Wld^(S),Nmnats and axon degeneration-progress in the past two decades

A chimeric protein called Wallerian degeneration slow(Wld^(S))was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration.This provides a useful tool in elucidating the mechanisms of axon degeneration.Over-expression of WldS attenuates the axon degeneration that is associated with several neurodegenerative disease models,suggesting a new logic for developing a potential protective strategy.At molecular level,although Wld^(S)is a fusion protein,the nicotinamide mononucleotide adenylyl transferase 1(Nmnat1)is required and sufficient for the protective effects of Wld^(S),indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration.These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration.In this review,we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.

Chemotactic signaling and beyond: link between interleukin-16 and axonal degeneration in multiple sclerosis

Multiple sclerosis（MS）is a progressive inflammatory,and chronic demyelinating,neurodegenerative disease of central nervous system（CNS）.Autoimmune responses to myelin and other CNS antigens mediated by cluster of differentiation 4（CD4＋）T cells are critical for initiation and progression of disease.Migration of autoimmune T cells from the peripheral lymph organs into CNS parenchyma leads to inflammation,demyelination and damage of axonal cytoskeleton, which manifest in decreased impulse conduction velocity of motor and sensory nerves. Myelin and axonal pathology causes motor, sensory and autonomic nerve dysfunction, including optic nerve damage leading to double or distorted vision; paresis and paralysis of extremities, painful sensations, and bladder sphincter dysfunction, manifested as bladder incontinence. Gray matter pa- thology in cortical and subcortical regions, including cerebellum and hippocampus underlies cognitive and behavioral dysfunction consisting of memory deficits, depression, and ataxic gait and tremor.

Diffusion tensor imaging as a tool to detect presymptomatic axonal degeneration in a preclinical spinal cord model of amyotrophic lateral sclerosis

The G93A-SOD1 mice model and MRI diffusion as a preclinical tool to study amyotrophic lateral sclerosis （ALS）： ALS is a progressive neurological disease characterized primarily by the development of limb paralysis, which eventually leads to lack of control on muscles under voluntary control and death within 3–5 years. Genetic heterogeneity and environmental factors play a critical role in the rate of disease progression and patients display faster declines once the symptoms have manifested. Since its original discovery, ALS has been associated with pathological alterations in motor neurons located in the spinal cord （SC）, where neuronal loss by a mutation in the protein superoxide dismutase in parenthesis （mSOD1） and impairment in axonal connectivity, have been linked to early functional impairments. In addition,mechanisms of neuroinflammation, apoptosis, necroptosis and autophagy have been also implicated in the development of this disease. Among different animal models developed to study ALS, the transgenic G93A-SOD1 mouse has become recognized as a benchmark model for preclinical screening of ALS therapies. Furthermore, the progressive alterations in the locomotor phenotype expressed in this model closely resemble the progressive lower limb dysfunction of ALS patients. Among other imaging tools, MR diffusion tensor imaging （DTI） has emerged as a crucial, noninvasive and real time neuroimaging tool to gather information in ALS. One of the current concerns with the use of DTI is the lack of biological validation of the microstructural information given by this technique. Although clinical studies using DTI can provide a remarkable insight on the targets of neurodegeneration and disease course,they lack histological correlations. To address these shortcomings, preclinical models can be designed to validate the microstructural information unveiled by this particular MRI technique. Thus, the scope of this review is to describe how MRI diffusion and optical microscopy evaluate axonal structural changes at early stages of the disease in a preclinical model of ALS.

SARM1 participates in axonal degeneration and mitochondrial dysfunction in prion disease

Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss,axonal degeneration,and mitochondrial dysfunction.Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1.We found that depletion or dysfunctional mutation of SARM1 protected against NAD+loss,axonal degeneration,and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126.NAD+supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect.NAD+supplementation in PrP106-126-incubated N2a cells,SARM1 depletion,and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival.Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP^(106-126) are partially dependent on SARM1 NADase activity.This pathway has potential as a therapeutic target in the early stages of prion disease.

