Microtubules, Membranes, and Movement: New Roles for Stathmin-2 in Axon Integrity.

Neurons establish functional connections responsible for how we perceive and react to the world around us. Communication from a neuron to its target cell occurs through a long projection called an axon. Axon distances can exceed 1 m in length in humans and require a dynamic microtubule cytoskeleton for growth during development and maintenance in adulthood. Stathmins are microtubule-associated proteins that function as relays between kinase signaling and microtubule polymerization. In this review, we describe the prolific role of Stathmins in microtubule homeostasis with an emphasis on emerging roles for Stathmin-2 (Stmn2) in axon integrity and neurodegeneration. Stmn2 levels are altered in Amyotrophic Lateral Sclerosis and loss of Stmn2 provokes motor and sensory neuropathies. There is growing potential for employing Stmn2 as a disease biomarker or even a therapeutic target. Meeting this potential requires a mechanistic understanding of emerging complexity in Stmn2 function. In particular, Stmn2 palmitoylation has a surprising contribution to axon maintenance through undefined mechanisms linking membrane association, tubulin interaction, and axon transport. Exploring these connections will reveal new insight on neuronal cell biology and novel opportunities for disease intervention.

Pyrrole adducts mediated mitochondrial dysfunction activates SARM1-dependent axon degeneration in 2,5-hexanedione-induced neuropathy.

2,5-hexanedione (HD) is the γ-diketone metabolite of industrial organic solvent n-hexane, primarily responsible for n-hexane neurotoxicity. Previous studies have shown that the formation of pyrrole adducts (PAs) is crucial for the toxic axonopathy induced by HD. However, the exact mechanism underlying PAs-induced axonal degeneration remains unclear. Recently, Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) has been identified as the central executor of axon degeneration. This study was designed to investigate the role of SARM1-mediated axon degeneration in rats exposed to HD. Furthermore, the causal relationship between PAs and SARM1-mediated axon degeneration was further explored using Sarm1 KO mice. Our findings suggest that HD causes axon degeneration and neuronal loss in animals. Mechanistic studies revealed that HD activates SARM1-dependent axonal degeneration machinery. In contrast, Sarm1 KO attenuates motor dysfunction and rescues neuron loss following HD exposure. Interestingly, the PAs formed by the binding of HD to proteins primarily accumulate on mitochondria, leading to mitochondrial dysfunction. This dysfunction serves as an upstream event in HD-induced nerve injuries. Our findings highlight the crucial role of PAs formation in the major pathological changes during n-hexane poisoning, providing a potential therapeutic target for n-hexane neuropathy.

HDAC6 inhibition as a mechanism to prevent neurodegeneration in the mSOD1G93A mouse model of ALS.

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One emerging dichotomy is that protection of the neuronal cell soma does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of individual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a mechanism to stabilize axons, prevent axonal loss and neurodegeneration in ALS. This study used an orally dosed potent HDAC6 inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1G93A mouse model of ALS. Co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1G93A mice, reduced lower motor neuron degeneration in female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS.

Recruitment, regulation, and release: Control of signaling enzyme localization and function by reversible S-acylation.

An ever-growing number of studies highlight the importance of S-acylation, a reversible protein-lipid modification, for diverse aspects of intracellular signaling. In this review, we summarize the current understanding of how S-acylation regulates perhaps the best-known class of signaling enzymes, protein kinases. We describe how S-acylation acts as a membrane targeting signal that localizes certain kinases to specific membranes, and how such membrane localization in turn facilitates the assembly of signaling hubs consisting of an S-acylated kinase's upstream activators and/or downstream targets. We further discuss recent findings that S-acylation can control additional aspects of the function of certain kinases, including their interactions and, surprisingly, their activity, and how such regulation might be exploited for potential therapeutic gain. We go on to describe the roles and regulation of de-S-acylases and how extracellular signals drive dynamic (de)S-acylation of certain kinases. We discuss how S-acylation has the potential to lead to "emergent properties" that alter the temporal profile and/or salience of intracellular signaling events. We close by giving examples of other S-acylation-dependent classes of signaling enzymes and by discussing how recent biological and technological advances should facilitate future studies into the functional roles of S-acylation-dependent signaling.

Beyond hypertrophy: Changing views of astrocytes in glaucoma.

