TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment.

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0g/kg) for 2weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.

Early short-term PXT3003 combinational therapy delays disease onset in a transgenic rat model of Charcot-Marie-Tooth disease 1A (CMT1A).

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.

The heme precursor delta-aminolevulinate blocks peripheral myelin formation.

Delta-aminolevulinic acid (delta-ALA) is a heme precursor implicated in neurological complications associated with porphyria and tyrosinemia type I. Delta-ALA is also elevated in the urine of animals and patients treated with the investigational drug dichloroacetate (DCA). We postulated that delta-ALA may be responsible, in part, for the peripheral neuropathy observed in subjects receiving DCA. To test this hypothesis, myelinating cocultures of Schwann cells and sensory neurons were exposed to delta-ALA (0.1-1 mM) and analyzed for the expression of neural proteins and lipids and markers of oxidative stress. Exposure of myelinating samples to delta-ALA is associated with a pronounced reduction in the levels of myelin-associated lipids and proteins, including myelin protein zero and peripheral myelin protein 22. We also observed an increase in protein carbonylation and the formation of hydroxynonenal and malondialdehyde after treatment with delta-ALA. Studies of isolated Schwann cells and neurons indicate that glial cells are more vulnerable to this pro-oxidant than neurons, based on a selective decrease in the expression of mitochondrial respiratory chain proteins in glial, but not in neuronal, cells. These results suggest that the neuropathic effects of delta-ALA are attributable, at least in part, to its pro-oxidant properties which damage myelinating Schwann cells.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Erythropoietin-mediated preservation of the white matter in rat spinal cord injury.

We investigated the effect of a single administration of recombinant human erythropoietin (rhEPO) on the preservation of the ventral white matter of rats at 4 weeks after contusive spinal cord injury (SCI), a time at which functional recovery is significantly improved in comparison to the controls [Gorio A, Necati Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Enver Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455; Gorio A, Madaschi L, Di Stefano B, Carelli S, Di Giulio AM, De Biasi S, Coleman T, Cerami A, Brines M (2005) Methylprednisolone neutralizes the beneficial effects of erythropoietin in experimental spinal cord injury. Proc Natl Acad Sci U S A 102:16379-16384]. Specifically, we examined, by morphological and cytochemical methods combined with light, confocal and electron microscopy, i) myelin preservation, ii) activation of adult oligodendrocyte progenitors (OPCs) identified for the expression of NG2 transmembrane proteoglycan, iii) changes in the amount of the chondroitin sulfate proteoglycans neurocan, versican and phosphacan and of their glycosaminoglycan component labeled with Wisteria floribunda lectin, and iv) ventral horn density of the serotonergic plexus as a marker of descending motor control axons. Injured rats received either saline or a single dose of rhEPO within 30 min after SCI. The results showed that the significant improvement of functional outcome observed in rhEPO-treated rats was associated with a better preservation of myelin in the ventral white matter. Moreover, the significant increase of both the number of NG2-positive OPCs and the labeling for Nogo-A, a marker of differentiated oligodendrocytes, suggested that rhEPO treatment could result in the generation of new myelinating oligodendrocytes. Sparing of fiber tracts in the ventral white matter was confirmed by the increased density of the serotonergic plexus around motor neurons. As for chondroitin sulfate proteoglycans, only phosphacan, increased in saline-treated rats, returned to normal levels in rhEPO group, probably reflecting a better maintenance of glial-axolemmal relationships along nerve fibers. In conclusion, this investigation expands previous studies supporting the pleiotropic neuroprotective effect of rhEPO on secondary degenerative response and its therapeutic potential for the treatment of SCI and confirms that the preservation of the ventral white matter, which contains descending motor pathways, may be critical for limiting functional deficit.

Neuroactive steroids influence peripheral myelination: a promising opportunity for preventing or treating age-dependent dysfunctions of peripheral nerves.

