In Vivo Imaging of Secondary Neurodegeneration Associated With Phosphatidylserine Externalization Along Axotomized Axons.

Purpose: Axonal degeneration in acute and chronic disorders is well-characterized, comprising retrograde (proximal) and Wallerian (distal) degeneration, but the mechanism of propagation remains less understood. Methods: Laser injury with a diode-pumped solid-state 532 nm laser was used to axotomize retinal ganglion cell axons. We used confocal in vivo imaging to demonstrate that phosphatidylserine externalization is a biomarker of early axonal degeneration after selective intraretinal axotomy. Results: Quantitative dynamic analysis revealed that the rate of axonal degeneration was fastest within 40 minutes, then decreased exponentially afterwards. Axonal degeneration was constrained within the same axotomized axonal bundles. Remarkably, axon degeneration arising from the site of injury induced a secondary degeneration of distal normal axons. Conclusions: Axonal degeneration in vivo is a progressive process associated with phosphatidylserine externalization, which can propagate not only along the axon but to adjacent uninjured axons. This finding has implications for acute and chronic neurodegenerative disorders associated with axonal injury.

[Resting tremor of Parkinson's disease changing into Holmes' tremor by cerebellar hemorrhage: an examination of the pathophysiological mechanism of tremor].

A 71-year-old male who suffered from Hoehn and Yahr stage III Parkinson's disease with bradykinesia, rigidity and a 5-6-Hz tremor at rest in the right extremities was admitted to our hospital due to the sudden onset of vertigo. Right cerebellar hemorrhage was confirmed by CT. The patient's resting tremor in the right extremities disappeared immediately following the cerebellar hemorrhage. Six days later, MRI showed Wallerian degeneration in the cerebello-rubro-thalamic tract. Approximately 5 months later, a 2-3-Hz Holmes' tremor gradually appeared in the right upper extremity. This tremor was improved by increasing L-dopa doses. Case reports of the disappearance of Parkinson's resting tremor and subsequent emergence of Holmes' tremor due to cerebellar lesion are rare. Furthermore, the Wallerian degeneration of the cerebello-rubro-thalamic tract identified on MRI between tremors of the different frequencies is very rare. We hypothesize that the cause of the tremor frequency change was simultaneous damage to the nigro-striatal network and the cerebello-thalamo-cerebral network.

Iron role paradox in nerve degeneration and regeneration.

Iron accumulates in the neural tissue during peripheral nerve degeneration. Some studies have already been suggested that iron facilitates Wallerian degeneration (WD) events such as Schwann cell de-differentiation. On the other hand, intracellular iron levels remain elevated during nerve regeneration and gradually decrease. Iron enhances Schwann cell differentiation and axonal outgrowth. Therefore, there seems to be a paradox in the role of iron during nerve degeneration and regeneration. We explain this contradiction by suggesting that the increase in intracellular iron concentration during peripheral nerve degeneration is likely to prepare neural cells for the initiation of regeneration. Changes in iron levels are the result of changes in the expression of iron homeostasis proteins. In this review, we will first discuss the changes in the iron/iron homeostasis protein levels during peripheral nerve degeneration and regeneration and then explain how iron is related to nerve regeneration. This data may help better understand the mechanisms of peripheral nerve repair and find a solution to prevent or slow the progression of peripheral neuropathies.

Early histopathological changes of secondary degeneration in the spinal cord after total MCA territory stroke.

Previous research has not demonstrated secondary degeneration of the spinal cord (SpC) motoneurons after cerebral infarct. The aim of the present study is to investigate the involvement of the anterior horn cells (AHC) in the early post-stroke period using histomorphological and immunohistochemical methods. Post-mortem analysis of the 6th cervical segment was performed in 7 patients who had total MCA stroke within 1 month before death. Nissl-stained sections were used for morphometry, while CD68 and synaptophysin (SYP) immunohistochemistry to monitor microglial activation and synaptic changes in the anterior horn (AH), respectively. Contralateral to the cerebral lesion (contralesional side), cells were smaller after 3 days and larger after 1 week of stroke, especially regarding the large alpha motoneurons. CD68 density increased mainly on the contralesional Rexed's IX lamina of the SpC. SYP coverage of the large motoneurons was reduced on the contralesional side. Early microglial activation in the AH and electrophysiological signs has suggested the possibility of impairment of anterior horn cells (AHC-s). Our study supported that early microglial activation in the contralesional side of the SpC may primarily affect the area corresponding to the location of large motoneurons, and is accompanied by a transient shrinkage followed by increase in size of the large AHC-s with a reduction of their synaptic coverage. After MCA stroke, early involvement of the SpC motoneurons may be suspected by their morphological and synaptic changes and by the pattern of microglial activation.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

PGC-1α Inhibits Schwann Cell Dedifferentiation and Delays Peripheral Nerve Degeneration by Targeting PON1.