Astrocytes serve multiple roles in helping to maintain homeostatic physiology of central nervous system tissue, ranging from metabolic support to coupling between vascular and neural elements. Astrocytes are especially critical in axonal tracts such as the optic nerve, where axons propagate energy-demanding action potentials great distances. In disease, astrocyte remodeling is a dynamic, multifaceted process that is often over-simplified between states of quiescence and reactivity. In glaucoma, axon degeneration in the optic nerve is characterized by progressive stages. So too is astrocyte remodeling. Here, using quantitative analysis of light and electron micrographs of myelinated optic nerve sections from the DBA/2J mouse model of glaucoma, we offer further insight into how astrocyte organization reflects stages of degeneration. This analysis indicates that even as axons degenerate, astrocyte gliosis in the nerve increases without abject proliferation, similar to results in the DBA/2J retina. Gliosis is accompanied by reorganization. As axons expand prior to frank degeneration, astrocyte processes retract from the extra-axonal space and reorient towards the nerve edge. After a critical threshold of expansion, axons drop out, and astrocyte processes distribute more evenly across the nerve reflecting gliosis. This multi-stage process likely reflects local rather than global cues from axons and the surrounding tissue that induce rapid reorganization to promote axon survival and extend functionality of the nerve.

Dissecting the neuroprotective interaction between the BH4 domain of BCL-w and the IP3 receptor.

BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection.

Hypothermia protects the integrity of corticospinal tracts and alleviates mitochondria injury after intracerebral hemorrhage in mice.

Disruption of corticospinal tracts (CST) is a leading factor for motor impairments following intracerebral hemorrhage (ICH) in the striatum. Previous studies have shown that therapeutic hypothermia (HT) improves outcomes of ICH patients. However, whether HT has a direct protection effect on the CST integrity and the underlying mechanisms remain largely unknown. In this study, we employed a chemogenetics approach to selectively activate bilateral warm-sensitive neurons in the preoptic areas to induce a hypothermia-like state. We then assessed effects of HT treatment on the integrity of CST and motor functional recovery after ICH. Our results showed that HT treatment significantly alleviated axonal degeneration around the hematoma and the CST axons at remote midbrain region, ultimately promoted skilled motor function recovery. Anterograde and retrograde tracing revealed that HT treatment protected the integrity of the CST over an extended period. Mechanistically, HT treatment prevented mitochondrial swelling in degenerated axons around the hematoma, alleviated mitochondrial impairment by reducing mitochondrial ROS accumulation and improving mitochondrial membrane potential in primarily cultured cortical neurons with oxyhemoglobin treatment. Serving as a proof of principle, our study provided novel insights into the application of HT to improve functional recovery after ICH.

Label-Free Visualization and Morphological Profiling of Neuronal Differentiation and Axonal Degeneration through Quantitative Phase Imaging.

Understanding the intricate processes of neuronal growth, degeneration, and neurotoxicity is paramount for unraveling nervous system function and holds significant promise in improving patient outcomes, especially in the context of chemotherapy-induced peripheral neuropathy (CIPN). These processes are influenced by a broad range of entwined events facilitated by chemical, electrical, and mechanical signals. The progress of each process is inherently linked to phenotypic changes in cells. Currently, the primary means of demonstrating morphological changes rely on measurements of neurite outgrowth and axon length. However, conventional techniques for monitoring these processes often require extensive preparation to enable manual or semi-automated measurements. Here, a label-free and non-invasive approach is employed for monitoring neuronal differentiation and degeneration using quantitative phase imaging (QPI). Operating on unlabeled specimens and offering little to no phototoxicity and photobleaching, QPI delivers quantitative maps of optical path length delays that provide an objective measure of cellular morphology and dynamics. This approach enables the visualization and quantification of axon length and other physical properties of dorsal root ganglion (DRG) neuronal cells, allowing greater understanding of neuronal responses to stimuli simulating CIPN conditions. This research paves new avenues for the development of more effective strategies in the clinical management of neurotoxicity.

An axon-T cell feedback loop enhances inflammation and axon degeneration.

