Cerebroside synthesis as a measure of the rate of remyelination following cuprizone-induced demyelination in brain.

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12-week time-course, control animals showed slight increases in the content of oligodendroglial-specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of (3)H(2)O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down-regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl-CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin-related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.

Parameters related to lipid metabolism as markers of myelination in mouse brain.

Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end-points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time-consuming to establish, requiring a complete developmental study with labor-intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of (3)H(2)O to label body water pools, followed by determination of label in the myelin-specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin-specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination.

Diurnal and dietary-induced changes in cholesterol synthesis correlate with levels of mRNA for HMG-CoA reductase.

We determined the extent to which diurnal variation in cholesterol synthesis in liver is controlled by steady-state mRNA levels for the rate-limiting enzyme in the pathway, hydroxymethylglutaryl (HMG)-CoA reductase. Rats 30 days of age and maintained on a low-cholesterol diet since weaning were injected intraperitoneally with (3)H(2)O. The specific radioactivity of the whole-body water pool soon became constant, allowing for expression of values for incorporation of label into cholesterol as absolute rates of cholesterol synthesis. In liver, there was a peak of cholesterol synthesis from 8 pm to midnight, a 4-fold increase over synthesis rates from 8 am to noon. Increases in synthesis were quantitatively in lock step with increases in mRNA levels for HMG-CoA reductase occurring 4 h earlier. In a parallel experiment, rats received 1% cholesterol in the diet from weaning to 30 days of age. Basal levels of hepatic cholesterol synthesis were greatly diminished and there was little diurnal variation of cholesterol synthesis or of levels of mRNA for HMG-CoA reductase. Levels of mRNA for the low density lipoprotein receptor and scavenger receptor-B1 (putative high density lipoprotein receptor) showed little diurnal variation, regardless of diet. This suggests that diurnal variation of hepatic cholesterol synthesis is driven primarily by varying the steady-state mRNA levels for HMG-CoA reductase. Other tissues were also examined. Adrenal gland also showed a 4-fold diurnal increase in accumulation of recently synthesized cholesterol. In contrast to liver, however, there was little corresponding change in mRNA expression for HMG-CoA reductase. Much of this newly synthesized cholesterol may be of hepatic origin, imported into adrenal by SR-B1, whose mRNA was up-regulated 2-fold. In brain, there was no diurnal variation in either cholesterol synthesis or mRNA expression, and no influence of high- or low-cholesterol diets on synthesis rates or HMG-CoA reductase mRNA levels.

Peripheral nerve regeneration and cholesterol reutilization are normal in the low-density lipoprotein receptor knockout mouse.

Following peripheral nerve injury, cholesterol from degenerating myelin is retained locally within macrophages and subsequently reutilized by Schwann cells for synthesis of new myelin during nerve regeneration. Substantial evidence indicates this conservation and reutilization of cholesterol is accomplished via lipoprotein-mediated intercellular transport, although the identities of the lipoproteins and their receptors are unresolved. Because Schwann cells in regenerating nerve are reported to express the low-density lipoprotein (LDL) receptor (LDLR), we used the LDLR knockout mouse to examine the potential role of this receptor in cholesterol reutilization. Sciatic nerves were crushed in knockout and wild-type mice and examined 3 days to 10 weeks later. Morphometric analyses and measures of mRNA levels for myelin protein P(0), indicate that axon regeneration and myelination proceed normally in the LDLR knockout mouse. We therefore measured hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase activity and mRNA levels to determine whether Schwann cells compensated for the absence of the LDLR by upregulating cholesterol synthesis. Unexpectedly, these measures remained at the same downregulated levels found in regenerating nerves of wild-type animals. The apparently normal nerve regeneration, coupled with the lack of any compensatory upregulation of cholesterol synthesis in the LDLR knockout mice, indicates that other lipoprotein receptors must be primarily involved in cholesterol uptake by Schwann cells.

Molecular Probes for PNS Neurotoxicity, Degeneration, and Regeneration.

