Axon degeneration is a hallmark of neurodegenerative disease and neural injury. Axotomy activates an intrinsic pro-degenerative axon death signaling cascade involving loss of the NAD biosynthetic enzyme Nmnat/Nmnat2 in axons, activation of dSarm/Sarm1, and subsequent Sarm-dependent depletion of NAD. Here we identify Axundead (Axed) as a mediator of axon death. axed mutants suppress axon death in several types of axons for the lifespan of the fly and block the pro-degenerative effects of activated dSarm in vivo. Neurodegeneration induced by loss of the sole fly Nmnat ortholog is also fully blocked by axed, but not dsarm, mutants. Thus, pro-degenerative pathways activated by dSarm signaling or Nmnat elimination ultimately converge on Axed. Remarkably, severed axons morphologically preserved by axon death pathway mutations remain integrated in circuits and able to elicit complex behaviors after stimulation, indicating that blockade of axon death signaling results in long-term functional preservation of axons.

Urate is the end product of purine metabolism in humans, owing to the evolutionary disruption of the gene encoding urate oxidase (UOx). Elevated urate can cause gout and urolithiasis and is associated with cardiovascular and other diseases. However, urate also possesses antioxidant and neuroprotective properties. Recent convergence of epidemiological and clinical data has identified urate as a predictor of both reduced risk and favorable progression of Parkinson's disease (PD). In rodents, functional UOx catalyzes urate oxidation to allantoin. We found that UOx KO mice with a constitutive mutation of the gene have increased concentrations of brain urate. By contrast, UOx transgenic (Tg) mice overexpressing the enzyme have reduced brain urate concentrations. Effects of the complementary UOx manipulations were assessed in a mouse intrastriatal 6-hydroxydopamine (6-OHDA) model of hemiparkinsonism. UOx KO mice exhibit attenuated toxic effects of 6-OHDA on nigral dopaminergic cell counts, striatal dopamine content, and rotational behavior. Conversely, Tg overexpression of UOx exacerbates these morphological, neurochemical, and functional lesions of the dopaminergic nigrostriatal pathway. Together our data support a neuroprotective role of endogenous urate in dopaminergic neurons and strengthen the rationale for developing urate-elevating strategies as potential disease-modifying therapy for PD.U rate, the anionic component of uric acid, predominates at physiological pH. As an apparent consequence of mutations in the urate oxidase (UOx) gene during primate evolution, urate constitutes the enzymatic end product of purine metabolism in humans (1). There remains controversy over how the loss of UOx activity and the resultant high urate concentrations in hominoids may have been beneficial and whether it still is. On one hand, urate is considered a pathogenic factor in gout, urolithiasis, and nephropathy, and hyperuricemia is associated with other medical conditions, such as hypertension, cardiovascular disease, and metabolic syndrome (2). On the other hand, the loss of UOx activity through multiple independent mutations in hominoids presumably conferred evolutionary advantages. Urate possesses potent antioxidant properties. High urate levels may have provided an antioxidant defense against aging and cancer, thereby contributing to a prolonged hominoid life span (3). In addition, increased urate may mediate blood pressure homeostasis in low-salt environments. Furthermore, higher urate has been suggested to enhance human intelligence or motivational behaviors or promote neuronal integrity and function (4).Recently a series of population and clinical epidemiology studies have lent support to a potential neuroprotective effect of urate (5,6). These studies demonstrated a robust inverse link between urate levels and both the risk and clinical progression of Parkinson's disease (PD), one of the most common neurodegenerative diseases. Given the putative role of oxidative stress in the pathogenesis of PD (7), these studies have i...

Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFPlabeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.neurodegeneration | glial response W idespread axonal degeneration and synapse loss occurs during neurodegenerative disease and after neural trauma. These degradative events result in disruption of neural circuit connectivity and ultimately functional impairment of the nervous system. Identifying molecular cascades that actively promote axonal self-destruction is a key goal. However, despite decades of work, remarkably little is known about the molecular pathways that drive the degeneration of neurites or synapses in any context (1, 2).Axotomy-induced axon degeneration (termed Wallerian degeneration, WD) serves as a useful model to study the mechanisms of axonal self-destruction. When axons are severed, the portion of the axon distal to the injury site and its synapses undergo catastrophic fragmentation after a defined latent phase, and the resulting debris is eventually cleared by surrounding glial cells. The discovery of the spontaneous Wallerian degeneration slow (Wld S ) mouse revealed, surprisingly, that severed axons can in fact survive for weeks in the absence of a cell body (3). It also led to the proposal that "axon death" signaling cascades might exist, akin to apoptotic cell death programs, which actively drive the destruction of the axon (4).Interestingly, Wld S provides significant suppression in mouse models of progressive motor neuron disease and glaucoma (5-8), and moderate protection from chemotherapy-induced axon degeneration (9). These observations argue that defining the molecular mechanisms of axon degeneration in the context of WD could have an important therapeutic impact on the treat...