Inflammation is closely associated with many neurodegenerative disorders. Yet, whether inflammation causes, exacerbates, or responds to neurodegeneration has been challenging to define because the two processes are so closely linked. Here, we disentangle inflammation from the axon damage it causes by individually blocking cytotoxic T cell function and axon degeneration. We model inflammatory damage in mouse skin, a barrier tissue that, despite frequent inflammation, must maintain proper functioning of a dense array of axon terminals. We show that sympathetic axons modulate skin inflammation through release of norepinephrine, which suppresses activation of γδ T cells via the ß2 adrenergic receptor. Strong inflammatory stimulation-modeled by application of the Toll-like receptor 7 agonist imiquimod-causes progressive γδ T cell-mediated, Sarm1-dependent loss of these immunosuppressive sympathetic axons. This removes a physiological brake on T cells, initiating a positive feedback loop of enhanced inflammation and further axon damage.

The Axonal Actin Filament Cytoskeleton: Structure, Function, and Relevance to Injury and Degeneration.

Early investigations of the neuronal actin filament cytoskeleton gave rise to the notion that, although growth cones exhibit high levels of actin filaments, the axon shaft exhibits low levels of actin filaments. With the development of new tools and imaging techniques, the axonal actin filament cytoskeleton has undergone a renaissance and is now an active field of research. This article reviews the current state of knowledge about the actin cytoskeleton of the axon shaft. The best understood forms of actin filament organization along axons are axonal actin patches and a submembranous system of rings that endow the axon with protrusive competency and structural integrity, respectively. Additional forms of actin filament organization along the axon have also been described and their roles are being elucidated. Extracellular signals regulate the axonal actin filament cytoskeleton and our understanding of the signaling mechanisms involved is being elaborated. Finally, recent years have seen advances in our perspective on how the axonal actin cytoskeleton is impacted by, and contributes to, axon injury and degeneration. The work to date has opened new venues and future research will undoubtedly continue to provide a richer understanding of the axonal actin filament cytoskeleton.

Deregulated mitochondrial quality control, the heel of Achilles in elucidating the role of autophagy in SARM1-mediated axon degeneration.

Autophagic dysfunction in neurodegenerative diseases is being extensively studied, yet the exact mechanism of macroautophagy/autophagy in axon degeneration is still elusive. A recent study by Kim et al. links autophagic stress to the sterile α and toll/interleukin 1 receptor motif containing protein 1 (SARM1)-dependent core axonal degeneration program, providing a new insight into the role of autophagy in axon degeneration. In the classical Wallerian axon degeneration model of axotomy, disruption of axonal transport destroys the coordinated activity of pro-survival and pro-degenerative factors in the axoplasm and activates the NADase activity of SARM1, thus triggering the axonal self-destruction program. However, the mechanism for SARM1 activation in the chronic neurodegenerative disorders is more complex. Mitochondrial defects and oxidative stress contribute to the activation of SARM1, while mitophagy can inhibit mitochondrial dysfunction and promote the clearance of SARM1 on mitochondria, thus protecting against neuronal degeneration. Therefore, in-depth elucidation of the underlying mechanisms of mitophagy during axonal degeneration can help develop promising strategies for the prevention and treatment of various neurodegenerative disorders.

A phase transition reduces the threshold for nicotinamide mononucleotide-based activation of SARM1, an NAD(P) hydrolase, to physiologically relevant levels.

Axonal degeneration is a hallmark feature of neurodegenerative diseases. Activation of the NAD(P)ase sterile alpha and toll-interleukin receptor motif containing protein 1 (SARM1) is critical for this process. In resting neurons, SARM1 activity is inhibited, but upon damage, SARM1 is activated and catalyzes one of three NAD(P)+ dependent reactions: (1) NAD(P)+ hydrolysis to form ADP-ribose (ADPR[P]) and nicotinamide; (2) the formation of cyclic-ADPR (cADPR[P]); or (3) a base exchange reaction with nicotinic acid (NA) and NADP+ to form NA adenine dinucleotide phosphate. Production of these metabolites triggers axonal death. Two activation mechanisms have been proposed: (1) an increase in the nicotinamide mononucleotide (NMN) concentration, which leads to the allosteric activation of SARM1, and (2) a phase transition, which stabilizes the active conformation of the enzyme. However, neither of these mechanisms have been shown to occur at the same time. Using in vitro assay systems, we show that the liquid-to-solid phase transition lowers the NMN concentration required to activate the catalytic activity of SARM1 by up to 140-fold. These results unify the proposed activation mechanisms and show for the first time that a phase transition reduces the threshold for NMN-based SARM1 activation to physiologically relevant levels. These results further our understanding of SARM1 activation and will be important for the future development of therapeutics targeting SARM1.