PPARγ coactivator-1 alpha (PGC-1α) is an essential transcription factor co-activator that regulates gene transcription and neural regeneration. Schwann cells, which are unique glial cells in peripheral nerves that dedifferentiate after peripheral nerve injury (PNI) and are released from degenerative nerves. Wallerian degeneration is a series of stereotypical events that occurs in response to nerve fibers after PNI. The role of PGC-1α in Schwann cell dedifferentiation and Wallerian degeneration is not yet clear. As Wallerian degeneration plays a crucial role in PNI, we conducted a study to determine whether PGC-1α has an effect on peripheral nerve degeneration after injury. We examined the expression of PGC-1α after sciatic nerve crush or transection using Western blotting and found that PGC-1α expression increased after PNI. Then we utilized ex vivo and in vitro models to investigate the effects of PGC-1α inhibition and activation on Schwann cell dedifferentiation and nerve degeneration. Our findings indicate that PGC-1α negatively regulates Schwann cell dedifferentiation and nerve degeneration. Through the use of RNA-seq, siRNA/plasmid transfection and reversal experiments, we identified that PGC-1α targets inhibit the expression of paraoxonase 1 (PON1) during Schwann cell dedifferentiation in degenerated nerves. In summary, PGC-1α plays a crucial role in preventing Schwann cell dedifferentiation and its activation can reduce peripheral nerve degeneration by targeting PON1. PGC-1α inhibits Schwann cell dedifferentiation and peripheral nerve degeneration. PGC-1α negatively regulates Schwann cell dedifferentiation and peripheral nerve degeneration after injury by targeting PON1.

NAD+, Axonal Maintenance, and Neurological Disease.

Significance: The remarkable geometry of the axon exposes it to unique challenges for survival and maintenance. Axonal degeneration is a feature of peripheral neuropathies, glaucoma, and traumatic brain injury, and an early event in neurodegenerative diseases. Since the discovery of Wallerian degeneration (WD), a molecular program that hijacks nicotinamide adenine dinucleotide (NAD+) metabolism for axonal self-destruction, the complex roles of NAD+ in axonal viability and disease have become research priority. Recent Advances: The discoveries of the protective Wallerian degeneration slow (WldS) and of sterile alpha and TIR motif containing 1 (SARM1) activation as the main instructive signal for WD have shed new light on the regulatory role of NAD+ in axonal degeneration in a growing number of neurological diseases. SARM1 has been characterized as a NAD+ hydrolase and sensor of NAD+ metabolism. The discovery of regulators of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) proteostasis in axons, the allosteric regulation of SARM1 by NAD+ and NMN, and the existence of clinically relevant windows of action of these signals has opened new opportunities for therapeutic interventions, including SARM1 inhibitors and modulators of NAD+ metabolism. Critical Issues: Events upstream and downstream of SARM1 remain unclear. Furthermore, manipulating NAD+ metabolism, an overdetermined process crucial in cell survival, for preventing the degeneration of the injured axon may be difficult and potentially toxic. Future Directions: There is a need for clarification of the distinct roles of NAD+ metabolism in axonal maintenance as contrasted to WD. There is also a need to better understand the role of NAD+ metabolism in axonal endangerment in neuropathies, diseases of the white matter, and the early stages of neurodegenerative diseases of the central nervous system. Antioxid. Redox Signal. 39, 1167-1184.

Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration.

The cellular and molecular underpinnings of Wallerian degeneration have been robustly explored in laboratory models of successful nerve regeneration. In contrast, there is limited interrogation of failed regeneration, which is the challenge facing clinical practice. Specifically, we lack insight on the pathophysiologic mechanisms that lead to the formation of neuromas-in-continuity (NIC). To address this knowledge gap, we have developed and validated a novel basic science model of rapid-stretch nerve injury, which provides a biofidelic injury with NIC development and incomplete neurologic recovery. In this study, we applied next-generation RNA sequencing to elucidate the temporal transcriptional landscape of pathophysiologic nerve regeneration. To corroborate genetic analysis, nerves were subject to immunofluorescent staining for transcripts representative of the prominent biological pathways identified. Pathophysiologic nerve regeneration produces substantially altered genetic profiles both temporally and in the mature neuroma microenvironment, in contrast to the coordinated genetic signatures of Wallerian degeneration and successful regeneration. To our knowledge, this study presents as the first transcriptional study of NIC pathophysiology and has identified cellular death, fibrosis, neurodegeneration, metabolism, and unresolved inflammatory signatures that diverge from pathways elaborated by traditional models of successful nerve regeneration.

Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve.

Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.

Zebrafish cutaneous injury models reveal that Langerhans cells engulf axonal debris in adult epidermis.

Somatosensory neurons extend enormous peripheral axons to the skin, where they detect diverse environmental stimuli. Somatosensory peripheral axons are easily damaged due to their small caliber and superficial location. Axonal damage results in Wallerian degeneration, creating vast quantities of cellular debris that phagocytes must remove to maintain organ homeostasis. The cellular mechanisms that ensure efficient clearance of axon debris from stratified adult skin are unknown. Here, we established zebrafish scales as a tractable model to study axon degeneration in the adult epidermis. Using this system, we demonstrated that skin-resident immune cells known as Langerhans cells engulf the majority of axon debris. In contrast to immature skin, adult keratinocytes did not significantly contribute to debris removal, even in animals lacking Langerhans cells. Our study establishes a powerful new model for studying Wallerian degeneration and identifies a new function for Langerhans cells in maintenance of adult skin homeostasis following injury. These findings have important implications for pathologies that trigger somatosensory axon degeneration.

Regulation of the V-ATPase subunit ATP6V0D2 and its role in demyelination after peripheral nerve injury.

After peripheral nerve injury, demyelinating Schwann cells discharge myelin debris and macrophages execute myelin degradation, leading to demyelination of degenerating axons, which is essential for efficient nerve regeneration. In this study, we show that vacuolar-type proton ATPase subunit d2 (Atp6v0d2) is among the most highly upregulated genes in degenerating mouse sciatic nerves after nerve injury using microarray analysis. ATP6V0D2 is mostly expressed in macrophages of injured nerves. Atp6v0d2 knockout mice display delayed peripheral nerve demyelination and significantly attenuated myelin lipid digestion after nerve injury. However, macrophage recruitment and Schwann cell dedifferentiation are unaffected by loss of Atp6v0d2 expression. Taken together, these data demonstrate that ATP6V0D2 in macrophages is specifically required for demyelination during Wallerian degeneration.

Traumatic axonopathy in spinal tracts after impact acceleration head injury: Ultrastructural observations and evidence of SARM1-dependent axonal degeneration.

Traumatic axonal injury (TAI) and the associated axonopathy are common consequences of traumatic brain injury (TBI) and contribute to significant neurological morbidity. It has been previously suggested that TAI activates a highly conserved program of axonal self-destruction known as Wallerian degeneration (WD). In the present study, we utilize our well-established impact acceleration model of TBI (IA-TBI) to characterize the pathology of injured myelinated axons in the white matter tracks traversing the ventral, lateral, and dorsal spinal columns in the mouse and assess the effect of Sterile Alpha and TIR Motif Containing 1 (Sarm1) gene knockout on acute and subacute axonal degeneration and myelin pathology. In silver-stained preparations, we found that IA-TBI results in white matter pathology as well as terminal field degeneration across the rostrocaudal axis of the spinal cord. At the ultrastructural level, we found that traumatic axonopathy is associated with diverse types of axonal and myelin pathology, ranging from focal axoskeletal perturbations and focal disruption of the myelin sheath to axonal fragmentation. Several morphological features such as neurofilament compaction, accumulation of organelles and inclusions, axoskeletal flocculation, myelin degeneration and formation of ovoids are similar to profiles encountered in classical examples of WD. Other profiles such as excess myelin figures and inner tongue evaginations are more typical of chronic neuropathies. Stereological analysis of pathological axonal and myelin profiles in the ventral, lateral, and dorsal columns of the lower cervical cord (C6) segments from wild type and Sarm1 KO mice at 3 and 7 days post IA-TBI (n = 32) revealed an up to 90% reduction in the density of pathological profiles in Sarm1 KO mice after IA-TBI. Protection was evident across all white matter tracts assessed, but showed some variability. Finally, Sarm1 deletion ameliorated the activation of microglia associated with TAI. Our findings demonstrate the presence of severe traumatic axonopathy in multiple ascending and descending long tracts after IA-TBI with features consistent with some chronic axonopathies and models of WD and the across-tract protective effect of Sarm1 deletion.

