Multipotential neural precursors transplanted into the metachromatic leukodystrophy brain fail to generate oligodendrocytes but contribute to limit brain dysfunction.

Neural stem cells appear to be best suited for regenerative therapy in neurological diseases. However, the effects of high levels of potentially toxic substances such as sulfatides--which accumulate in metachromatic leukodystrophy (MLD)--on this regenerative ability are still largely unclear. To start addressing this question, in vitro and in vivo experiments were used to examine the behavior of multipotential neural precursors exposed to abnormally high levels of sulfatides. Following transplantation of dissociated neurospheres into the brain of presymptomatic MLD pups, the majority of donor-derived cells were distributed in a caudal to rostral direction, with higher numbers in the cortex. Most if not all of the donor cells acquired an astroglial phenotype. We found no evidence of oligodendrocyte or neuronal commitment of transplanted cells in long-term-treated MLD mice (e.g. up to 1.5 years of age). This was in line with our in vitro findings of sulfatides blocking oligodendrocyte formation after induction of differentiation in sulfatide-treated epidermal growth factor/fibroblast growth factor responsive neurospheres. Transplanted MLD mice showed an improved arylsulfatase A (ARSA) activity and a significant amelioration of sulfatide metabolism, neurodegeneration and motor-learning/memory deficits. Furthermore, transplanted cells were shown to act as a source of ARSA enzyme that accumulated in endogenous brain cells, indicating the occurrence of enzyme cross-correction between transplanted and host cells. These results provide a first insight into the effect of sulfatides on the stemness properties of neural stem cells and on the effects of the MLD environment on the in vivo expectations of using neural stem cells in cell therapy.

Human IgM anti-GM1 autoantibodies modulate intracellular calcium homeostasis in neuroblastoma cells.

Increased titers of IgM anti-GM1 antibodies are present in some patients with Lower Motor Neuron Disease (LMND) or Motor Neuropathy (MN), but their pathogenic role and the mechanism of action are unclear. Previous studies have shown that the B subunit of Cholera Toxin (CT), which binds and crosslinks ganglioside GM1, modulate intracellular calcium in murine neuroblastoma cells via the activation of L-type voltage-dependent calcium channels (VGCC). Therefore, using a fluorimetric approach, we have examined the hypothesis that the pentameric IgM anti-GM1 antibodies, could similarly alter calcium concentration in N18 neuroblastoma cells. Sera with human IgM anti-GM1 antibodies were obtained from 5 patients with LMND and 2 patients with MN. Human IgG anti-GM1, IgM anti-Myelin Associated Glycoprotein (MAG), IgM anti-sulfatide antibodies and lectin peanut agglutinin (PNA), that recognizes specifically the Gal(betal-3)GalNAc epitope, were used as control sera. Direct application of either human IgM anti-GM1 antibodies or the B subunit of CT to N18 neuroblastoma cells induced a sustained influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. Furthermore, the dihydropyridine L-type channel antagonists completely inhibited the manganese influx, suggesting that it is due to activation of an L-type VGCC. The magnitude of the influx was correlated with antibody titers. None of human IgG anti-GM1, IgM anti-MAG, IgM anti-sulfatide antibodies or PNA induce an ion influx, pointing to the selective participation of the pentameric IgM isotype of anti-GM1 in the modulation of L-type calcium channels opening. Given that L-type calcium channels are present on motor neurons, the modulation of L-type calcium channels by IgM GM1 antisera may have important implications in diseases such as LMND and MN.

Role of integrins in the peripheral nervous system.

Integrins, a subgroup of adhesion receptors, are transmembrane glycoproteins that mediate interactions between cytoplasm and the extracellular environment. These interactions influence, among others, events such as cell migration, proliferation, and differentiation. Differential expression of integrins is developmentally regulated in the peripheral nervous system (PNS) and is associated with crucial events in both physiological and pathological processes. Preliminary studies suggest that integrin expression influences neural crest cell migration, axonal outgrowth, and Schwann cell differentiation. Similarly, the abnormal expression of integrins or their ligands, is associated with degenerative, inflammatory, and malignant disorders of the PNS. Finally, integrins participate in the complex interactions that promote repair of the PNS. A better comprehension of the role of integrins in the PNS, their protein interactions and transducing signals is being achieved by selected biochemical and genetic experiments. Here we review a large bias of evidence suggesting the key functions for integrins in the PNS.