Exploratory use of ultrasound to determine whether demyelination following carpal tunnel syndrome co-exists with axonal degeneration

Carpal tunnel syndrome （CTS） accompanied by secondary axonal degeneration cannot be clearly dis- criminated using the current cross-validated ultrasound severity classification system. This study aimed at exploring cut-off values of ultrasound parameters, including wrist cross-sectional area （W-CSA）, wrist perimeter （W-P）, ratio of cross-sectional area （R-CSA） and perimeter （R-P）, changes of CSA and P from wrist to one third distal forearm （△CSA＆AP） for differentiation. Seventy-three patients （13 male and 60 female） were assigned into group A （demyelination only, n = 40） and group B （demyelination with secondary axonal degeneration, n = 33） based on the outcomes of nerve conduction studies （NCS）. Receiver Operative Characteristics （ROC） curves were plotted to obtain sensitivity, specificity, and accuracy of cut- off values for all the ultrasound parameters. The overall identified cut-off values （W-CSA 12.0 mm2, W-P 16.27 mm, R-CSA 1.85, R-P 1.48, △CSA 6.98 mm2, △P 5.77 mm） had good sensitivity （77.1-88.6%）, fair specificity （40-62.2%） and fair-to-good accuracy （0.676-0.758）. There were also significant differences in demographics （age and severity gradation, P 〈 0.001）, NCS findings （wrist motor latency and conduction velocity, P 〈 0.0001; wrist motor amplitude, P 〈 0.05; distal sensory latency, P 〈 0.05; sensory amplitude, P 〈 0.001） and ultrasound measurements （W-CSA, W-P, R-CSA, R-P, △CSA＆△P, P 〈 0.05） between groups. These findings suggest that ultrasound can be potentially used to differentiate demyelinating CTS with sec- ondary axonal degeneration and provide better treatment guidance.

Potential utility of aldose reductase-deficient Schwann cells IKARS1 for the study of axonal degeneration and regeneration

Diabetic peripheral neuropathy（DPN）is one of the most common and intractable complications of diabetes mellitus.Its irritating symptoms,such as paresthesia,hyperalgesia and allodynia,can be causes of insomnia and depression;whereas its progression to more advanced stages can result in serious consequences,such as lower limb amputations and lethal arrhythmias.

Ca^(2+)-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca^(2+)entry after spinal cord injury

The formation of axonal spheroid is a common feature following spinal cord injury.To further understand the source of Ca^(2+)that mediates axonal spheroid formation,we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca^(2+).We performed twophoton excitation imaging of spinal cords isolated from Thy1YFP+transgenic mice and applied the lipophilic dye,Nile red,to record dynamic changes in dorsal column axons and their myelin sheaths respectively.We selectively released Ca^(2+)from internal stores using the Ca^(2+)ionophore ionomycin in the presence or absence of external Ca^(2+).We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 m M Ca^(2+)artificial cerebrospinal fluid.In contrast,removal of external Ca^(2+)significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment.Using mice that express a neuron-specific Ca^(2+)indicator in spinal cord axons,we confirmed that ionomycin induced significant increases in intra-axonal Ca^(2+),but not in the absence of external Ca^(2+).Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation.Pretreatment with YM58483(500 n M),a well-established blocker of store-operated Ca^(2+)entry,significantly decreased myelin injury and axonal spheroid formation.Collectively,these data reveal that ionomycin-induced depletion of internal Ca^(2+)stores and subsequent external Ca^(2+)entry through store-operated Ca^(2+)entry contributes to pathological changes in myelin and axonal spheroid formation,providing new targets to protect central myelinated fibers.

Potential physiological and pathological roles for axonal ryanodine receptors

Clinical disability following trauma or disease to the spinal cord often involves the loss of vital white matter elements including axons and glia.Although excessive Cais an established driver of axonal degeneration,therapeutically targeting externally sourced Cato date has had limited success in both basic and clinical studies.Contributing factors that may underlie this limited success include the complexity of the many potential sources of Caentry and the discovery that axons also contain substantial amounts of stored Cathat if inappropriately released could contribute to axonal demise.Axonal Castorage is largely accomplished by the axoplasmic reticulum that is part of a continuous network of the endoplasmic reticulum that provides a major sink and source of intracellular Cafrom the tips of dendrites to axonal terminals.This“neuron-within-a-neuron”is positioned to rapidly respond to diverse external and internal stimuli by amplifying cytosolic Calevels and generating short and long distance regenerative Cawaves through Cainduced Carelease.This review provides a glimpse into the molecular machinery that has been implicated in regulating ryanodine receptor mediated Carelease in axons and how dysregulation and/or overstimulation of these internodal axonal signaling nanocomplexes may directly contribute to Ca-dependent axonal demise.Neuronal ryanodine receptors expressed in dendrites,soma,and axonal terminals have been implicated in synaptic transmission and synaptic plasticity,but a physiological role for internodal localized ryanodine receptors remains largely obscure.Plausible physiological roles for internodal ryanodine receptors and such an elaborate internodal binary membrane signaling network in axons will also be discussed.

Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure

High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases,yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown.Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel(Healaflow■).Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure.Our results identified a total of 12 cell types,namely retinal pigment epithelial cells,rod-photoreceptor cells,bipolar cells,Müller cells,microglia,cone-photoreceptor cells,retinal ganglion cells,endothelial cells,retinal progenitor cells,oligodendrocytes,pericytes,and fibroblasts.The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells,with ganglion cells decreased by 23%.Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure.We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression.We found upregulation of the B3gat2 gene,which is associated with neuronal migration and adhesion,and downregulation of the Tsc22d gene,which participates in inhibition of inflammation.This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure.These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

Calcium-dependent proteasome activation is required for axonal neurofilament degradation

Even though many studies have identified roles of proteasomes in axonal degeneration, the mo- lecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were sig- nificantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.

Axonal remodeling of the corticospinal tract during neurological recovery after stroke

Stroke remains the leading cause of long-term disability.Hemiparesis is one of the most common post-stroke motor deficits and is largely attributed to loss or disruption of the motor signals from the affected motor cortex.As the only direct descending motor pathway,the corticospinal tract(CST)is the primary pathway to innervate spinal motor neurons,and thus,forms the neuroanatomical basis to control the peripheral muscles for voluntary movements.Here,we review evidence from both experimental animals and stroke patients,regarding CST axonal damage,functional contribution of CST axonal integrity and remodeling to neurological recovery,and therapeutic approaches aimed to enhance CST axonal remodeling after stroke.The new insights gleaned from preclinical and clinical studies may encourage the development of more rational therapeutics with a strategy targeted to promote axonal rewiring for corticospinal innervation,which will significantly impact the current clinical needs of subacute and chronic stroke treatment.

Calcium channel inhibition-mediated axonal stabilization improves axonal regeneration after optic nerve crush

Axonal projections are specialized neuronal compartments and the longest parts of neurons.Axonal degeneration is a common pathological feature in many neurodegenerative disorders,such as Parkinson’s disease,amyotrophic lateral sclerosis,glaucoma,as well as in traumatic lesions of the central nervous system（CNS）,such as spinal cord injury.

Autophagy in degenerating axons following spinal cord injury: evidence for autophagosome biogenesis in retraction bulbs

Macroautophagy （here autophagy） is a catabolic mechanism responsible for the degradation of bulk cytoplasm, long-lived proteins and organeUes. During autophagy, the cargos are engulfed by double-membrane structures named phagophores, which expand to form the autophagosomes. Subsequently, these autophagosomes fuse with lysosomes, in which the cytoplasmic cargos are degraded. Autophagy is a constitutive pro- cess, which plays an important role in cellular homeostasis. In primary neurons autophagosome formation occurs continuously and preferentially at the distal end of axons. On the other hand, autophagy is increased by different stresses, and its dysregulation or excessive induction may lead to detrimental effects. Many neurological disorders have been associated with alterations in the autophagic pathway and an increase in autophagy during axonal degeneration was described.

CMT1A current gene therapy approaches and promising biomarkers

Charcot-Marie-Tooth neuropathies(CMT)constitute a group of common but highly heterogeneous,non-syndromic genetic disorders affecting predominantly the peripheral nervous system.CMT type 1A(CMT1A)is the most frequent type and accounts for almost~50%of all diagnosed CMT cases.CMT1A results from the duplication of the peripheral myelin protein 22(PMP22)gene.Overexpression of PMP22 protein overloads the protein folding apparatus in Schwann cells and activates the unfolded protein response.This leads to Schwann cell apoptosis,dys-and de-myelination and secondary axonal degeneration,ultimately causing neurological disabilities.During the last decades,several different gene therapies have been developed to treat CMT1A.Almost all of them remain at the pre-clinical stage using CMT1A animal models overexpressing PMP22.The therapeutic goal is to achieve gene silencing,directly or indirectly,thereby reversing the CMT1A genetic mechanism allowing the recovery of myelination and prevention of axonal loss.As promising treatments are rapidly emerging,treatment-responsive and clinically relevant biomarkers are becoming necessary.These biomarkers and sensitive clinical evaluation tools will facilitate the design and successful completion of future clinical trials for CMT1A.