The functional unit of the peripheral nervous system (PNS) is the neuron, with its long axon enveloped either by Schwann cells (unmyelinated axons) or by the multilamellar myelin sheath formed and maintained by these cells (myelinated axons) (Fig. 1 A). Neuronal cell bodies may be located within the CNS (e.g., motor neurons in the ventral horn of the spinal cord) or within the PNS itself (e.g., sensory neurons in the dorsal root ganglia), but in either case, bundles of long axons course through various peripheral nerves to their target tissues (Fig. 1 B). Normal operation of the PNS depends on the integrity of both neurons and glial (Schwann) cells. Additionally, however, function is critically dependent on intricate interactions between these two cell types at molecular, structural, and functional levels. As an example, the intimate structural relationship between large axons and the myelin sheath that surrounds them is an absolute requirement for the rapid and efficient "saltatory conuction" of nervous impulses (1,2). Damage or insult to either the neuronal or glial component of this functional unit affects the other component, and most often has deleterious effects on normal nervous system function. In some cases, damage may be so severe that it is readily apparent as gross functional or morphological alterations, but in many cases the initial insult, although eventually leading to serious pathological consequences, may be subtle and difficult to detect. In recent years, it has become possible to utilize molecular biological approaches as rapid and sensitive probes for neurotoxic insults. Fig. 1. (A) The neuron-Schwann cell functional unit of the peripheral nervous system. The functional integrity of the PNS depends on both the neuron and its ensheathing Schwann cells (which may or may not produce a myelin sheath), as well as on vital interactions between these two components. The axon is actually several orders of magnitude longer (in relation to its cell body) than is shown in the diagram, and each axon branches into numerous nerve endings. (B) Generalized diagram of the peripheral nervous system. The peripheral nervous system contains both myelinated and unmyelinated axons. Unmyelinated axons are also enveloped by Schwann cells (not shown in the figure). Neuronal cell bodies from which PNS axons arise are located in either the dorsal root ganglia (sensory neurons) or in the ventral horn of the spinal cord (motor neurons). Sympathetic ganglia with their associated neurons, also part of the peripheral nervous system, are not shown. The sciatic nerve is an easily accessible, major component of the peripheral nervous system.

Gene expression in brain during cuprizone-induced demyelination and remyelination.

When C57BL/6J mice, 8 weeks of age, received 0.2% Cuprizone in their diet, extensive demyelination in corpus callosum was detectable after 3 weeks, and there was massive demyelination by 4 weeks. As expected, the accumulation of phagocytically active microglia/macrophages correlated closely with demyelination. When Cuprizone was removed from the diet, remyelination was soon initiated; after 6 weeks of recovery, myelin levels were near-normal and phagocytic cells were no longer prominent. Steady-state levels of mRNA for myelin-associated glycoprotein, myelin basic protein, and ceramide galactosyltransferase were already profoundly depressed after 1 week of Cuprizone exposure and were only 10-20% of control values after 2 weeks. Unexpectedly, upregulation of mRNA for these myelin genes did not correlate with initiation of remyelination but rather with accumulation of microglia/macrophages. After 6 weeks of exposure to Cuprizone, mRNA levels were at control levels or higher-in the face of massive demyelination. This suggests that in addition to effecting myelin removal, microglia/macrophages may simultaneously push surviving oligodendroglia or their progenitors toward myelination.

Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve.

Following injury to the peripheral nervous system, circulating monocytes/macrophages are recruited to the damaged tissue, where they play vital roles during both nerve degeneration and subsequent regeneration. Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C or beta-chemokine family, is a powerful leukocyte recruitment/activation factor that is relatively specific for monocytes/macrophages. Because these are the predominant leukocyte type recruited by injured nerve, we hypothesized that upregulation of MCP-1 expression is involved in recruitment of these cells. Indeed, assay of steady-state levels of MCP-1 mRNA in rat sciatic nerve during tellurium-induced primary demyelination indicated up-regulation of this chemokine with a peak after 3 days of tellurium exposure, preceding the peak of accumulation of phagocytic macrophages (assayed as lysozyme mRNA levels) by 6 days. Increasing levels of MCP-1 mRNA expression, induced by increasing levels of tellurium exposure, resulted in corresponding increases in subsequent recruitment of macrophages. In situ hybridization suggested that MCP-1 mRNA was localized in Schwann cells. No expression of MIP-2, which is a C-X-C or alpha-chemokine that is specific for recruitment of neutrophils, was detected, consistent with the lack of recruitment of significant numbers of these cells. In addition, we also investigated the response seen following nerve transection (axonal degeneration and secondary demyelination with no subsequent regeneration) and nerve crush (degeneration followed by regeneration). In these latter two nerve injury models, there was also a marked, early up-regulation of MCP-1 mRNA, with a time course that is compatible with a role for this chemokine in macrophage recruitment. We conclude that MCP-1 is involved in recruiting monocytes/macrophages to injured peripheral nerve and that the specificity of leukocyte types recruited results from specificity of chemokine production.