Epitope-tagged P(0) glycoprotein causes Charcot-Marie-Tooth-like neuropathy in transgenic mice.

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P(0)) glycoprotein. Point mutations in this region of P(0) cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P(0) modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P(0)myc retained partial adhesive function, and suggested that the transgene inhibits P(0)-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P(0) ECD mutants and the normal interactions of P(0) in the myelin sheath.

A novel P0 glycoprotein transgene activates expression of lacZ in myelin-forming Schwann cells.

P0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P0 promoter-based transgenes has been inconsistent. As much as 9 kb of 5' flanking sequence fused to lacZ never yielded detectable levels of beta-galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P0 gene, including 6 kb of 5' flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin-forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2-kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P0 mRNA. These data suggest that intragenic or 3' flanking sequences are necessary to generate the remarkable levels of endogenous P0 gene expression.

Laminin receptor alpha6beta4 integrin is highly expressed in ENU-induced glioma in rat.

Laminins and their receptors influence neoplastic growth and invasiveness. We recently reported the abnormal expression of a laminin receptor, alpha6beta4 integrin, in human astrocytomas. To further investigate the role of alpha6beta4 in gliomas, we produced an experimental model of glioma in rat by transplacental ethylnitrosourea (ENU) administration. This animal model allowed us to study the timing of alpha6beta4 expression during tumor development and the topography of expression in the tumor and the surrounding tissue. Immunohistochemistry, in situ hybridization, and immunoprecipitation studies demonstrated that alpha6beta4 heterodimer forms in experimental gliomas, and confirmed that alpha6beta4 is expressed diffusely in neoplastic cells and reactive astrocytes, but not in normal glia surrounding the tumors. Interestingly, alpha6beta4 was expressed from the early phases of tumor development, and more highly expressed by cells in the proliferative centers of the tumors. Both neoplastic cells and reactive astrocytes also expressed the glial growth factor (neuregulin) receptors, Erb-B2 and Erb-B3. Finally, alpha6beta4 expression was reduced in a subset of tumor blood vessels. Thus, this study suggests a potential role for alpha6beta4 in the pathogenesis of gliomas. Furthermore, this is the first description of altered integrin expression in experimental gliomas; transplacental ENU-induced gliomas in rat will provide a useful model to study the role of altered adhesion in the pathogenesis of human gliomas.

Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer arylsulfatase A to myelinating glia and deficient cells in vitro.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, caused by deficiency of arylsulfatase A (ASA), that manifests primarily in the white matter of the nervous system. Currently, no specific treatment exists that will reverse its fatal outcome. Replacement therapy has been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymphocytes, that are able to traverse the BBB or other membranes of the CNS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodeficiency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibroblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expressed 30 times higher ASA activity when compared with control PBLs. Moreover, cell culture experiments demonstrated that transduced fibroblasts could efficiently transfer ASA to deficient cells across a transwell barrier, whereas transduced MLD lymphocytes could transfer ASA to deficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possible short-term strategies for transfer of ASA into the CNS via transduced autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.

A minimal human MBP promoter-lacZ transgene is appropriately regulated in developing brain and after optic enucleation, but not in shiverer mutant mice.

Previous studies, both in vitro and in vivo, suggest that small portions of the mouse myelin basic protein (MBP) promoter are sufficient to activate regulated expression of MBP. To confirm our previous in vitro studies, we prepared transgenic mice with short regions of the human MBP promoter fused to the lacZ reporter gene. We found that 750 nucleotides of the proximal human MBP promoter is sufficient to activate oligodendrocyte-specific, developmentally regulated expression of lacZ in three independent lines. This promoter, however, does not activate expression of lacZ in Schwann cells in peripheral nerve or in adult mouse brain. The relative levels of beta-galactosidase specific activity, mRNA, and transcription parallel those of MBP mRNA during myelinogenesis. Thus, we exploited this transgene as a quantitative tool to evaluate the response to stimuli known to affect myelination. Transgene expression is reduced 75 % after optic enucleation, as previously reported for levels of MBP mRNA, indicating that axons signal to this portion of the proximal MBP promoter to fully activate MBP expression during myelinogenesis. Instead, in adult shiverer mice, another setting in which MBP transcription is modulated, transgene expression is not increased, in contrast to the increased transcriptional activation of MBP previously reported in these mice. These data suggest that the regulatory region that mediates transcriptional activation of the MBP gene is modular, since discrete subregions are required for activation in Schwann cells, during myelinogenesis in oligodendrocytes, during maintenance myelination in adult brain, and in the dysmyelinating mutant shiverer mouse.

Axonal neuropathy associated with interferon-alpha treatment for hepatitis C: HLA-DR immunoreactivity in Schwann cells.

A 44-year-old man developed a peripheral neuropathy during treatment with interferon-alpha for chronic hepatitis C. The onset was insidious, beginning symmetrically in the hands with paresthesia. Neurophysiological investigation revealed a predominantly sensory axonal neuropathy. A sural nerve biopsy confirmed primary axonal damage. Immunofluorescence studies showed increased expression of HLA-DR molecules prevalently on Schwann cells of non-myelin-forming type.

Atypical hereditary neuropathy with liability to pressure palsies (HNPP): the value of direct DNA diagnosis.

We report two patients with suspected hereditary liability to pressure palsies. Neurophysiological studies showed a mixed axonal-demyelinating sensory-motor polyneuropathy with focal slowing of conduction velocities at the common sites of entrapment. Morphological studies on sural nerve biopsy from the proband showed active axonal regeneration without typical tomacula. Molecular analysis confirmed the presence of a deletion of chromosome 17p11.2 in both patients. Our observation confirms the heterogeneity of hereditary liability to pressure palsies and the relevance of DNA testing for the diagnosis of this hereditary neuropathy.

Alpha6 beta4 and alpha6 beta1 integrins in astrocytomas and other CNS tumors.

Laminin may alter the biological behavior of gliomas. Therefore, we investigated the expression of two laminin receptors, alpha6 beta1 and alpha6 beta4 integrins in normal brain, astrogliotic brain, and astrocytomas as compared to other central nervous system (CNS) tumors. In most CNS tumors, the expression of these integrins was unchanged in neoplastic as compared to normal counterpart cells. In contrast, increased numbers of reactive and neoplastic astrocytes expressed beta4 integrin as compared to normal astrocytes, whereas alpha6 and beta1 integrin expression did not change. Conversely, lower numbers of astrocytoma blood vessels expressed beta4, whereas all blood vessels in normal brain expressed beta4. These data suggest that the profile of laminin receptors changes in neoplastic astrocytes and in astrocytoma blood vessels; this change may play an important role in astrocytoma pathogenesis.

Axon-Schwann cell interactions regulate the expression of c-jun in Schwann cells.

The transcription factor c-jun is selectively expressed by non-myelinating Schwann cells in normal peripheral nerve, and be "denervated," previously myelinatng Schwann cells, after axotomy. When axons regenerate into the distal nerve-stump, the expression of c-jun declines as Schwann cells remyelinate axons. Treating cultured Schwann cells with forskolin, a drug that mimics many of the effects of axon-Schwann cell interactions, decreases the expression of myelin-specific genes. Overexpressing c-jun in cultured Schwann cells, however, does not decrease the expression of a myelin basic protein promoter-reporter construct, indicating that c-jun expression may not directly regulate myelin-specific gene expression. These data suggest that c-jun is invloved in regulating the phenotype of non-myelinating and denervated Schwann cells.

Regulation of neurofibromin expression in rat sciatic nerve and cultured Schwann cells.