Highlights d Autoinhibited (3.3 Å ) and active (6.8 Å ) structures of prodegenerative NADase SARM1 solved d Identification of a critical autoinhibitory lock d Lock mutations convert inactive SARM1 to an active, neurotoxic state d Enzymatic studies explain SARM1's functional dependence on local metabolic environment

Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. Here we identify a role for the CH zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with mutants, linking to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. Our findings identify a transcription factor that regulates axon death signaling, and mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.

Urate is the end product of purine metabolism and a major antioxidant circulating in humans. Recent data link higher levels of urate with a reduced risk of developing Parkinson’s disease (PD) and with a slower rate of its progression. In this study, we investigated the role of astrocytes in urate-induced protection of dopaminergic cells in a cellular model of PD. In mixed cultures of dopaminergic cells and astrocytes oxidative stress-induced cell death and protein damage were reduced by urate. By contrast, urate was not protective in pure dopaminergic cell cultures. Physical contact between dopaminergic cells and astrocytes was not required for astrocyte-dependent rescue as shown by conditioned medium experiments. Urate accumulation in dopaminergic cells and astrocytes was blocked by pharmacological inhibitors of urate transporters expressed differentially in these cells. The ability of a urate transport blocker to prevent urate accumulation into astroglial (but not dopaminergic) cells predicted its ability to prevent dopaminergic cell death. Transgenic expression of uricase reduced urate accumulation in astrocytes and attenuated the protective influence of urate on dopaminergic cells. These data indicate that urate might act within astrocytes to trigger release of molecule(s) that are protective for dopaminergic cells.

Urate is a major antioxidant as well as the enzymatic end product of purine metabolism in humans. Higher levels correlate with a reduced risk of developing Parkinson's disease (PD) and with a slower rate of PD progression. In this study we investigated the effects of modulating intracellular urate concentration on 1-methyl-4-phenyl-pyridinium (MPP+)-induced degeneration of dopaminergic neurons in cultures of mouse ventral mesencephalon prepared to contain low (neuron-enriched cultures) or high (neuron-glial cultures) percentage of astrocytes. Urate, added to the cultures 24 hours before and during treatment with MPP+, attenuated the loss of dopaminergic neurons in neuron-enriched cultures and fully prevented their loss and atrophy in neuron-astrocyte cultures. Exogenous urate was found to increase intracellular urate content in cortical neuronal cultures. To assess the effect of reducing cellular urate content on MPP+-induced toxicity, mesencephalic neurons were prepared from mice over-expressing urate oxidase (UOx). Transgenic UOx expression decreased endogenous urate content both in neurons and astrocytes. Dopaminergic neurons expressing UOx were more susceptible to MPP+ in mesencephalic neuron-enriched cultures and to a greater extent in mesencephalic neuron-astrocyte cultures. Our findings correlate intracellular urate content in dopaminergic neurons with their toxin resistance in a cellular model of PD and suggest a facilitative role for astrocytes in the neuroprotective effect of urate.

Background: Increasing evidence suggests that urate may play an important role in neurodegenerative disease. In Parkinson's disease (PD) higher, but still normal, levels of blood and cerebrospinal fluid urate have been associated with a lower rate of disease progression. Objective: We explored the hypothesis that lower levels of urate and its purine precursors in brain may be associated with PD and related neurodegenerative disorders, including Alzheimer's disease (AD) and Lewy body dementia (DLB). Methods: Human postmortem brain tissues were obtained from PD, AD, and DLB patients and non-neurodegenerative disease controls. We measured urate and other purine pathway analytes in the frontal and temporal cortex, striatum, and cerebellum, using high-performance liquid chromatography with electrochemical and ultraviolet detection. Results: Age was well-matched among groups. Mean postmortem interval for samples was 16.3 ± 9.9 h. Urate levels in cortical and striatal tissue trended lower in PD and AD compared to controls in males only. These findings correlated with increased urate in male versus female control tissues. By contrast, in DLB urate levels were significantly elevated relative to PD and AD. Measurement of urate precursors suggested a decrease in xanthine in PD compared to AD in females only, and relative increases in inosine and adenosine in DLB and AD samples among males. Xanthine and hypoxanthine were more concentrated in striatal tissue than in other brain regions. Conclusions: Though limited in sample size, these findings lend support to the inverse association between urate levels and PD, as well as possibly AD. The finding of increased urate in DLB brain tissue is novel and warrants further study.