Control of cholesterol biosynthesis in Schwann cells.

Cholesterol accounts for over one-fourth of total myelin lipids. We found that, during development of the rat sciatic nerve, expression of mRNA for hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, was up-regulated in parallel with mRNA for P0, the major structural protein of PNS myelin, and with ceramide galactosyltransferase (CGT), the rate-limiting enzyme in cerebroside biosynthesis. To help establish the nature of this coordinate regulation of myelin-related genes, we examined their steady-state mRNA levels in cultured primary Schwann cells. We also assayed synthesis of cholesterol and cerebroside to distinguish how much control of synthetic activity for these two myelin lipids involved mRNA levels for HMG-CoA reductase and CGT, and how much involved post-mRNA control mechanisms. Addition of forskolin to cells cultured in media supplemented with normal calf serum resulted in up-regulation of P0 and CGT mRNA expression and cerebroside synthesis, without corresponding increases in HMG-CoA reductase mRNA or cholesterol synthesis. Cholesterol synthesis increased approximately threefold in Schwann cells cultured with lipoprotein-deficient serum, without any increase in HMG-CoA reductase mRNA. Furthermore, addition of either serum lipoproteins or 25-hydroxycholesterol decreased cholesterol synthesis without altering HMG-CoA reductase mRNA levels. We conclude that, as in other tissues, cholesterol synthesis in Schwann cells is regulated primarily by intracellular sterol levels. Much of this regulation occurs at posttranscriptional levels. Thus, the in vivo coordinate up-regulation of HMG-CoA reductase gene expression in myelinating Schwann cells is secondary to intracellular depletion of cholesterol, as it is compartmentalized within the myelin. It is probably not due to coordinate control at the level of mRNA expression.

Regenerating sciatic nerve does not utilize circulating cholesterol.

The rapid accumulation of myelin in the peripheral nervous system during the early postnatal period requires large amounts of cholesterol, a major myelin lipid. All of the cholesterol accumulating in the developing rat sciatic nerve is synthesized locally within the nerve, rather than being derived from the supply in lipoproteins in the systemic circulation (Jurevics and Morell, J. Lipid Res. 5:112-120; 1994). Since this lack of utilization of circulating cholesterol may relate to exclusion by the blood-nerve barrier, we examined the sources of cholesterol needed for regeneration following nerve injury, when the blood-nerve barrier is breached. One sciatic nerve was crushed or transected, and at various times later, the rate of cholesterol accumulation was compared with the rate of local in vivo synthesis of cholesterol within the nerve, utilizing intraperitoneally injected 3H2O as precursor. The accumulation of additional cholesterol in nerve during regeneration and remyelination could all be accounted for by that locally synthesized within the nerve. There was also an increase in cholesterol esters in injured nerve segments; in crushed nerves, these levels decreased during regeneration and remyelination, consistent with reutilization of cholesterol originally salvaged by phagocytic macrophages and Schwann cells. Thus, regeneration and remyelination following injury in sciatic nerve utilizes both salvaged cholesterol and cholesterol synthesized locally within the nerve, but not cholesterol from the circulation.

Carbon disulfide neurotoxicity in rats: IV. Increased mRNA expression of low-affinity nerve growth factor receptor--a sensitive and early indicator of PNS damage.