Promising application of a new ulnar nerve compound muscle action potential measurement montage in amyotrophic lateral sclerosis:a prospective cross-sectional study

Previous studies have shown that ulnar nerve compound muscle action potential recorded by the conventional“belly-tendon”montage does not accurately and completely reflect the action potential of the ulnar nerve dominating the abductor digiti minimi muscle due to the effects of far-field potentials of intrinsic hand muscles.A new method of ulnar nerve compound muscle action potential measurement was developed in 2020,which adjusts the E2 electrode from the distal tendon of the abductor digitorum to the middle of the back of the proximal wrist.This new method may reduce the influence of the reference electrode and better reflect the actual ulnar nerve compound muscle action potential.In this prospective cross-sectional study,we included 64 patients with amyotrophic lateral sclerosis and 64 age-and sex-matched controls who underwent conventional and novel ulnar nerve compound muscle action potential measurement between April 2020 and May 2021 in Peking University Third Hospital.The compound muscle action potential waveforms recorded by the new montage were unimodal and more uniform than those recorded by traditional montage.In the controls,no significant difference in the compound muscle action potential waveforms was found between the traditional montage and new montage recordings.In amyotrophic lateral sclerosis patients presenting with abductor digiti minimi spontaneous activity and muscular atrophy,the amplitude of compound muscle action potential-pE2 was significantly lower than that of compound muscle action potential-dE2(P<0.01).Using the new method,damaged axons were more likely to exhibit more severe amplitude decreases than those measured with the traditional method,in particular for patients in early stage amyotrophic lateral sclerosis.In addition,the decline in compound muscle action potential amplitude measured by the new method was correlated with a decrease in Revised Amyotrophic Lateral Sclerosis Functional Rating Scale scores.These findings suggest that the new ulnar nerve compound muscle action potential measurement montage reduces the effects of the reference electrode through altering the E2 electrode position,and that this method is more suitable for monitoring disease progression than the traditional montage.This method may be useful as a biomarker for longitudinal follow-up and clinical trials in amyotrophic lateral sclerosis.

Current landscape in motoneuron regeneration and reconstruction for motor cranial nerve injuries

The intricate anatomy and physiology of cranial nerves have inspired clinicians and scientists to study their roles in the nervous system. Damage to motor cranial nerves may result from a variety of organic or iatrogenic insults and causes devastating functional impairment and disfigurement. Surgical innovations directed towards restoring function to injured motor cranial nerves and their associated organs have evolved to include nerve repair, grafting, substitution, and muscle transposition. In parallel with this progress, research on tissue-engineered constructs, development of bioelectrical interfaces, and modulation of the regenerative milieu through cellular, immunomodulatory, or neurotrophic mechanisms has proliferated to enhance the available repertoire of clinically applicable reconstructive options. Despite these advances, patients continue to suffer from functional limitations relating to inadequate cranial nerve regeneration, aberrant reinnervation, or incomplete recovery of neuromuscular function. These shortfalls have profound quality of life ramifications and provide an impetus to further elucidate mechanisms underlying cranial nerve denervation and to improve repair. In this review, we summarize the literature on reconstruction and regeneration of motor cranial nerves following various injury patterns. We focus on seven cranial nerves with predominantly efferent functions and highlight shared patterns of injuries and clinical manifestations. We also present an overview of the existing reconstructive approaches, from facial reanimation, laryngeal reinnervation, to variations of interposition nerve grafts for reconstruction. We discuss ongoing endeavors to promote nerve regeneration and to suppress aberrant reinnervation and the development of synkinesis. Insights from these studies will shed light on recent progress and new horizons in understanding the biomechanics of peripheral nerve neurobiology, with emphasis on promising strategies for optimizing neural regeneration and identifying future directions in the field of motor cranial neuron research.

