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1.
Neuron ; 110(22): 3711-3726.e16, 2022 11 16.
Article in English | MEDLINE | ID: mdl-36087583

ABSTRACT

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.


Subject(s)
Armadillo Domain Proteins , NAD , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , NAD/metabolism , Neuroprotection , Cytoskeletal Proteins/metabolism , Axons/metabolism , Hydrolases/metabolism
2.
Cell Rep ; 32(5): 107999, 2020 08 04.
Article in English | MEDLINE | ID: mdl-32755591

ABSTRACT

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.


Subject(s)
Armadillo Domain Proteins/chemistry , Armadillo Domain Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , NAD/metabolism , Animals , Cell Death , Cell Line, Tumor , Cryoelectron Microscopy , Female , HEK293 Cells , Humans , Mice, Inbred C57BL , Models, Molecular , Neurons/cytology , Nicotinamide Mononucleotide/metabolism , Protein Domains
3.
Proc Natl Acad Sci U S A ; 115(6): 1358-1363, 2018 02 06.
Article in English | MEDLINE | ID: mdl-29295933

ABSTRACT

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 Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila 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. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 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 peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.


Subject(s)
Axons/pathology , Drosophila Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Wallerian Degeneration/pathology , Animals , Animals, Genetically Modified , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , Axons/metabolism , Cholinergic Neurons/pathology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Wallerian Degeneration/genetics , Wallerian Degeneration/metabolism , Wings, Animal/innervation , Wings, Animal/metabolism , Zinc Fingers/genetics
4.
Neuron ; 95(1): 78-91.e5, 2017 Jul 05.
Article in English | MEDLINE | ID: mdl-28683272

ABSTRACT

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.


Subject(s)
Armadillo Domain Proteins/genetics , Axons/metabolism , Cytoskeletal Proteins/genetics , Drosophila Proteins/genetics , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Wallerian Degeneration/genetics , Animals , Animals, Genetically Modified , Armadillo Domain Proteins/metabolism , Arthropod Antennae/injuries , Arthropod Antennae/innervation , Axotomy , Behavior, Animal , Blotting, Western , Cell Line , Cytoskeletal Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Grooming , Immunity, Active , NAD/metabolism , Neurons/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Optogenetics , Wallerian Degeneration/metabolism , Wings, Animal/injuries , Wings, Animal/innervation
6.
Proc Natl Acad Sci U S A ; 111(27): 9965-70, 2014 Jul 08.
Article in English | MEDLINE | ID: mdl-24958874

ABSTRACT

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., GFP-labeled, 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.


Subject(s)
Axons , Drosophila/genetics , Alleles , Animals , Genes, Dominant , Genes, Insect , Genes, Recessive , Mosaicism , Wings, Animal/blood supply
7.
Proc Natl Acad Sci U S A ; 110(1): 300-5, 2013 Jan 02.
Article in English | MEDLINE | ID: mdl-23248282

ABSTRACT

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.


Subject(s)
Brain/metabolism , Parkinsonian Disorders/metabolism , Urate Oxidase/metabolism , Uric Acid/metabolism , Allantoin/metabolism , Analysis of Variance , Animals , Blotting, Western , Dopamine/metabolism , Dopaminergic Neurons/drug effects , Immunohistochemistry , Mice , Mice, Knockout , Mice, Transgenic , Movement/physiology , Oxidopamine/toxicity , Urate Oxidase/genetics
8.
Biomed Chromatogr ; 27(1): 122-9, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22674671