Expression of the low-affinity nerve growth factor receptor (NGF-R) in the peripheral nervous system is regulated by Schwann cell-axonal contact. Steady-state mRNA levels for NGF-R are very low in the mature peripheral nervous system, but are markedly upregulated in sciatic nerve during both primary demyelination (tellurium exposure) and secondary demyelination (Wallerian degeneration). Upregulation also occurs in various subdegenerative axonopathy models where there is axonal atrophy, suggesting its usefulness as a marker for subtle perturbations in normal axon-Schwann cell interactions (Roberson et al., Mol Brain Res 1995; 28:231-238). To further test this hypothesis, we examined NGF-R mRNA expression in sciatic nerves of rats exposed to carbon disulfide (CS2), a toxicant known to cause a distal axonopathy. Adult rats were exposed to CS2 gas (50, 500, or 800 ppm, 6 hr/day, 5 days/wk) for 2-13 weeks. RNA was isolated from sciatic nerves and levels of mRNA for NGF-R determined by Northern blot analysis. NGF-R mRNA expression increased in a dose- and time-dependent manner. Message levels were already increased after 2 wks of exposure to 800 ppm CS2, and increased further with continued exposure. Morphological alterations were not apparent in the sciatic nerve, even at the highest dosage levels with the longest exposure times. Upregulation of NGF-R mRNA is thus an indicator of subtle alterations in the normal axon-Schwann cell relationship and provides a sensitive measure of CS2 neurotoxicity. Assay of this marker may also be useful as a rapid and very sensitive general screen for other compounds which are potentially toxic to the peripheral nervous system.

Tellurium causes dose-dependent coordinate down-regulation of myelin gene expression.

Exposure of developing rats to a diet containing elemental tellurium systemically inhibits cholesterol synthesis at the level of squalene epoxidase. At high tellurium exposure levels (> 0.1% in the diet), there is an associated segmental demyelination of the PNS. Low levels of dietary tellurium (0.0001%) led to in vivo inhibition of squalene epoxidase activity in sciatic nerve, and inhibition increased with increasing exposure levels. With increasing dose and increasing exposure times, there was an increasing degree of demyelination and increasing down-regulation of mRNA levels for myelin P0 protein, ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis), and HMG-CoA reductase (rate-limiting enzyme in cholesterol synthesis). Because these were all down-regulated in parallel, we conclude there is coordinate regulation of the entire program for myelin synthesis in Schwann cells. An anomaly was that at early time points and low tellurium levels, mRNA levels for HMG-CoA reductase were slightly elevated, presumably in response to tellurium-induced sterol deficits. We suggest the eventual down-regulation relates to a separate mechanism by which Schwann cells regulate cholesterol synthesis, related to the need for coordinate synthesis of myelin components. Levels of mRNA for the low-affinity nerve growth factor receptor (indicator of alterations in axon-Schwann cell interactions) and for lysozyme (marker for phagocytic macrophages) were both up-regulated in a dose- and time-dependent manner which correlated with the presence of segmental demyelination. Levels of mRNA coding for myelin-related proteins were down-regulated at low tellurium exposure levels, without demyelination or up-regulation of nerve growth factor receptor. This suggests the down-regulation is related to the tellurium-induced cholesterol deficit, and not to the loss of axonal contact associated with early stages of demyelination or to the entry of activated macrophages.

Alterations in gene expression associated with primary demyelination and remyelination in the peripheral nervous system.

Primary demyelination is an important component of a number of human diseases and toxic neuropathies. Animal models of primary demyelination are useful for isolating processes involved in myelin breakdown and remyelination because the complicating events associated with axonal degeneration and regeneration are not present. The tellurium neuropathy model has proven especially useful in this respect. Tellurium specifically blocks synthesis of cholesterol, a major component of PNS myelin. The resulting cholesterol deficit in myelin-producing Schwann cells rapidly leads to sychronous primary demyelination of the sciatic nerve, which is followed by rapid synchronous remyelination when tellurium exposure is discontinued. Known alterations in gene expression for myelin proteins and for other proteins involved in the sequence of events associated with demyelination and subsequent remyelination in the PNS are reviewed, and new data regarding gene expression changes during tellurium neuropathy are presented and discussed.

Tissue-specific coordinate regulation of enzymes of cholesterol biosynthesis: sciatic nerve versus liver.