Loss of function mutations at the NF1 locus may act intrinsically in Schwann cells to cause the formation of benign Schwann cell tumors (neurofibromas) in patients with type 1 neurofibromatosis. To identify contexts in Schwann cells in which such mutations may play an important role, we measured the levels of NF1 mRNA and neurofibromin in rat sciatic nerve during development, after axotomy, and in cultured rat Schwann cells. NF1 mRNA was present in developing sciatic nerve throughout the period of active Schwann cell proliferation and myelination. After nerve transection, no alteration in NF1 message level was detected, but neurofibromin levels increased, as assessed by immunohistochemistry and Western blotting, suggesting that, in vivo, neurofibromin expression in Schwann cells is post-transcriptionally induced during Wallerian degeneration. Cultured rat Schwann cells constitutively expressed NF1 mRNA and neurofibromin. Schwann cell proliferation induced by exposure to serum and forskolin was not associated with changes in NF1 mRNA or neurofibromin expression, whereas Schwann cell proliferation induced by extracts of embryonic brain membranes was associated with increased NF1 message and neurofibromin expression. Thus, Schwann cells, both in vivo and in vitro, express NF1 mRNA constitutively; the expression of NF1 mRNA and neurofibromin is modulated by only some mitogenic stimuli in Schwann cells.

Expression of growth-associated protein-43 kD in Schwann cells is regulated by axon-Schwann cell interactions and cAMP.

We have examined the regulation of growth-associated protein 43 kD (GAP-43) in rat Schwann cells. In unlesioned adult nerves, GAP-43-immunoreactivity was restricted to non-myelinating Schwann cells and unmyelinated axons. When adult nerves were transected to cause permanent axotomy, previously myelinating Schwann cells expressed progressively more GAP-43-immunoreactivity over 3 weeks, and GAP-43 mRNA levels increased over a similar time course. The peak level of GAP-43 mRNA occurred at least 2 weeks later than that of nerve growth factor receptor, another marker of denervated Schwann cells. In contrast, after nerve-crush, which allows axonal regeneration, many fewer Schwann cells had GAP-43-immunoreactivity, and the amount of GAP-43 mRNA was markedly lower than in transected nerves. Forskolin, a drug that activates adenylate cyclase and mimics many effects of axon-Schwann cell interactions, markedly reduced GAP-43-immunoreactivity and mRNA expression in cultured Schwann cells, whereas interleukin-1 had no effect. These data demonstrate that axon-Schwann cell interactions inhibit the expression of GAP-43 in Schwann cells and that this effect is mimicked by forskolin.

A novel cyclic AMP response element, CACTTGATC, mediates forskolin induction of the myelin basic protein promoter in the rat Schwannoma line, D6P2T.

The rat Schwannoma cell line D6P2T constitutively expresses the mRNA encoding the major myelin protein, P0, but only expresses the mRNA encoding myelin basic protein (MBP) after exposure to forskolin or other substances that raise the levels of intracellular cyclic AMP. In this study we have investigated the molecular basis for forskolin induction of MBP transcription in D6P2T cells. We have found that a 9-bp sequence element, CACTTGATC, located between nucleotides -85 and -77 in the MBP promoter, is necessary for forskolin induction of chloramphenicol acetyltransferase (CAT) expression after transient transfection of MBP promoter-CAT fusion constructs into D6P2T cells. Although similar DNase I footprints, one of which is located within the above 9-bp sequence element, are produced by nuclear extracts prepared from both forskolin-treated and untreated cells, this same sequence can be shown to interact with a forskolin-inducible protein complex using an electrophoretic mobility shift assay. In addition, mutation of this 9-bp sequence abolishes both formation of this new protein--DNA complex and forskolin-inducible CAT expression from the heterologous SV40 promoter. Finally, we have shown that the appearance of this forskolin-inducible protein--DNA complex precedes that of MBP mRNA. Taken together, these data strongly support the notion that the induction of MBP transcription by forskolin in D6P2T cells is mediated by the binding of a forskolin-inducible protein complex to the MBP promoter sequence CACTTGATC.

