Biology subjects

DiAntonio, A.

Publications and source records attributed to DiAntonio, A..

9 recordsLinked to original sources

bioRxiv2021

Defining hierarchical protein interaction networks from spectral analysis of bacterial proteomes

Cellular phenotypes emerge from a hierarchy of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be extracted from the statistical pattern of proteome variation as measured across thousands of bacteria and that these hierarchies reflect the emergence of complex bacterial phenotypes. We describe the mathematics underlying our statistical approach and validate our results through gene-set enrichment analysis and comparison to existing experimentally-derived hierarchical databases. We demonstrate the biological utility of our unbiased hierarchical models by creating a model of motility in Pseudomonas aeruginosa and using it to discover a previously unappreciated genetic effector of twitch-based motility. Overall, our approach, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), predicts hierarchies of protein interaction networks describing emergent biological function using only the statistical pattern of bacterial proteome variation.

systems biology↗

bioRxiv2021

FKBPL and FKBP8 regulate DLK degradation and neuronal responses to axon injury

DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

neuroscience↗

bioRxiv2021

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders. HighlightsO_LINaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase C_LIO_LINaMN inhibits SARM1 activation by stabilizing its open, inactive structure C_LIO_LINMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN C_LI

neuroscience↗

bioRxiv2021

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

neuroscience↗

bioRxiv2021

Live Imaging Reveals the Cellular Events Downstream of SARM1 Activation

SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here we used live imaging with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.

neuroscience↗

bioRxiv2021

Constitutively active SARM1 variants found in ALS patients induce neuropathy

In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD+ hydrolase SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively-active SARM1 enzymes that promote degeneration when expressed in cultured neurons. To investigate whether naturally-occurring human variants might similarly disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of 29 rare SARM1 alleles identified among 8,507 amyotrophic lateral sclerosis (ALS) patients. Ten missense variants or small in-frame deletions exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. Expression of these constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1V184G, the constitutively active variant found most frequently in the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.

neuroscience↗

bioRxiv2021

Activation of Sarm1 produces cADPR to increase intra-axonal calcium and promote axon degeneration in CIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating CIPN. HIGHLIGHTSO_LIPaclitaxel induces intra-axonal calcium flux C_LIO_LISarm1-dependent cADPR production promotes axonal calcium elevation and degeneration C_LIO_LIAntagonizing cADPR signaling pathway protects against paclitaxel-induced peripheral neuropathy in vitro and in vivo C_LI

neuroscience↗

bioRxiv2020

Bidirectional regulation of glial potassium buffering: glioprotection versus neuroprotection

Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A SIK3-regulated gene expression program controls the glial capacity to buffer K+ and water, however upstream regulatory mechanisms are unknown. Here we identify an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic GPCR Oct{beta}R to upregulate glial buffering capacity, while under pathological K+ stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway in inactivated. Re-activation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened lifespan in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability.

cell biology↗

bioRxiv2020

SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration.

Leber congenital amaurosis type 9 is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

neuroscience↗