Exposure of weanling rats to a diet containing the element tellurium results in specific inhibition of squalene epoxidase, an obligate enzyme in cholesterol biosynthesis. Liver responds to the resulting intracellular sterol deficit by up-regulating, in parallel and to the same extent, expression of mRNA for squalene epoxidase and for HMG-CoA reductase, the major rate-limiting enzyme in the pathway. This increased mRNA expression, coupled with additional translational and posttranslational activation of the pathway allows normal levels of cholesterol synthesis in liver despite tellurium-induced inhibition of squalene epoxidase. The response to tellurium challenge in sciatic nerve is very different. In this tissue, cholesterol synthesis is prominent because of the large amount of cholesterol required for synthesis and maintenance of myelin. Although nerve shows an initial (at 1 day) up-regulation of mRNA expression for both enzymes in response to tellurium exposure, this is followed quickly by parallel down-regulation of both enzymes, in concert with down-regulation of mRNA expression for myelin proteins. We suggest that the tellurium-induced deficit in sterols leads to a coordinate down-regulation of synthesis of myelin components. The initial early up-regulation of cholesterol biosynthesis in sciatic nerve due to the cholesterol deficit is countered by down-regulation which is coordinated with overall control of the program of myelin assembly. This tissue-specific control of cholesterol synthesis in sciatic nerve is a point of vulnerability to toxicants, and may be related to the need for coordinate synthesis of all components of myelin.

Schwann cells as targets for neurotoxicants.

Schwann cells subserve a variety of roles in the peripheral nervous system (PNS), including ionic homeostasis, and protection and possible metabolic support of axons. It is, however, the myelinating subtype of these glia which appear most sensitive to toxic insults. Myelinating Schwann cells must synthesize large amounts of myelin proteins (P0 is the major myelin protein) and lipids (cholesterol is most prominent) within a short, tightly-programmed developmental window. Schwann cells are preferentially vulnerable to neurotoxic insults during this period of maximal metabolic stress. The hydrophobicity of myelin (reservoir for lipid-soluble toxicants) and possible specialized energy-requiring mechanisms for maintenance of myelin structure are points of vulnerability for the mature myelin sheath. Fortunately, Schwann cells are highly plastic; they dedifferentiate to more primitive precursor cells following a demyelinating insult, but are able to redifferentiate and remyelinate axons during subsequent nerve regeneration. For study of such processes, a useful model system is exposure of developing rats to the element tellurium; this produces a highly synchronous primary demyelination of PNS which is followed closely by rapid remyelination. Interpretation of the metabolic events involved in simplified by the nearly complete lack of axonal degeneration. We have uncovered the primary lesion (block in cholesterol biosynthesis) and elucidated some of the steps involved in the demyelination-remyelination response. Particularly useful have been studies of gene expression of certain proteins (nerve growth factor receptor, myelin proteins, macrophage-specific lysozyme) which have enabled us to define some of the cellular responses to this toxicant-induced injury. A generally applicable result that has emerged from these and other similar studies is that upregulation of NGF-R mRNA is a sensitive marker of nerve damage; it may be useful as a screen for potentially neurotoxic compounds.

Tellurite specifically affects squalene epoxidase: investigations examining the mechanism of tellurium-induced neuropathy.

A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene expoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 microM tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo(a)pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo(a)pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.

NGFR-mRNA expression in sciatic nerve: a sensitive indicator of early stages of axonopathy.

Expression of the low-affinity nerve growth factor receptor (NGFR) in the sciatic nerve (particularly Schwann cells) is high during development but is downregulated upon establishment of the mature axon-Schwann cell relationship. NGFR is re-expressed by Schwann cells if this relationship is altered by degeneration of axons (axotomy) or myelin (tellurium intoxication). To determine the sensitivity of NGFR expression to axonal injury, we have assayed NGFR-mRNA levels in proximal and distal regions of nerves exposed to the axonopathic agents acrylamide and isoniazid, as well as in proximal and distal stumps of axotomized nerves. NGFR-mRNA was elevated in all three models and correlated regionally with sites of axonal perturbation. In distal regions of acrylamide- and isoniazid-intoxicated nerves, NGFR-mRNA was elevated at least 2 days prior to visible signs of axonal degeneration as assayed by morphological techniques utilizing light microscopy. NGFR-mRNA was also elevated in proximal regions of axotomized and acrylamide-intoxicated nerves prior to signs of axonal degeneration. In these models, increased mRNA expression correlated with alterations in the size distribution of axonal cross sections. The common response in all of these situations indicates that NGFR expression, in addition to being a marker for axonal degeneration, is also a sensitive indicator of less profound perturbations in normal axon-Schwann cell interactions, including early stages of axonopathy. We suggest that assay for NGFR-mRNA may be utilized as a rapid and simple method (relative to more labor-intensive morphological methods) to screen for peripheral neurotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage recruitment in different models of nerve injury: lysozyme as a marker for active phagocytosis.