The process of aging deeply influences morphological and functional parameters of peripheral nerves. The observations summarized here indicate that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the levels of the major peripheral myelin proteins [e.g., glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22)]. Neuroactive steroids, such as progesterone (PROG), dihydroprogesterone (5alpha-DH PROG), and tetrahydroprogesterone (3alpha,5alpha-TH PROG), are able to stimulate the low expression of these two myelin proteins present in the sciatic nerve of aged male rats. Since Po and PMP22 play an important physiological role in the maintenance of the multilamellar structure of PNS myelin, we have evaluated the effect of PROG and its neuroactive derivatives, 5alpha-DH PROG and 3alpha,5alpha-TH PROG, on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24-month-old male rats. Data obtained clearly indicate that neuroactive steroids are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve.

Aging in peripheral nerves: regulation of myelin protein genes by steroid hormones.

The process of aging deeply influences morphological and functional parameters of the peripheral nerves. Interestingly, recent observations performed in our laboratory on the rat sciatic nerves have indicated that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the messenger levels of the major peripheral myelin proteins (glycoprotein Po, myelin basic protein and peripheral myelin protein 22). At least in the case of the Po, the low levels of its messengers and of the protein itself found in aged animals are increased by the treatment with a physiological progesterone derivative like dihydroprogesterone. It has also been found that in normal adult male rats the levels of the messengers for Po in the sciatic nerve are increased by progesterone, dihydroprogesterone and tetrahydroprogesterone; surprisingly, the gene expression of peripheral myelin protein 22 is stimulated only by tetrahydroprogesterone. These observations have been confirmed in parallel studies performed on Schwann cell cultures. Since tetrahydroprogesterone does not bind to the progesterone receptor but is a ligand for the GABAA receptor, the hypothesis has been put forward that part of the steroidal effects reported might occur not through the classical progesterone receptor, but rather via an interaction with the GABAA receptor. In other experiments it has been found that the gene expression of Po may be decreased by orchidectomy and restored by treatment with the androgen dihydrotestosterone. Altogether, these observations suggest the future use of physiological and/ or synthetic steroid hormones as a possible therapeutic approach for some pathological situations occurring in peripheral nerves during aging and demyelinating diseases.

Asynchronous therapy targeting Nogo-A enhances neurobehavioral recovery by reducing neuronal loss and promoting neurite outgrowth after cerebral ischemia in mice.

Therapeutics targeting the Nogo-A signal pathway hold promise to promote recovery following brain injury. Based on the temporal characteristics of Nogo-A expression in the process of cerebral ischemia and reperfusion, we tested a novel asynchronous treatment, in which TAT-M9 was used in the early stage to decrease neuronal loss, and TAT-NEP1-40 was used in the delayed stage to promote neurite outgrowth after bilateral common carotid artery occlusion (BCCAO) in mice. Both TAT-M9 and TAT-NEP1-40 were efficiently delivered into the brains of mice by intraperitoneal injection. TAT-M9 treatment promoted neuron survival and inhibited neuronal apoptosis. Asynchronous therapy with TAT-M9 and TAT-NEP1-40 increased the expression of Tau, GAP43 and MAP-2 proteins, and enhanced short-term and long-term cognitive functions. In conclusion, the asynchronous treatment had a long-term neuroprotective effect, which reduced neurologic injury and apoptosis, promoted neurite outgrowth and enhanced functional recovery after ischemia. It suggests that this asynchronous treatment could be a promising therapy for cerebral ischemia in humans.

Structure and stability of internodal myelin in mouse models of hereditary neuropathy.