Protecting axons in grandchildren.

Prenatal exposure to environmental agents can influence the fitness of not only the fetus, but also subsequent generations. In a recent study, Wang et al. demonstrated that feeding ursolic acid (UA), a plant-derived compound, to Caenorhabditis elegans mothers during their reproductive period prevented neurodegeneration in not only their offspring, but also the F2 progeny.

A nerve-wracking buzz: lessons from Drosophila models of peripheral neuropathy and axon degeneration.

The degeneration of axons and their terminals occurs following traumatic, toxic, or genetically-induced insults. Common molecular mechanisms unite these disparate triggers to execute a conserved nerve degeneration cascade. In this review, we will discuss how models of peripheral nerve injury and neuropathy in Drosophila have led the way in advancing molecular understanding of axon degeneration and nerve injury pathways. Both neuron-intrinsic as well as glial responses to injury will be highlighted. Finally, we will offer perspective on what additional questions should be answered to advance these discoveries toward clinical interventions for patients with neuropathy.

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell-neuron cocultures.

Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.

Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR.

Traumatic brain injury (TBI) elicits neuronal loss at the site of injury and progressive neuronal loss in the penumbra. However, the consequences of TBI on afferent neurons projecting to the injured tissue from distal locations is unknown. Basal forebrain cholinergic neurons (BFCNs) extend long projections to multiple brain regions including the cortex, regulate many cognitive functions, and are compromised in numerous neurodegenerative disorders. To determine the consequence of cortical injury on these afferent neurons, we used the fluid percussion injury model of traumatic brain injury and assessed the effects on BFCN survival and axon integrity in male and female mice. Survival or death of BF neurons can be regulated by neurotrophins or proneurotrophins, respectively. The injury elicited an induction of proNGF and proBDNF in the cortex and a loss of BFCNs ipsilateral to the injury compared with sham uninjured mice. The p75NTR knock-out mice did not show loss of BFCN neurons, indicating a retrograde degenerative effect of the cortical injury on the afferent BFCNs mediated through p75NTR. In contrast, locus ceruleus neurons, which also project throughout the cortex, were unaffected by the injury, suggesting specificity in retrograde degeneration after cortical TBI. Proneurotrophins (proNTs) provided directly to basal forebrain axons in microfluidic cultures triggered retrograde axonal degeneration and cell death, which did not occur in the absence of p75NTR. This study shows that after traumatic brain injury, proNTs induced in the injured cortex promote BFCN axonal degeneration and retrograde neuron loss through p75NTR.

Adult-onset depletion of sulfatide leads to axonal degeneration with relative myelin sparing.

3-O-sulfogalactosylceramide (sulfatide) constitutes a class of sphingolipids that comprise about 4% of myelin lipids in the central nervous system. Previously, our group characterized a mouse with sulfatide's synthesizing enzyme, cerebroside sulfotransferase (CST), constitutively disrupted. Using these mice, we demonstrated that sulfatide is required for establishment and maintenance of myelin, axoglial junctions, and axonal domains and that sulfatide depletion results in structural pathologies commonly observed in Multiple Sclerosis (MS). Interestingly, sulfatide is reduced in regions of normal appearing white matter (NAWM) of MS patients. Sulfatide reduction in NAWM suggests depletion occurs early in disease development and consistent with functioning as a driving force of disease progression. To closely model MS, an adult-onset disease, our lab generated a "floxed" CST mouse and mated it against the PLP-creERT mouse, resulting in a double transgenic mouse that provides temporal and cell-type specific ablation of the Cst gene (Gal3st1). Using this mouse, we demonstrate adult-onset sulfatide depletion has limited effects on myelin structure but results in the loss of axonal integrity including deterioration of domain organization accompanied by axonal degeneration. Moreover, structurally preserved myelinated axons progressively lose the ability to function as myelinated axons, indicated by the loss of the N1 peak. Together, our findings indicate that sulfatide depletion, which occurs in the early stages of MS progression, is sufficient to drive the loss of axonal function independent of demyelination and that axonal pathology, which is responsible for the irreversible loss of neuronal function that is prevalent in MS, may occur earlier than previously recognized.

Deletion of Transferrin Receptor 1 in Parvalbumin Interneurons Induces a Hereditary Spastic Paraplegia-Like Phenotype.