Macrophages play critical roles in both degenerative and regenerative processes following peripheral nerve injury. These include phagocytosis of debris, stimulation of Schwann cell dedifferentiation and proliferation, and salvage of myelin lipids for reutilization during regeneration. To better define the role of macrophages, we studied models of primary demyelination (tellurium intoxication) and secondary demyelination (nerve crush and cut). Sections of paraformaldehyde-fixed rat sciatic nerves at various stages of demyelination were stained with monoclonal antibody ED1, a standard macrophage marker, and a polyclonal antiserum specific for lysozyme (LYS). Near the peak of demyelination in all three models, LYS immunoreactivity colocalized with ED1 staining. Macrophages present in nerve after the period of maximal phagocytosis of myelin were much less immunoreactive for LYS. These results suggest LYS is a good marker for macrophages which are active in phagocytosis. Tellurium intoxication, which causes synchronous demyelination and subsequent remyelination of only about 25% of myelin internodes, recruited more macrophages (and induced more lysozyme expression) than either nerve crush or cut, which cause demyelination of all internodes distal to the injury site. This suggests that Schwann cells may recruit macrophages soon after metabolic insult and prior to actual demyelination. The final signal for macrophage recruitment is not directly related to the amount of damaged myelin. In the models listed above, steady state mRNA levels for apolipoprotein E (ApoE; possible mediator of cholesterol salvage), LYS, and P0 (major structural protein of PNS myelin), were analyzed by Northern blot analysis. LYS mRNA levels peaked sharply in all models, with a temporal pattern consistent with the expected presence of activated, phagocytic macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression during tellurium-induced primary demyelination.

A compound may be "developmentally neurotoxic" because it interferes with a metabolic step exclusively or preferentially expressed during development in a particular class of neural cells. The initial metabolic specificity is often complicated by: (1) secondary responses in the affected cells, (2) involvement of other functionally-related cell types, and (3) the presence of compensatory and/or regenerative responses. In this context we study tellurium, which systemically blocks cholesterol biosynthesis at the squalene epoxidase step. Because of the high demand in developing peripheral nerves for newly synthesized cholesterol required for myelin assembly, this metabolic block leads to demyelination of the sciatic nerve. This insult is confounded by the fact that the myelin-forming Schwann cells do not upregulate their cholesterol biosynthetic pathway. This is contrary to expectations; liver (the main source of cholesterol for many tissues outside the nervous system) upregulates synthesis of cholesterol and overcomes the metabolic block. The shortage of cholesterol in Schwann cells results in an immediate secondary response down-regulation of steady-state mRNA levels for specific myelin proteins. Remyelination occurs after cessation of tellurium exposure. This model of primary demyelination allows study of Schwann-cell specific responses during the processes of myelin breakdown and subsequent steps leading to remyelination, without the complications of axonal degeneration and regeneration. Because tellurium specifically blocks the synthesis of a major required membrane component, it is also well suited for examining the coordinate control of membrane synthesis and assembly at the genomic level.

Neurofilament and tubulin mRNA expression in Schwann cells.

Feeding of elemental tellurium to weanling rats blocks synthesis of cholesterol (a major component of myelin), and causes demyelination of the sciatic nerve. Expression of mRNA for myelin-specific genes in Schwann cells is downregulated. We now demonstrate specificity for Schwann cell injury in that expression of mRNAs for neurofilament subunits and for class II beta-tubulin (parameters sensitive to axonal injury) is unaltered in neurons of the dorsal root ganglia. An unexpected result was that in tellurium-treated rats there was marked upregulation of expression of mRNAs coding for the light and medium neurofilament subunits ("neuron-specific" proteins) as well as that for class II beta-tubulin (the major neuronal beta-tubulin isotype) in Schwann cells. Expression of these "neuronal" mRNA species was also detected in distal stumps of transected nerves at times when Schwann cells were undergoing dedifferentiation.

Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor.

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium-induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady-state mRNA levels (expressed on a "per nerve" basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring.