A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria

Axon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor wt and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting. Foretinib delayed chemotherapy-induced and Wallerian axonal degeneration in culture by preventing axotomy-induced local energy deficit and preserving mitochondria, and peripheral Wallerian degeneration in vivo. These findings identify a new axon degeneration pathway and a potentially clinically useful therapeutic drug.

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Neural regulation of intestinal smooth muscle growth in vitro

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.3.g511 ◽

2000 ◽

Vol 279 (3) ◽

pp. G511-G519 ◽

Cited By ~ 16

Author(s):

M. G. Blennerhassett ◽

S. Lourenssen

Keyword(s):

Smooth Muscle ◽

Sympathetic Neurons ◽

Muscle Growth ◽

Scorpion Venom ◽

Enteric Neurons ◽

Intestinal Smooth Muscle ◽

Early Event ◽

Thymidine Uptake

The loss of intrinsic neurons is an early event in inflammation of the rat intestine that precedes the growth of intestinal smooth muscle cells (ISMC). To study this relationship, we cocultured ISMC and myenteric plexus neurons from the rat small intestine and examined the effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141 ± 13% ( n = 6) and the uptake of [3H]thymidine in response to mitogenic stimulation was nearly doubled. Atropine caused a dose-dependent increase in [3H]thymidine uptake in cocultures, suggesting the involvement of neural stimulation of cholinergic receptors in regulation of ISMC growth. In contrast, coculture of ISMC with sympathetic neurons increased [3H]thymidine uptake by 45–80%, which was sensitive to propranolol (30 μM) and was lost when the neurons were separated from ISMC by a permeable filter. Western blotting showed that coculture with myenteric neurons increased α-smooth muscle-specific actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may initiate or modulate ISMC hyperplasia.

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Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration

10.1101/683342 ◽

2019 ◽

Author(s):

Andrea Loreto ◽

Ciaran S. Hill ◽

Victoria L. Hewitt ◽

Giuseppe Orsomando ◽

Carlo Angeletti ◽

...

Keyword(s):

Wallerian Degeneration ◽

Sympathetic Neurons ◽

Axon Degeneration ◽

Loss Of Function ◽

Mitochondrial Uncoupling ◽

Charcot Marie Tooth Disease ◽

Mitochondrial Impairment ◽

Metabolic Fingerprint ◽

Mitochondrial Defects ◽

Tooth Disease

ABSTRACTWallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson’s disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

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Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS

eLife ◽

10.7554/elife.41973 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 29

Author(s):

Éric Martineau ◽

Adriana Di Polo ◽

Christine Vande Velde ◽

Richard Robitaille

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Axonal Degeneration ◽

Synapse Formation ◽

Neuromuscular Junctions ◽

Motor Units ◽

Early Event ◽

Motor Neuron Degeneration ◽

Single Motor Units

Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.

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Single-axon-resolution intravital imaging reveals a rapid onset form of Wallerian degeneration in the adult neocortex

10.1101/391425 ◽

2018 ◽

Author(s):

A.J. Canty ◽

J.S. Jackson ◽

L. Huang ◽

A. Trabalza ◽

C. Bass ◽

...

Keyword(s):

Cortical Neurons ◽

Wallerian Degeneration ◽

Rapid Onset ◽

Therapeutic Window ◽

Mammalian Brain ◽

Axon Degeneration ◽

Single Axon ◽

Threshold Length ◽

Longitudinal Imaging

ABSTRACTDespite the widespread occurrence of axon degeneration in the injured and diseased nervous system, the mechanisms of the degenerative process remain incompletely understood. In particular, the factors that regulate how individual axons degenerate within their native environment in the mammalian brain are unknown. Longitudinal imaging of >120 individually injured cortical axons revealed a threshold length below which injured axons undergo a rapid-onset form of Wallerian degeneration (ROWD). ROWD consistently starts 10 times earlier and is executed 4 times slower than classic Wallerian degeneration (WD). ROWD is dependent on synaptic density, unlike WD, but is independent of axon complexity. Finally, we provide both pharmacological and genetic evidence that a Nicotinamide Adenine Dinucleotide (NAD+)-dependent pathway controls cortical axon ROWD independent of transcription in the damaged neurons. Thus, our data redefine the therapeutic window for intervention to maintain neurological function in injured cortical neurons, and support the use of in vivo optical imaging to gain unique insights into the mechanisms of axon degeneration in the brain.

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A Liquid-to-Solid Phase Transition Enhances the Catalytic Activity of SARM1

10.1101/2020.08.28.272377 ◽

2020 ◽

Author(s):

Heather S. Loring ◽

Paul R. Thompson

Keyword(s):

Phase Transition ◽

Traumatic Brain Injury ◽

Brain Injury ◽

Wallerian Degeneration ◽

Axonal Degeneration ◽

Solid Phase ◽

Neurological Diseases ◽

Promising Therapeutic Target ◽

Solid Phase Transition

ABSTRACTSterile alpha and toll/interleukin receptor (TIR) motif–containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to various stressors. The consequent depletion of NAD+ triggers axonal degeneration (i.e., Wallerian degeneration), which is a characteristic feature of neurological diseases, including peripheral neuropathies and traumatic brain injury. Notably, SARM1 knockout mice show minimal degeneration in models of peripheral neuropathy and traumatic brain injury, making SARM1 a promising therapeutic target. However, the development of SARM1 inhibitors has been challenging as the purified enzyme is largely inactive. Herein, we report that SARM1 activity is increased ∼2000–fold by a liquid-to-solid phase transition. These findings provide critical insights into SARM1 biochemistry with important implications for the situation in vivo. Moreover, they will facilitate the discovery of novel SARM1–targeted therapeutics.Graphical Abstract