Hereditary spastic paraplegia (HSP) is a severe neurodegenerative movement disorder, the underlying pathophysiology of which remains poorly understood. Mounting evidence has suggested that iron homeostasis dysregulation can lead to motor function impairment. However, whether deficits in iron homeostasis are involved in the pathophysiology of HSP remains unknown. To address this knowledge gap, we focused on parvalbumin-positive (PV+) interneurons, a large category of inhibitory neurons in the central nervous system, which play a critical role in motor regulation. The PV+ interneuron-specific deletion of the gene encoding transferrin receptor 1 (TFR1), a key component of the neuronal iron uptake machinery, induced severe progressive motor deficits in both male and female mice. In addition, we observed skeletal muscle atrophy, axon degeneration in the spinal cord dorsal column, and alterations in the expression of HSP-related proteins in male mice with Tfr1 deletion in the PV+ interneurons. These phenotypes were highly consistent with the core clinical features of HSP cases. Furthermore, the effects on motor function induced by Tfr1 ablation in PV+ interneurons were mostly concentrated in the dorsal spinal cord; however, iron repletion partly rescued the motor defects and axon loss seen in both sexes of conditional Tfr1 mutant mice. Our study describes a new mouse model for mechanistic and therapeutic studies relating to HSP and provides novel insights into iron metabolism in spinal cord PV+ interneurons and its role in the regulation of motor functions.SIGNIFICANCE STATEMENT Iron is crucial for neuronal functioning. Mounting evidence suggests that iron homeostasis dysregulation can induce motor function deficits. Transferrin receptor 1 (TFR1) is thought to be the key component in neuronal iron uptake. We found that deletion of Tfr1 in parvalbumin-positive (PV+) interneurons in mice induced severe progressive motor deficits, skeletal muscle atrophy, axon degeneration in the spinal cord dorsal column, and alterations in the expression of hereditary spastic paraplegia (HSP)-related proteins. These phenotypes were highly consistent with the core clinical features of HSP cases and partly rescued by iron repletion. This study describes a new mouse model for the study of HSP and provides novel insights into iron metabolism in spinal cord PV+ interneurons.

Neuroprotection in glaucoma: Mechanisms beyond intraocular pressure lowering.

Glaucoma is a common, complex, multifactorial neurodegenerative disease characterized by progressive dysfunction and then loss of retinal ganglion cells, the output neurons of the retina. Glaucoma is the most common cause of irreversible blindness and affects ∼80 million people worldwide with many more undiagnosed. The major risk factors for glaucoma are genetics, age, and elevated intraocular pressure. Current strategies only target intraocular pressure management and do not directly target the neurodegenerative processes occurring at the level of the retinal ganglion cell. Despite strategies to manage intraocular pressure, as many as 40% of glaucoma patients progress to blindness in at least one eye during their lifetime. As such, neuroprotective strategies that target the retinal ganglion cell and these neurodegenerative processes directly are of great therapeutic need. This review will cover the recent advances from basic biology to on-going clinical trials for neuroprotection in glaucoma covering degenerative mechanisms, metabolism, insulin signaling, mTOR, axon transport, apoptosis, autophagy, and neuroinflammation. With an increased understanding of both the basic and clinical mechanisms of the disease, we are closer than ever to a neuroprotective strategy for glaucoma.

SARM1-Dependent Axon Degeneration: Nucleotide Signaling, Neurodegenerative Disorders, Toxicity, and Therapeutic Opportunities.

Axons are an essential component of the nervous system, and axon degeneration is an early feature of many neurodegenerative disorders. The NAD+ metabolome plays an essential role in regulating axonal integrity. Axonal levels of NAD+ and its precursor NMN are controlled in large part by the NAD+ synthesizing survival factor NMNAT2 and the pro-neurodegenerative NADase SARM1, whose activation triggers axon destruction. SARM1 has emerged as a promising axon-specific target for therapeutic intervention, and its function, regulation, structure, and role in neurodegenerative diseases have been extensively characterized in recent years. In this review, we first introduce the key molecular players involved in the SARM1-dependent axon degeneration program. Next, we summarize recent major advances in our understanding of how SARM1 is kept inactive in healthy neurons and how it becomes activated in injured or diseased neurons, which has involved important insights from structural biology. Finally, we discuss the role of SARM1 in neurodegenerative disorders and environmental neurotoxicity and its potential as a therapeutic target.