Adenosine 5′-Triphosphate (ATP) Inhibits Schwann Cell Demyelination During Wallerian Degeneration

Adenosine 5′-triphosphate (ATP) is implicated in intercellular communication as a neurotransmitter in the peripheral nervous system. In addition, ATP is known as lysosomal exocytosis activator. In this study, we investigated the role of extracellular ATP on demyelination during Wallerian degeneration (WD) using ex vivo and in vivo nerve degeneration models. We found that extracellular ATP inhibited myelin fragmentation and axonal degradation during WD. Furthermore, metformin and chlorpromazine, lysosomal exocytosis antagonists blocked the effect of ATP on the inhibition of demyelination. Thus, these findings indicate that ATP-induced-lysosomal exocytosis may be involved in demyelination during WD.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

Incorporation of plasma radiophosphate into the inorganic, organic acid-soluble and phospholipid phosphate fractions of the normal rabbit sciatic nerve and during Wallerian degeneration]

Inorganic phosphate exchanges between plasma and sciatic nerve have been measured in the rabbit using a 32PO4 tracer technique. Inorganic phosphate is taken up at the rate of 0.13 microng per hour and per 100 mg fresh weight. Incorporation of plasma radiophosphate is markedly increased into the inorganic and organic acid soluble phosphate fractions of the distal part of the sectioned sciatic nerve. This increase is already signficant within one hour after surgical division, spreading at least 3 cm distally within 6 hours. This high level of incorporation persists until the 29th day of degeneration. These results favour the hypothesis that the axonal continuity maintains the metabolic activity of the Schwann cells at an inframaximal level. We confirm the rapid decrease in total phospholipid concentration in the nerve undergoing Wallerian degeneration as well as the marked increase in their specific activity. We show however that this increase in specific activity is due partly to the increased specific activities of the precursors (organic acid soluble phosphates), partly to the disappearance of a metabolically insert pool (myelin phospholipids). The Schwann cells of the nerve undergoing Wallerian degeneration do not have a more active phospholipid metabolism than their normal counterparts.     

Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose- specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM- CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Calcium-Mediated Degeneration of the Axonal Cytoskeleton in the Ola Mouse

Abstract: The C57BL/Ola (Ola) mouse is a mutant substrain in which transected axons undergo very slow Wallerian degeneration. Because axonal degradation during Wallerian degeneration is calcium dependent, we tested whether Ola axons are susceptible to calcium-mediated axonal degeneration by comparing neuro-filament degradation between Ola and C57BL/6 mice in sciatic nerve explants. Using immunoblot analysis of neurofilament degradation and electron microscopy we found that as in normal axons, axonal degeneration in the Ola is calcium dependent. However, when compared with normal animals, higher levels of calcium were required for complete degradation of neurofilaments in Ola nerve, suggesting a relative insensitivity to calcium-mediated degeneration in the Ola. We conclude that calcium-activated proteases are present and active in Ola axons but that higher levels of calcium are required to accomplish complete axonal degradation. These results suggest a possible mechanism for prolonged survival of transected Ola axons and provide potential insight into the pathophysiology of axonal degeneration in injury and disease.     

Hydrogen sulfide is essential for Schwann cell responses to peripheral nerve injury

Hydrogen sulfide (H₂S) functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H₂S is produced from substances by three enzymes: cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE), and 3‐mercaptopyruvate sulfurtransferase (MST). In human tissues, these enzymes are involved in tissue‐specific biochemical pathways for H₂S production. For example, CBS and cysteine aminotransferase/MST are present in the brain, but CSE is not. Thus, we examined the expression of H₂S production‐related enzymes in peripheral nerves. Here, we found that CSE and MST/cysteine aminotransferase, but not CBS, were present in normal peripheral nerves. In addition, injured sciatic nerves in vivo up‐regulated CSE in Schwann cells during Wallerian degeneration (WD); however, CSE was not up‐regulated in peripheral axons. Using an ex vivo sciatic nerve explant culture, we found that the inhibition of H₂S production broadly prevented the process of nerve degeneration, including myelin fragmentation, axonal degradation, Schwann cell dedifferentiation, and Schwann cell proliferation in vitro and in vivo. Thus, these results indicate that H₂S signaling is essential for Schwann cell responses to peripheral nerve injury.

     

Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that beta1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1

Sciatic nerve axons in cyclin D1 knockout mice develop normally, become properly ensheathed by Schwann cells, and appear to function normally. However, in the Wallerian degeneration model of nerve injury, the mitotic response of Schwann cells is completely inhibited. The mitotic block is Schwann cell autonomous and developmentally regulated. Rescue analysis (by "knockin" of cyclin E) indicates that D1 protein, rather than regulatory elements of the D1 gene, provides the essential Schwann cell function. Genetic inhibition of the Schwann cell cycle shows that neuronal responses to nerve injury are surprisingly independent of Schwann cell mitotic responses. Even axonal regrowth into the distal zone of a nerve crush injury is not markedly impaired in cyclin D1-/- mice.     

PATTERN OF RNA SYNTHESIS IN THE SCIATIC NERVE OF THE HEN DURING WALLERIAN DEGENERATION

The pattern of synthesis of rapidly-labelled RNA of hen sciatic nerve was studied during Wallerian degeneration. At 2,4,8, 16 and 30 days of degeneration the proximal and distal stumps of the severed nerve as well as the intact contralateral sciatic nerve (functional control) were excised and incubated with either [5-³H]uridine or [2-¹⁴C]uridine for 0.5 h. The electrophoretic pattern of RNA from the normal adult sciatic nerve showed that most of the radioactivity was incorporated into RNA species migrating between the 18 S and 4 S components of the bulk RNA. The synthesis of RNA was sensitive to actinomycin-D, an indication that it was directed by a DNA template. The electrophoretic patterns of the rapidly-labelled RNA in the proximal and distal nerve stumps demonstrated a change following nerve section. After 2–4 days of Wallerian degeneration the degenerating distal nerves incorporated more radioactivity in the 4 S region than the corresponding controls, but at 8 and 16-days after degeneration relatively more label appeared in higher molecular weight RNA species. In the intact sciatic nerve of the operated hens progressively more radioactivity was detected in the 4 S region with increasing time after the contralateral nerve section. At each stage of Wallerian degeneration the specific radioactivities of RNA in the control nerves from experimental hens were higher than those of the normal adult sciatic nerve. These results indicated a change of RNA metabolism in increased functional activity and during Wallerian degeneration.     

Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression

The peripheral nerve contains both nonmyelinating and myelinating Schwann cells. The interactions between axons, surrounding myelin, and Schwann cells are thought to be important for the correct functioning of the nervous system. To get insight into the genes involved in human myelination and maintenance of the myelin sheath and nerve, we performed a serial analysis of gene expression of human sciatic nerve and cultured Schwann cells. In the sciatic nerve library, we found high expression of genes encoding proteins related to lipid metabolism, the complement system, and the cell cycle, while cultured Schwann cells showed mainly high expression of genes encoding extracellular matrix proteins. The results of our study will assist in the identification of genes involved in maintenance of myelin and peripheral nerve and of genes involved in inherited peripheral neuropathies.     

A mitogen for Schwann cells is derived from myelin basic protein

Evidence is presented that a mitogen can be produced from myelin basic protein (MBP) which may be related to the Schwann cell proliferation characteristic of Wallerian degeneration. Myelin derived from the shiverer mutant which is devoid of MBP is also devoid of mitogenic activity. Absorption of the mitogen with a polyclonal antisera to MBP abolishes the mitogenic effect. In addition, only liposomes containing MBP are mitogenic to cultured Schwann cells; liposomes containing other myelin-specific proteins do not stimulate Schwann cell division. These results directly demonstrate the MBP origin of a mitogen for Schwann cells.     

Lesion-Induced Changes in the Production of Newly Synthesized and Secreted Apo-E and Other Molecules Are Independent of the Concomitant Recruitment of Blood-Borne Macrophages into Injured Peripheral Nerves

Peripheral nerve injury produces Wallerian degeneration characterized by a change in the composition of resident nonneuronal cells: macrophages are recruited from the circulation to join Schwann, fibroblast, and endothelial cells. At the same time, the nonneuronal cell population exhibits, as a whole, alterations in synthesis and secretion of diffusible molecules, some of which are instrumental in nerve repair mechanisms. In this study, we determined whether changes in the production of secreted molecules depend on the concomitant modification in cell composition. Therefore, we studied the secretion of newly synthesized molecules by defined cell populations of intact nerves, intact nerve explants undergoing in vitro axonal degeneration, in vivo degenerating nerves, and recruited cells. Nerves were incubated in serum-free, [35S]methionine-containing media. Secreted, radioactively labeled proteins were precipitated from the medium and analyzed by gel electrophoresis. Reduced production of 43-, 46-, and 48-kDa proteins and increased production of 33-34-, 37-, 49-, 59-, and 67-kDa proteins were detected in in situ degenerating nerves. High-density ultracentrifugation and immunoblot analysis revealed that the 33-34-kDa protein is apolipoprotein-E (apo-E). Similar alterations in the production of these molecules were detected in intact nerve explants from which blood-borne cells were excluded. Apo-E, 37-, 49-, 59-, and 67-kDa proteins were also produced in frozen nerves that lacked the intact nerve nonneuronal cell population. Instead, these preparations contained blood-borne cells, primarily macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early events of peripheral nerve regeneration

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon-SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry,including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels.Regrowth is 'staggered' such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow.'Naked' axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of beta III tubulin, PGP(9 x 5) and GAP(43)/B(50) were complex, implying a pause in their regrowth, and were most prominent at the proximal stump-regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges.Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.