NMNAT2: An important metabolic enzyme affecting the disease progression.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an evolutionarily conserved nicotinamide adenine dinucleotide (NAD+) synthase located in the cytoplasm and Golgi apparatus. NMNAT2 has an important role in neurodegenerative diseases, malignant tumors, and other diseases that seriously endanger human health. NMNAT2 exerts a neuroprotective function through its NAD synthase activity and chaperone function. Among them, the NMNAT2-NAD+-Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) axis is closely related to Wallerian degeneration. Physical injury or pathological stimulation will cause a decrease in NMNAT2, which activates SARM1, leading to axonal degeneration and the occurrence of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, peripheral neuropathy, and other neurodegenerative diseases. In addition, NMNAT2 exerts a cancer-promoting role in solid tumors, including colorectal cancer, lung cancer, ovarian cancer, and glioma, and is closely related to tumor occurrence and development. This paper reviews the chromosomal and subcellular localization of NMNAT2 and its basic biological functions. We also summarize the NMNAT2-related signal transduction pathway and the role of NMNAT2 in diseases. We aimed to provide a new perspective to comprehensively understand the relationship between NMNAT2 and its associated diseases.

Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury.

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

The injured sciatic nerve atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration.

Upon trauma, the adult murine peripheral nervous system (PNS) displays a remarkable degree of spontaneous anatomical and functional regeneration. To explore extrinsic mechanisms of neural repair, we carried out single-cell analysis of naïve mouse sciatic nerve, peripheral blood mononuclear cells, and crushed sciatic nerves at 1 day, 3 days, and 7 days following injury. During the first week, monocytes and macrophages (Mo/Mac) rapidly accumulate in the injured nerve and undergo extensive metabolic reprogramming. Proinflammatory Mo/Mac with a high glycolytic flux dominate the early injury response and rapidly give way to inflammation resolving Mac, programmed toward oxidative phosphorylation. Nerve crush injury causes partial leakiness of the blood-nerve barrier, proliferation of endoneurial and perineurial stromal cells, and entry of opsonizing serum proteins. Micro-dissection of the nerve injury site and distal nerve, followed by single-cell RNA-sequencing, identified distinct immune compartments, triggered by mechanical nerve wounding and Wallerian degeneration, respectively. This finding was independently confirmed with Sarm1-/- mice, in which Wallerian degeneration is greatly delayed. Experiments with chimeric mice showed that wildtype immune cells readily enter the injury site in Sarm1-/- mice, but are sparse in the distal nerve, except for Mo. We used CellChat to explore intercellular communications in the naïve and injured PNS and report on hundreds of ligand-receptor interactions. Our longitudinal analysis represents a new resource for neural tissue regeneration, reveals location- specific immune microenvironments, and reports on large intercellular communication networks. To facilitate mining of scRNAseq datasets, we generated the injured sciatic nerve atlas (iSNAT): https://cdb-rshiny.med.umich.edu/Giger_iSNAT/.

A photo-switchable assay system for dendrite degeneration and repair in Drosophila melanogaster.

Neurodegeneration arising from aging, injury, or diseases has devastating health consequences. Whereas neuronal survival and axon degeneration have been studied extensively, much less is known about how neurodegeneration affects dendrites, in part due to the limited assay systems available. To develop an assay for dendrite degeneration and repair, we used photo-switchable caspase-3 (caspase-Light-Oxygen-Voltage-sensing [caspase-LOV]) in peripheral class 4 dendrite arborization (c4da) neurons to induce graded neurodegeneration by adjusting illumination duration during development and adulthood in Drosophila melanogaster. We found that both developing and mature c4da neurons were able to survive while sustaining mild neurodegeneration induced by moderate caspase-LOV activation. Further, we observed active dendrite addition and dendrite regeneration in developing and mature c4da neurons, respectively. Using this assay, we found that the mouse Wallerian degeneration slow (WldS) protein can protect c4da neurons from caspase-LOV-induced dendrite degeneration and cell death. Furthermore, our data show that WldS can reduce dendrite elimination without affecting dendrite addition. In summary, we successfully established a photo-switchable assay system in both developing and mature neurons and used WldS as a test case to study the mechanisms underlying dendrite regeneration and repair.

Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma.

Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood-nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.