ABSTRACT

The purine metabolic pathway has been implicated in neurodegeneration and neuroprotection. High-performance liquid chromatography (HPLC) is widely used to determine purines and metabolites. However, methods for analysis of multiple purines in a single analysis have not been standardized, especially in brain tissue. We report the development and validation of a reversed-phase HPLC method combining electrochemical and UV detection after a short gradient run to measure seven purine metabolites (adenosine, guanosine, inosine, guanine, hypoxanthine, xanthine and urate) from the entire purine metabolic pathway. The limit of detection (LoD) for each analyte was determined. The LoD using UV absorption was 0.001 mg/dL for hypoxanthine (Hyp), inosine (Ino), guanosine (Guo) and adenosine (Ado), and those using coulometric electrodes were 0.001 mg/dL for guanine (Gua), 0.0001 mg/dL for urate (UA) and 0.0005 mg/dL for xanthine (Xan). The intra- and inter-day coefficient of variance was generally <8%. Using this method, we determined basal levels of these metabolites in mouse brain and serum, as well as in post-mortem human brain. Peak identities were confirmed by enzyme degradation. Spike recovery was performed to assess accuracy. All recoveries fell within 80-120%. Our HPLC method provides a sensitive, rapid, reproducible and low-cost method for determining multiple purine metabolites in a single analysis in serum and brain specimens.


Subject(s)
Chromatography, High Pressure Liquid/methods , Corpus Striatum/chemistry , Purines/analysis , Animals , Electrochemical Techniques , Humans , Male , Mice , Mice, Inbred C57BL , Purines/blood , Purines/chemistry , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Ultraviolet
9.
J Neurochem ; 123(1): 172-81, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22671773

ABSTRACT

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 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 Parkinson's disease. 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.


Subject(s)
Antioxidants/metabolism , Antioxidants/pharmacology , Astrocytes/metabolism , Dopaminergic Neurons/drug effects , Uric Acid/metabolism , Uric Acid/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Cell Survival , Cells, Cultured , Chromatography, High Pressure Liquid , Coculture Techniques , Culture Media, Conditioned/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Hydrogen Peroxide/toxicity , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nitrites/metabolism , Oxidants/toxicity , Protein Carbonylation/drug effects , Reactive Oxygen Species/metabolism , Urate Oxidase/genetics
10.
PLoS One ; 7(5): e37331, 2012.
Article in English | MEDLINE | ID: mdl-22606360

ABSTRACT

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.


Subject(s)
Parkinsonian Disorders/metabolism , Uric Acid/metabolism , 1-Methyl-4-phenylpyridinium/toxicity , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Mesencephalon/drug effects , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Nerve Degeneration/prevention & control , Parkinsonian Disorders/genetics , Parkinsonian Disorders/pathology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tissue Culture Techniques , Urate Oxidase/genetics , Urate Oxidase/metabolism , Uric Acid/pharmacology
11.
Brain Res ; 1367: 310-8, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-20828543

ABSTRACT

Adenosine A(2A) receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson's disease (PD). Clinical trials of A(2A) antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A1 receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A1 and/or A(2A) receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A1, A(2A) and double A1-A(2A) knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A1 and A(2A) receptors) or saline were treated daily for 18-21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A1 and A(2A) KOs, but not in A1-A(2A) KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A1 or A(2A) receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear.


Subject(s)
Antiparkinson Agents/adverse effects , Dyskinesia, Drug-Induced/genetics , Dyskinesia, Drug-Induced/prevention & control , Levodopa/adverse effects , Receptor, Adenosine A1/deficiency , Receptor, Adenosine A2A/deficiency , Adrenergic Agents/toxicity , Animals , Caffeine/administration & dosage , Corpus Striatum/drug effects , Disease Models, Animal , Dynorphins/genetics , Dynorphins/metabolism , Dyskinesia, Drug-Induced/etiology , Enkephalins/genetics , Enkephalins/metabolism , Gene Expression Regulation/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidopamine/toxicity , Parkinson Disease/drug therapy , Parkinson Disease/etiology , Parkinson Disease/genetics , Protein Precursors/genetics , Protein Precursors/metabolism , Purinergic P1 Receptor Antagonists/administration & dosage , RNA, Messenger/metabolism , Statistics, Nonparametric , Time Factors
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