A quantitative electron microscopic analysis of myelination in the optic nerve of suckling rats treated with an inhibitor of cholesterol biosynthesis

Summary The effect of AY-9944, an inhibitory cholesterol biosynthesis, on the myelination of the optic nerve of rats was studied. Suckling rats were injected intraperitoneally with the drug every other day from birth, and were sacrificed at 10, 20 and 30 days of age together with littermate controls. The analysis is based on counting, at the electron-microscope level, the number of unmyelinated axons and the number of myelin lamellae surrounding each myelinating axon. The results indicate that a decrease in endogenous cholesterol by AY 9944, induced an overall retardation of the myelination process in the optic nerve: a larger proportion of myelinated axons and smaller number of myelin lamellae around the myelinating axons, when compared with the littermate controls, was observed. Exogenous cholesterol from the maternal milk did not compensate for a lack in endogenous cholesterol.Degenerating myelin sheaths were frequently seen in the experimental optic nerves at 20 and 30 days of age. Numerous membranous, intracytoplasmic drug-induced inclusions were found at all ages studied. Acknowledgements. The author is particularly indebted to Dr. B. G. Uzman and Dr. G. M. Villegas for their valuable discussion and suggestions. He wishes also to thank Mr. F. Paredes, Mr. J. Aristimuño and Miss Marcia Escala for their technical assistance; Mr. J. Bigorra for the photographic aid, and Miss Sonia Rodríguez for her secretarial help.     

FINE STRUCTURAL LOCALIZATION OF CHOLESTEROL-1,2-3H IN DEGENERATING AND REGENERATING MOUSE SCIATIC NERVE

The localization of ³H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level. Two types of experiments were carried out: (a) Cholesterol-1,2-³H was injected intraperitoneally into suckling mice. 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches. The animals were then sacrificed at various times after the operation. During degeneration, radioactivity was found over myelin debris and fat droplets. In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths. Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths. Radioactivity was also associated with the myelin sheaths of the unaltered fibers, (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches. Cholesterol-1,2-³H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers. The results from these experiments indicate that: (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration. Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin. (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration, (c) mature myelin maintains its ability to incorporate cholesterol.     

Localization of Phospholipid Synthesis to Schwann Cells and Axons

Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glycerol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent-shaped perinuclear cytoplasm of myelinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt-Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholipids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: myelinating Schwann cells dominate phospholipid and protein synthesis, myelinated axons selectively incorporate tritiated inositol, phospholipid precursors label myelin sheaths and myelinated axons better than proline.     

Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells.

Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies.     

Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination.     

Cerebral Vulnerability Is Associated with Selective Increase in Extracellular Glutamate Concentration in Experimental Thiamine Deficiency

Abstract: The concentration of apolipoprotein E (apoE), a high-affinity ligand for the low-density lipoprotein receptor, increases dramatically in peripheral nerve following injury. This endoneurial apoE is thought to play an important role in the redistribution of lipids from the degenerating axonal and myelin membranes to the regenerating axons and myelin sheaths. The importance of apoE in nerve repair was examined using mutant mice that lack apoE. We show that at 2 and 4 weeks following sciatic nerve crush, regenerating nerves in apoE-deficient mice were morphologically similar to regenerating nerves in control animals, indicating that apoE is not essential for peripheral nerve repair. Moreover, cholesterol synthesis was reduced in regenerating nerves of apoE-deficient mice as much as in regenerating nerves of control animals. These results suggest that the intraneural conservation and reutilization of cholesterol following nerve injury do not require apoE.     

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.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.     

Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells

In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in non-myelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31-kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.     

A role for Nogo receptor in macrophage clearance from injured peripheral nerve

We report a role for Nogo receptors (NgRs) in macrophage efflux from sites of inflammation in peripheral nerve. Increasing numbers of macrophages in crushed rat sciatic nerves express NgR1 and NgR2 on the cell surface in the first week after injury. These macrophages show reduced binding to myelin and MAG in vitro, which is reversed by NgR siRNA knockdown and by inhibiting Rho-associated kinase. Fourteen days after sciatic nerve crush, regenerating nerves with newly synthesized myelin have fewer macrophages than cut/ligated nerves that lack axons and myelin. Almost all macrophages in the cut/ligated nerves lie within the Schwann cell basal lamina, while in the crushed regenerating nerves the majority migrate out. Furthermore, crush-injured nerves of NgR1- and MAG-deficient mice and Y-27632-treated rats show impaired macrophage efflux from Schwann cell basal lamina containing myelinated axons. These data have implications for the resolution of inflammation in peripheral nerve and CNS pathologies.     

Epineurial adipocytes are dispensable for Schwann cell myelination

Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte‐specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte‐specific Lpin1 knockout mice. To evaluate the possibility that Lpin1‐negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast‐specific deletion of peroxisome proliferator‐activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator‐activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.     

A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells

Searching for specific markers of neural crest-derived cell lineages, we immunized mice with glycoproteins purified from adult quail peripheral myelin. We obtained a monoclonal antibody that reacts with myelin and peripheral glial cells. This antibody, to Schwann cell myelin protein (SMP), is specific for the membranes of all Schwann cells, irrespective of whether they are associated with myelinated nerves. SMP persists on Schwann cells in long-term cultures in vitro, but is absent from satellite cells of peripheral ganglia, both in vivo and in vitro. The antigen (a protein doublet of Mr 75,000-80,000) is present in, but not restricted to, the myelin lamellae, since it is distributed along the whole myelinating Schwann cell membrane. In the CNS, SMP appears as a single band of Mr 80,000. SMP is first detectable by immunofluorescence at E6 in the quail, which is at least 6 days earlier than the first appearance of already described markers related to myelination.     