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GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration

The Journal of Cell Biology ◽

10.1083/jcb.201606020 ◽

2017 ◽

Vol 216 (2) ◽

pp. 477-493 ◽

Cited By ~ 28

Author(s):

Shuji Wakatsuki ◽

Shinji Tokunaga ◽

Megumi Shibata ◽

Toshiyuki Araki

Keyword(s):

Wallerian Degeneration ◽

Axonal Degeneration ◽

Physiological Function ◽

Glycogen Synthase ◽

Bcl2 Family ◽

Adenosine Triphosphate Production ◽

Bcl2 Family Member ◽

Catabolic Process ◽

Degenerating Axons

Macroautophagy is a catabolic process, in which portions of cytoplasm or organelles are delivered to lysosomes for degradation. Emerging evidence has indicated a pathological connection between axonal degeneration and autophagy. However, the physiological function and induction mechanism of autophagy in axons remain elusive. We herein show that, through activation of BECLIN1, glycogen synthase kinase 3B (GSK3B)–mediated phosphorylation of BCL2 family member MCL1 induces axonal autophagy and axonal degeneration. Phosphorylated MCL1 is ubiquitinated by the FBXW7 ubiquitin ligase and degraded by the proteasome, thereby releasing BECLIN1 to induce axonal autophagy. Axonal autophagy contributes to local adenosine triphosphate production in degenerating axons and the exposure of phosphatidylserine—an “eat-me” signal for phagocytes—on transected axons and is required for normal recruitment of phagocytes to axonal debris in vivo. These results suggest that GSK3B–MCL1 signaling to regulate autophagy might be important for the successful completion of Wallerian degeneration.

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Rapid depletion of ESCRT protein Vps4 underlies injury-induced autophagic impediment and Wallerian degeneration

Science Advances ◽

10.1126/sciadv.aav4971 ◽

2019 ◽

Vol 5 (2) ◽

pp. eaav4971 ◽

Cited By ~ 1

Author(s):

Haiqiong Wang ◽

Xuejie Wang ◽

Kai Zhang ◽

Qingyao Wang ◽

Xu Cao ◽

...

Keyword(s):

Mammalian Cells ◽

Wallerian Degeneration ◽

Axonal Degeneration ◽

Essential Gene ◽

Underlying Mechanism ◽

Autophagic Flux ◽

In Vivo Models ◽

Axon Injury

Injured axons undergo a controlled, self-destruction process, known as Wallerian degeneration. However, the underlying mechanism remains elusive. Using the Drosophila wing nerve as a model, we identify the ESCRT component Vps4 as a previously unidentified essential gene for axonal integrity. Up-regulation of Vps4 remarkably delays degeneration of injured axons. We further reveal that Vps4 is required and sufficient to promote autophagic flux in axons and mammalian cells. Moreover, using both in vitro and in vivo models, we show that the function of Vps4 in maintaining axonal autophagy and suppressing Wallerian degeneration is conserved in mammals. Last, we uncover that Vps4 protein is rapidly depleted in injured mouse axons, which may underlie the injury-induced autophagic impediment and the subsequent axonal degeneration. Together, Vps4 and ESCRT may represent a novel signal transduction mechanism in axon injury and Wallerian degeneration.

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Gene therapy targeting SARM1 blocks pathological axon degeneration in mice

Journal of Experimental Medicine ◽

10.1084/jem.20181040 ◽

2019 ◽

Vol 216 (2) ◽

pp. 294-303 ◽

Cited By ~ 32

Author(s):

Stefanie Geisler ◽

Shay X. Huang ◽

Amy Strickland ◽

Ryan A. Doan ◽

Daniel W. Summers ◽

...

Keyword(s):

Neurological Disorders ◽

Axonal Degeneration ◽

Dominant Negative ◽

Axon Degeneration ◽

Nerve Transection ◽

Adeno Associated Virus ◽

Cellular Models ◽

Promising Strategy ◽

Axon Loss

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus–mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for >10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss.

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The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease

10.1101/539700 ◽

2019 ◽

Cited By ~ 1

Author(s):

Maritza Oñate ◽

Alejandra Catenaccio ◽

Natalia Salvadores ◽

Cristian Saquel ◽

Alexis Martinez ◽

...

Keyword(s):

Cortical Neurons ◽

Axonal Degeneration ◽

Neuronal Loss ◽

Postmortem Brain ◽

Death Process ◽

Early Event ◽

Genetic Ablation ◽

Postmortem Brain Tissue

AbstractParkinson’s disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in vivo, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.

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Regulation of degenerative spheroids after injury

Scientific Reports ◽

10.1038/s41598-020-71906-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yu Yong ◽

Kanchana Gamage ◽

Courtny Cushman ◽

Anthony Spano ◽

Christopher Deppmann

Keyword(s):

Extracellular Space ◽

Wallerian Degeneration ◽

Neurodegenerative Disorder ◽

Sympathetic Neurons ◽

Calcium Flux ◽

Axon Degeneration ◽

Wild Type ◽

Spheroid Formation ◽

Axonal Spheroids ◽

Dependent Growth

Abstract Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1−/− and DR6−/−, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1−/− and DR6−/−, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

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