Ototoxic effects of mefloquine in cochlear organotypic cultures

Mefloquine is a widely used anti-malarial drug. Some clinical reports suggest that mefloquine may be ototoxic and neurotoxic, but there is little scientific evidence from which to draw any firm conclusion. To evaluate the ototoxic and neurotoxic potential of mefloquine, we treated cochlear organotypic cultures and spiral ganglion cultures with various concentrations of mefloquine. Mefloquine caused a dose-dependent loss of cochlear hair cells at doses exceeding 0.01 mM. Hair cell loss progressed from base to apex and from outer to inner hair cells with increasing dose. Spiral ganglion neurons and auditory nerve fibers were also rapidly destroyed by mefloquine in a dose-dependent manner. To investigate the mechanisms underlying mefloquine-induced cell death, cochlear cultures were stained with TO-Pro-3 to identify morphological changes in the nucleus, and with carboxyfluorescein FAM-labeled caspase inhibitor 8, 9 or 3 to determine caspase-mediated cell death. TO-Pro-3-labeled nuclei in hair cells, spiral ganglion neurons and supporting cells were shrunken or fragmented, morphological features characteristic of cells undergoing apoptosis. Both initiator caspase 8 (membrane damage) and caspase 9 (mitochondrial damage), along with executioner caspase 3, were heavily expressed in cochlear hair cells and spiral ganglions after mefloquine treatment. These three caspases were also expressed in support cells, although labeling was less widespread and less intense. These results indicate that mefloquine damages both the sensory and neural elements in the postnatal rat cochlea by initially activating cell death signaling pathways on the cell membrane and in mitochondria.

Modeling subcortical ischemic white matter injury in rodents:unmet need for a breakthrough in translational research

Subcortical ischemic white matter injury(SIWMI),pathological correlate of white matter hyperintensities or leukoaraiosis on magnetic resonance imaging,is a common cause of cognitive decline in elderly.Despite its high prevalence,it remains unknown how various components of the white matter degenerate in response to chronic ischemia.This incomplete knowledge is in part due to a lack of adequate animal model.The current review introduces various SIWMI animal models and aims to scrutinize their advantages and disadvantages primarily in regard to the pathological manifestations of white matter components.The SIWMI animal models are categorized into 1)chemically induced SIWMI models,2)vascular occlusive SIWMI models,and 3)SIWMI models with comorbid vascular risk factors.Chemically induced models display consistent lesions in predetermined areas of the white matter,but the abrupt evolution of lesions does not appropriately reflect the progressive pathological processes in human white matter hyperintensities.Vascular occlusive SIWMI models often do not exhibit white matter lesions that are sufficiently unequivocal to be quantified.When combined with comorbid vascular risk factors(specifically hypertension),however,they can produce progressive and definitive white matter lesions including diffuse rarefaction,demyelination,loss of oligodendrocytes,and glial activation,which are by far the closest to those found in human white matter hyperintensities lesions.However,considerable surgical mortality and unpredictable natural deaths during a follow-up period would necessitate further refinements in these models.In the meantime,in vitro SIWMI models that recapitulate myelinated white matter track may be utilized to study molecular mechanisms of the ischemic white matter injury.Appropriate in vivo and in vitro SIWMI models will contribute in a complementary manner to making a breakthrough in developing effective treatment to prevent progression of white matter hyperintensities.

Coexistent Charcot-Marie-Tooth type 1A and type 2 diabetes mellitus neuropathies in a Chinese family

Charcot-Marie-Tooth disease type 1A（CMT1A） is caused by duplication of the peripheral myelin protein 22（PMP22） gene on chromosome 17. It is the most common inherited demyelinating neuropathy. Type 2 diabetes mellitus is a common metabolic disorder that frequently causes predominantly sensory neuropathy. In this study, we report the occurrence of CMT1 A in a Chinese family affected by type 2 diabetes mellitus. In this family, seven individuals had duplication of the PMP22 gene, although only four had clinical features of polyneuropathy. All CMT1 A patients with a clinical phenotype also presented with type 2 diabetes mellitus. The other three individuals had no signs of CMT1 A or type 2 diabetes mellitus. We believe that there may be a genetic link between these two diseases.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1（TRPV1） provides the sensation of pain（nociception）. However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517（300 mg/kg）, a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.