Peripheral neuropathies often result in abnormalities in the structure of internodal myelin, including changes in period and membrane packing, as observed by electron microscopy (EM). Mutations in the gene that encodes the major adhesive structural protein of internodal myelin in the peripheral nervous system of humans and mice--P0 glycoprotein--correlate with these defects. The mechanisms by which P0 mutations interfere with myelin packing and stability are not well understood and cannot be provided by EM studies that give static and qualitative information on fixed material. To gain insights into the pathogenesis of mutant P0, we used x-ray diffraction, which can detect more subtle and dynamic changes in native myelin, to investigate myelin structure in sciatic nerves from murine models of hereditary neuropathies. We used mice with disruption of one or both copies of the P0 gene (models of Charcot-Marie-Tooth-like neuropathy [CMT1B] or Dejerine-Sottas-like neuropathy) and mice with a CMT1B resulting from a transgene encoding P0 with an amino terminal myc-tag. To directly test the structural role of P0, we also examined a mouse that expresses P0 instead of proteolipid protein in central nervous system myelin. To link our findings on unfixed nerves with EM results, we analyzed x-ray patterns from unembedded, aldehyde-fixed nerves and from plastic-embedded nerves. From the x-ray patterns recorded from whole nerves, we assessed the amount of myelin and its quality (i.e. relative thickness and regularity). Among sciatic nerves having different levels of P0, we found that unfixed nerves and, to a lesser extent, fixed but unembedded nerves gave diffraction patterns of sufficient quality to distinguish periods, sometimes differing by a few Angstroms. Certain packing abnormalities were preserved qualitatively by aldehyde fixation, and the relative amount and structural integrity of myelin among nerves could be distinguished. Measurements from the same nerve over time showed that the amount of P0 affected myelin's stability against swelling, thus directly supporting the hypothesis that packing defects underlie instability in "live" or intact myelin. Our findings demonstrate that diffraction can provide a quantitative basis for understanding, at a molecular level, the membrane packing defects that occur in internodal myelin in demyelinating peripheral neuropathies.

Solubilization of PNS myelin membrane proteins by detergents.

We have applied 25 different detergents to the PNS myelin membrane. The extracts produced were analyzed regarding the total amount of protein solubilized, and which myelin glycoproteins were solubilized. The degree of protein extraction was correlated with the critical micelle concentration. For some detergents the protein extraction ability depends clearly on critical micelle concentration. These detergents are more potent if critical micelle concentration is small. The other type of extraction is independent on the critical micelle concentration, but extractability of proteins is low. The best detergents for solubilization of myelin proteins are neutral of the alkyl chain length n =9-11, as follows: dodecyl-beta-D-maltopyranoside, decyl-beta-D-maltopyranoside, n-dodecylsulfobetaine. We expect that these detergents will also be suitable for crystallization of P0 and PASII/PMP22 glycoproteins.

Insulin-like growth factor-I influences the initiation of myelination: studies of the anterior commissure of transgenic mice.

The effects of insulin-like growth factor-I (IGF-I) on the initiation of myelination have been examined in the posterior portion of anterior commissure of the transgenic (Tg) mice that overexpress IGF-I in brain, and those which exhibit ectopic brain expression of IGF binding protein-1 (IGFBP-1), an inhibitor of IGF actions. In IGF-I Tg mice the number of ensheathed axons was increased compared to their normal littermates. The distribution of the axon size further showed that both small (< 300 nm) and large (> 700 nm) axons were more often ensheathed and myelinated. The opposite was true in IGFBP-1 Tg mice. These data indicate that IGF-I increases myelin ensheathment independent of axon diameter, and support the hypothesis that the absolute axon size is not the sole determinant for the initiation of myelination in CNS.

Regional heterogeneity of cuprizone-induced demyelination: topographical aspects of the midline of the corpus callosum.

The cuprizone model is a suitable animal model of de- and remyelination secondary to toxin-induced oligodendrogliopathy. From a pharmaceutical point of view, the cuprizone model is a valuable tool to study the potency of compounds which interfere with toxin-induced oligodendrocyte cell death or boost/inhibit remyelinating pathways and processes. The aim of this study was to analyze the vulnerability of neighboring white mater tracts (i.e., the fornix and cingulum) next to the midline of the corpus callosum which is the region of interest of most studies using this model. Male mice were fed cuprizone for various time periods. Different white matter areas were analyzed for myelin (anti-PLP), microglia (anti-IBA1), and astrocyte (anti-GFAP) responses by means of immunohistochemistry. Furthermore, Luxol fast blue-periodic acid Schiff stains were performed to validate loss of myelin-reactive fibers in the different regions. Cuprizone induced profound demyelination of the midline of the corpus callosum and medial parts of the cingulum that was paralleled by a significant astrocyte and microglia response. In contrast, lateral parts of the corpus callosum and the cingulum, as well as the fornix region which is just beneath the midline of the corpus callosum appeared to be resistant to cuprizone exposure. Furthermore, resistant areas displayed reduced astrogliosis and microgliosis. This study clearly demonstrates that neighboring white matter tracts display distinct vulnerability to toxin-induced demyelination. This important finding has direct relevance for evaluation strategies in this frequently used animal model for multiple sclerosis.

