicate that AR plays an important role in diabetes-associated impaired nerve regeneration, in part by affecting vascularization and macrophage invasion during Wallerian degeneration. The thy1-YFP mice are valuable tools for further investigation of the mechanism of diabetes-associated nerve regeneration.

Thirteen-month inhibition of aldose reductase by zenarestat prevents morphological abnormalities in the dorsal root ganglia of streptozotocin-induced diabetic rats.

The dorsal root ganglia (DRG) have been identified as the target tissue in diabetic somatosensory neuropathy. It has been reported that, in the chronically diabetic state, DRG sensory neurons may undergo morphological changes. In this study, we examined the effect of zenarestat, an aldose reductase inhibitor, on the morphological derangement of the DRG and the sural nerve of streptozotocin-induced diabetic rats (STZ rats) over a 13-month period. The cell area of the DRG in STZ rats was smaller than that in normal rats. A decrease in fiber size was apparent in the sural nerve of the STZ rats, and the fiber density was greater. These morphological changes were reversed in zenarestat-treated STZ rats. The data suggest that, in peripheral sensory diabetic neuropathy, hyperactivation of the polyol pathway induces abnormalities not only in peripheral nerve fiber, but also in the DRG, which is an aggregate of primary sensory afferent cell bodies.

Intracellular phospholipase A(2) group IVA and group VIA play important roles in Wallerian degeneration and axon regeneration after peripheral nerve injury.

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.

Characterization of NADPH-diaphorase-positive glial cells of the tench optic nerve after axotomy.

NADPH-diaphorase (ND) positive cell types were characterized throughout the optic nerve of the tench in normal conditions and after optic nerve transection with survival periods of 1, 3, 7, 14, 30, 60, 120 and 180 days. Astrocytic markers (S100 and glutamine synthetase) and the microglial marker tomato lectin were employed. In the control prechiasmatic optic nerve two types (types I and II) of ND-positive glial cells appeared. All type I cells showed S100 immunoreactivity, whereas only a subpopulation of them were positive to glutamine synthetase. Type II cells only presented S100 immunoreactivity. In the control anterior optic tract, all ND-positive glial cells (type III) presented immunolabeling to S100 and glutamine synthetase. After transection, types I and II did not show any changes in the staining patterns for the glial markers when observed. Two new types of ND-positive glial cells (types IV and V) were observed after axotomy. All type IV cells were S100-immunopositive, and a subpopulation presented glutamine synthetase immunolabeling. Only a subpopulation of type V cells showed glutamine synthetase immunostaining. The presence of type IV or V cells in the lesioned optic nerve occurred simultaneously with significant decreases or absence of type I cells. These data suggest that type I and III cells are astrocytes and type II cells are oligodendrocytes. Types IV and V cells are the result of the activation of type I cells after optic nerve section. The polymorphism observed in ND-positive cells may reflect different cell functions during degenerative and regenerative processes.

High-affinity choline uptake and acetylcholine-metabolizing enzymes in CNS white matter. A quantitative study.

The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25-31% of that in hippocampus. White matter choline acetyltransferase activity was 10-50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20-25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8-4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.

Differential effects of two ROCK inhibitors, Fasudil and Y-27632, on optic nerve regeneration in adult cats.

A ROCK inhibitor Fasudil is widely administered to relieve vasospasm in patients after subarachnoid hemorrhage in Japan. We investigated the difference of Fasudil and Y-27632, a common ROCK inhibitor, on neurite regeneration in culture and axonal regeneration after injuring the optic nerve (OpN) in cats. The optimal dose of Y-27632, determined by counting the number and length of neurites in retinal explants, was found to be 100 microM: the only effect of Fasudil was to promote extension of glial processes. We next examined the effects of Fasudil (10 microM-100 microM) and Y-27632 (10 microM-300 microM) on axonal regeneration in the crushed OpN model in vivo. Immediately after crushing the left OpN, Fasudil or Y-27632 was injected into the vitreous and the crushed site. Injection of 10 microM and 100 microM Y-27632 induced extension of the optic axons beyond the crush site, with the latter dosage giving stronger regeneration. Very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection, and no axons elongated in the OpN with Fasudil injection.