Hypomyelinating peripheral neuropathies and schwannomas in transgenic mice expressing SV40 T-antigen.

We have prepared transgenic mice carrying a temperature-sensitive mutant of the SV40 oncogene (tsA-1609) under the control of 5' flanking sequences from the Schwann cell-specific P0 gene. Four of six founder mice showed moderate to severe hypomyelination in peripheral nerves of tail biopsies, with only rare myelinated fibers. Offspring were obtained from three of these founders. Northern blot and immunohistochemical analyses showed that expression of T-antigen was restricted to the PNS. Mice expressing the highest levels of T-antigen exhibited the most severe hypomyelination. Mice expressing lower levels developed transient mild hypomyelination, but after long latencies developed sporadic schwannomas. An immortalized cell line exhibiting properties of Schwann cells at an arrested stage of differentiation, termed "SCT-1," was derived from one of these tumors.

Beta 4 integrin expression in myelinating Schwann cells is polarized, developmentally regulated and axonally dependent.

In developing and regenerating peripheral nerve, Schwann cells interact with axons and extracellular matrix in order to ensheath and myelinate axons. Both of these interactions are likely to be mediated by adhesion molecules, including integrins, which mediate cell-cell and cell-extracellular matrix interactions. Recently, the beta 4 integrin subunit was reported to be expressed by Schwann cells in peripheral nerve. We have examined the expression of beta 4, beta 1 and their common heterodimeric partner, the alpha 6 integrin subunit, in developing and regenerating rat peripheral nerve. beta 4 and alpha 6 are enriched in peripheral nerve and they co-localize at the abaxonal surface of myelinating Schwann cells, opposite the Schwann cell basal lamina, which contains possible ligands of alpha 6 beta 4. In contrast, beta 4 and alpha 6 are expressed in a different pattern in non-myelinating Schwann cells. The level of beta 4, but not alpha 6 or beta 1 mRNAs, increases progressively in developing nerves, reaching a peak in adult nerves well after the peak of the myelin-specific mRNAs. After axotomy, the expression of beta 4 mRNA and protein, but not alpha 6 or beta 1 mRNAs, fall rapidly but subsequently are reinduced by regenerating axons. Similarly, in cultured Schwann cells, the expression of beta 4 mRNA, but not alpha 6 mRNA, is significantly modulated by forskolin, a drug that elevates cAMP and mimics some of the effects of axonal contact. beta 4 integrin expression in Schwann cells, therefore, is regulated by Schwann cell-axon interactions, which are known to be critical in determining the Schwann cell phenotype. Furthermore, the polarized expression of alpha 6 beta 4 to the abaxonal surface of myelinating Schwann cells suggests that alpha 6 beta 4 may mediate in part the morphological changes required of Schwann cells in the process of myelination in the peripheral nervous system.

Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.

Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5' flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5' of the initiation of transcription was sufficient to direct oligodendrocyte-specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis-acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis-acting segment were identified. The distal segment behaved more negatively in Cos-7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis-acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos-7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos-7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte-specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin-specific promoters.

Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.

Developmental changes in the molecular weights of polypeptides in the human CNS that carry the HNK-1 epitope and bind Phaseolus vulgaris lectins.

The binding patterns of electrophoresed polypeptides from homogenates of human frontal lobe, cerebellum, and spinal cord obtained at various stages of development were determined for several lectins with specificities for a wide range of oligosaccharides. A discrete developmental change in the molecular-weight pattern was seen only among polypeptides binding the two Phaseolus vulgaris agglutinins, E-phytohemagglutinin (E-PHA) and L-PHA. With increasing maturity, the apparent molecular weights of the major polypeptides binding these two lectins progressively decreased. Furthermore, at all stages of development, E-PHA and L-PHA bound to the same polypeptides as the monoclonal antibody HNK-1, which recognizes a carbohydrate epitope on polypeptides that may play roles in cell adhesion. Based on the carbohydrate specificities of the two PHAs, we conclude that it is likely that the HNK-1 epitope resides on a triantennary N-linked oligosaccharide bisected by N-acetylglucosamine.