TAT-NEP1-40 as a novel therapeutic candidate for axonal regeneration and functional recovery after stroke.

Currently available therapeutics has been less effective in promoting functional recovery from stroke or other injuries in the central nervous system (CNS). Axonal damage is a characteristic pathology seen in CNS injuries. Previously, it was reported that Nogo-A extracellular peptide residues 1-40 (NEP1-40), a competitive antagonist of Nogo-66 receptor (NgR1), has the ability to promote axonal regrowth and functional recovery after CNS injury. However, delivery of the therapeutic proteins into the brain parenchyma is limited due to its inability to cross the blood-brain barrier (BBB). We first generated a biologically active NEP1-40 fusion protein containing the protein transduction domain (PTD) of the transactivator of transcription (TAT), TAT-NEP1-40, which crosses the BBB in vivo after systemic delivery. The TAT-NEP1-40 can protect PC12 cells against oxygen and glucose deprivation (OGD) and promote neurite outgrowth when added exogenously to culture medium. The TAT-NEP1-40 protein transduced into the brain continued to sustain biological activities and protected the brain against ischemia/reperfusion injury through inhibition of neuronal apoptosis. Collectively, our data suggest that TAT-NEP1-40 may be a novel therapeutic candidate for axonal regeneration and functional recovery from CNS injuries such as cerebral hypoxia-ischemia, cerebral hemorrhage, brain trauma, and also for spinal cord injury.

Treatment of stroke with a synthetic liver X receptor agonist, TO901317, promotes synaptic plasticity and axonal regeneration in mice.

In this study, we tested the hypothesis that TO901317 promotes synapse plasticity and axonal regeneration after stroke. Adult male C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAo) and treated with or without TO901317 starting 24 h after MCAo daily for 14 days. Axonal damage and regeneration were evaluated by immunostaining. TO901317 significantly increased synaptophysin expression and axonal regeneration, as well as decreased the expressions of amyloid betaA4 precursor protein and Nogo receptor (NgR) in the ischemic brain. To test whether TO901317 regulates the phosphorylation of phosphatidylinositol 3-kinase (p-PI3K) and Akt (p-Akt) activity in the ischemic brain, MCAo mice were treated with or without TO901317 starting 24 h after MCAo daily for 4 days and were then killed at 5 days after MCAo. TO901317 treatment significantly increased p-PI3K and p-Akt activity, but did not increase total PI3K expression in the ischemic brain. Using primary cortical neuron (PCN) culture, TO901317 significantly increased synaptophysin expression, p-PI3K activity, and decreased NgR expression compared with nontreated controls. TO901317 also significantly increased neurite outgrowth, and inhibition of the PI3K/Akt pathway by LY294002 decreased neurite outgrowth in both controls and TO901317-treated groups in cultured hypoxic PCN. These data indicate that TO901317 promotes synaptic plasticity and axonal regeneration, and that PI3K/Akt signaling activity contributes to neurite outgrowth.

Enhancement of Schwann cell myelin formation by K252a in the Trembler-J mouse dorsal root ganglion explant culture.