THE EFFECTS OF UNDERNUTRITION ON THE PROTEINS OF OPTIC AND SCIATIC NERVES DURING DEVELOPMENT

Abstract— Polyacrylamide gel electrophoresis has been used to assess the appearance of some optic and sciatic nerve proteins in normal developing rats and in undernourished rats. Of the myelin proteins, the'Wolfgram'proteolipid is already present about the time myelination begins. The basic myelin proteins appear later, first in sciatic and then in optic nerve. A non-myelin basic protein, assumed to be a histone, is present at high levels in both nerves before myelination begins. There is no apparent effect of undernutrition on the appearance and amount of myelin proteins at 12, 16 and 22 days of age. The'histone'protein is reduced in optic and sciatic nerves at times corresponding roughly to the transition periods from cellular proliferation to myelin formation. The possibilities are discussed that myelin basic proteins are synthesized as compact myelin formation occurs, and that there may be retarded cellular proliferation in nerves of undernourished rats.     

Alteration of ethanolamine glycerophospholipid turnover in trembler dysmyelinating mutant: An analysis of the sciatic nerve by biochemistry and radioautography

The turnover of phospholipids was compared in peripheral nerves of Trembler dysmelinating mutant and control mice, after intraperitoneal and local injection of labeled ethanolamine. In the mutant sciatic nerve, neurochemical analysis showed that [¹⁴C]ethanolamine is incorporated into EGP (ethanolamine glycerophospholipids) of the sciatic nerve at a much higher rate in Trembler mutant than in control mice. Furthermore the decay rate of ¹⁴C-labeled EGP is faster in Trembler than in normal animals. The accelerated turnover of EGP in Trembler sciatic nerve affects the diacyl-EGP while the renewal of the alkenylacyl-EGP (plasmalogens) is slower than in controls. Quantitative radioautographic study at the ultrastructural level corroborate that the initial increase of the label in Trembler nerve fibers was different in axons, Schwann cells and myelin sheaths. EM radioautographs reveal indeed that the high label content observed in Trembler axons takes place preferentially in the myelinated portions of axons and drops within 1 week. In both myelinated and unmyelinated segments of the axons, the majority of the radioactivity was contained in axolemma and smooth axoplasmic reticulum. The 10-fold increase of label found in the myelin sheath of Trembler nerve fibers at 1 day raises the question of the origin of the labeled EGP, either by a stimulated synthesis in Schwann cells or by transfer from axonally transported phospholipids. In contrast, the label of axons, Schwann cells and myelin sheaths of control nerve remains stable during the same period.     

A TIME-SEQUENCE AUTORADIOGRAPHIC STUDY OF THE IN VIVO INCORPORATION OF [1, 2-3H]CHOLESTEROL INTO PERIPHERAL NERVE MYELIN

A time-sequence study of the incorporation and distribution of cholesterol in peripheral nerve myelin was carried out by electron microscope autoradiography. [1,2-³H]Cholesterol was injected into 10-day old mice and the sciatic nerves were dissected out at 10, 20, 40, 60, 90, 120, and 180 min after the injection. 20 min after injection the higher densities of grains due to the presence of [³H]cholesterol were confined to the outer and inner edges of the myelin sheath. Practically no cholesterol was detected in the midzone of the myelin sheath. 1 ½ h after injection, cholesterol showed a wider distribution within the myelin sheath, the higher densities of grains occurring over the two peripheral myelin bands, each approximately 3,100 Å wide. Cholesterol was also present in the center of the myelin sheath but to a considerably lesser extent. 3 h after injection cholesterol appeared homogeneously distributed within the myelin sheath. Schwann cell and axon compartments were also labeled at each time interval studied beginning 20 min postinjection. These observations indicate that preformed cholesterol enters myelin first and almost simultaneously through the inner and outer edges of the sheath; only after 90 min does the density of labeled cholesterol in the central zone of myelin reach the same density as that in the outer and inner zones. These findings suggest that cholesterol used by the nerve fibers in the formation and maintenance of the myelin sheath enters the lamellae from the Schwann cell cytoplasm and from the axon. The possibility of a bidirectional movement of molecules, i.e. from the Schwann cell to the axon and from the axon to the Schwann cell through the myelin sheath, is noted. The results are discussed in the light of recent observations on the exchange, reutilization, and transaxonal movement of cholesterol.     

Cholesterol Synthesis Is Down-Regulated During Regeneration of Peripheral Nerve

Abstract: The discovery of apolipoprotein E synthesis and secretion by injured peripheral nerve led to the hypothesis that endoneurial apolipoprotein E serves to salvage degenerating myelin cholesterol. This salvaged cholesterol could then be reutilized by Schwann cells during remyelination via uptake through low-density lipoprotein receptors. As a test of this hypothesis, we measured the rate of cholesterol synthesis in rat sciatic nerve endoneurium during development and at various times following a crush injury at 50 days of age. In control nerves [¹⁴C]acetate incorporation into cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase activity were closely linked throughout development, indicating that reductase activity in nerve, as in other tissues, is a good indicator of cholesterol's synthetic rate. In the crushed nerves cholesterol synthesis fell to nearly zero during the first week after the crush. There was a partial recovery during the second to fourth weeks, but unlike that of other lipids, cholesterol synthesis remained well below control nerve values throughout most of the 15-week post-crush period examined. Thus, cholesterol synthesis is at very low levels during the myelination of regenerating axons. These results are consistent with a receptor-mediated down-regulation of cholesterol synthesis by lipoproteins, and would be expected if Schwann cells were utilizing an external source of cholesterol as postulated above.