The Trembler-J (TrJ) mouse, containing a point mutation in the peripheral myelin protein 22 gene, is characterized by severe hypomyelination and is a representative model of Charcot-Marie-Tooth 1A disease/Dejerine-Sottas Syndrome. Previous studies have shown that protein kinase inhibitor K252a enhances wild-type Schwann cell myelination in culture. We used a dorsal root ganglion (DRG) explant culture system from the heterozygous TrJ/+ mouse to investigate if myelination could be enhanced by K252a. The TrJ/+ DRG explant cultures replicated some important features of the TrJ/+ mouse, showing reduced myelin protein accumulation, thinner myelin sheaths, and shortened myelin internodes. K252a increased myelin protein accumulation and myelin sheath thickness but did not substantially increase myelin internode length. Furthermore, the TrJ/+ DRG explant culture and sciatic nerves continued to respond to K252a during the stage when myelination is complete in the wild type. A general tyrosine kinase inhibitor, genistein, but not inhibitors of serine/threonine protein kinase inhibitors, had a similar effect to K252a. K252a is therefore able to partially overcome hypomyelination by enhancing mutant Schwann cell myelin formation in the TrJ/+ mouse.

The effects of the growth factor-antagonist, trapidil, on remyelination in the CNS.

In this study we describe the effects of trapidil, a putative platelet-derived growth factor-antagonist, on spontaneously occurring remyelination in rat spinal cord. Demyelination was created in the dorsal funiculus of 6- and 11-week-old female rats by the direct injection of 1.0 microliter of 1% lysolecithin. The animals received daily intra-peritoneal injections of either trapidil or saline for 21 days, commencing on the day of lesion induction. The 11-week-old rats receiving trapidil (60 mg/kg) showed a significant decrease in the extent of oligodendrocyte remyelination. Moreover, those axons that were remyelinated by oligodendrocytes tended to have thinner myelin sheaths than axons remyelinated by oligodendrocytes in the control group. In the 6-week-old group, the dose of trapidil which inhibited oligodendrocyte remyelination in the 11-week-old animals had a minimal effect on the extent of oligodendrocyte remyelination and no effect on the quality of myelin sheath formation. A higher dose of trapidil (80 mg/kg) was required before significant impairment of oligodendrocyte remyelination was achieved in the younger age group, implying an age-dependent effect of growth factor-inhibition of CNS remyelination. These results indicate an important role for growth factors, and in particular PDGF, in the orchestration of oligodendrocyte remyelination in the rodent CNS.

Renaturation of the brain myelin proteins by octyl glucoside detergent.

The secondary structure of myelin proteins undergoes a deep change when the membrane is delipidated and suspended in an aqueous buffer containing phosphate and sulfate anions. However, when increasing concentrations of octyl glucoside are dissolved in this saline medium, proteins recover gradually its native secondary structure, reaching a maximum for a detergent/protein ratio which, in addition, is optimal for maximal membrane solubilization. Larger amounts of detergent, however, reverted the effect. Results are explained in terms of anion-lipid and detergent-lipid interactions. Quantitative estimates on the spectral profiles let us find the optimal detergent-protein stoichiometry for preserving almost completely the native secondary structure of myelin proteins while keeping maximal solubilization. These findings are of great importance for reconstitution experiments designed with the goal of determining the biological functions of myelin proteins.

Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination.

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

Ultrastructure of peripheral neuropathy induced in rabbits by 2',3'-dideoxycytidine.

The nucleoside analogue, 2',3'-dideoxycytidine (ddC), an inhibitor of human immunodeficiency virus reverse transcriptase, mediates virologic and immunologic improvements in acquired immunodeficiency syndrome patients. However, in clinical studies ddC treatment is associated with a dose-limiting peripheral neuropathy. The purpose of this study was to characterize the ultrastructural features of the peripheral neuropathy induced in rabbits. Rabbits received 0, 10, 50, or 100 mg/kg/day of ddC for 18 weeks. A prominent ultrastructural change induced by ddC in sciatic nerve and ventral root was separation of myelin lamellae at the intraperiod line and vacuolation and fragmentation of myelin sheaths. Many demyelinated and remyelinated axons were observed. Although pathologic alterations in Schwann cells were evident by the presence of lipid droplets and myelin figures, their formation was not considered a primary event. Axons containing abnormal cytoplasmic components were occasionally observed. Other changes included redundance of Schwann cell basal lamina and the presence of lipid droplets and/or myelin figures within endoneurial fibroblasts and macrophages. The results of this study indicate that ddC induces a myelinopathy in rabbits characterized by myelin splitting and intramyelinic edema and an axonopathy that may be secondary to the myelin changes.