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S S Scherer

Publications and source records attributed to S S Scherer.

At least 19 recordsLinked to original sources
PubMed1999

Regulation of fibronectin alternative splicing during peripheral nerve repair.

Wallerian degeneration following peripheral nerve injury is associated with increased production of fibronectin and other extracellular matrix molecules that are thought to enhance repair. We have shown previously that alternative splicing of the mRNA for fibronectin also changes following sciatic nerve lesions so as to reexpress forms of mRNA seen during embryogenesis. In the present study, we have examined the role of the regenerating axons in the regulation of this splicing. We have compared the patterns of fibronectin mRNA splicing seen in sciatic nerve development with that seen in cut nerves (that do not regenerate), crushed nerves (that regenerate successfully), and Schwann cells cultured in forskolin so as to mimic axonal signals. By using a reverse transcriptase polymerase chain reaction assay to examine all three regions of fibronectin mRNA splicing in a quantitative manner, we found that embryonic patterns of fibronectin mRNA splicing appear rapidly following injury and are not then altered by reestablishment of axons in the nerve. In addition, we found that forskolin has no effect on fibronectin mRNA splicing in cultured cells. We conclude that axonal signals do not regulate the pattern of fibronectin alternative splicing in peripheral nerve repair.

Alternative Splicing↗
PubMed1999

p200, a collagen secreted by Schwann cells, is expressed in developing nerves and in adult nerves following axotomy.

Previously we reported that cultured rat Schwann cells secrete p200, a collagen-like heparin-binding adhesive glycoprotein with a restricted pattern of expression. Here we report that p200 is secreted as a stable trimer, but only after treatment of Schwann cells with ascorbic acid, and was deposited in the fibrillar extracellular matrix. Heparin and heparitinase treatment inhibited incorporation of p200 into extracellular matrix, suggesting the involvement of Schwann cell heparan sulfate proteoglycans in this process. Pepsin digestion revealed that p200 secreted by ascorbate-treated cells contains a collagenous domain of approximately 140 kDa. Immunofluorescent staining of rat embryos at different ages showed that p200 first appeared between embryonic days 15 and 18, and was confined to peripheral nerves. Staining of adult peripheral nerve was negative, but p200 expression was induced in adult sciatic nerve following nerve transection. These data suggest that p200 carries out unique functions during peripheral nerve development and regeneration and that its expression by Schwann cells is regulated by axon-Schwann cell interaction.

Aging↗
PubMed1999

The role of the gap junction protein connexin32 in the pathogenesis of X-linked Charcot-Marie-Tooth disease.

Mutations in the gene encoding the gap junction protein connexin32 (Cx32; beta 1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX), a common form of inherited demyelinating neuropathy. Cx32 is localized to the paranodes and incisures of myelinating Schwann cells, and probably participates in the formation of gap junctions at these locations, thereby allowing the diffusion of ions and small molecules directly across the myelin sheath. In transfected cells different CMTX mutations have different effects on the ability of the mutant protein to form functional gap junctions; some mutant proteins cannot be detected within the cell, other mutant proteins accumulate within the cell but do not reach the cell membrane, while other mutants reach the cell membrane and some of these form functional gap junctions. In transgenic mice two mutants, R142W and 175 frameshift, have similar effects on protein trafficking as in transfected cells: the R142W mutant protein remains in the perinuclear region and does not reach the paranodes or incisures, and the 175 frameshift protein cannot be detected. Thus, different CMTX mutations have different effects on Cx32 protein, and these differences may help to explain the phenotypic differences seen in CMTX kindreds.

Amino Acid Sequence↗
PubMed1999

Studies in transgenic mice indicate a loss of connexin32 function in X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is an inherited demyelinating neuropathy caused by mutations in the gene encoding the gap junction protein connexin32 (Cx32). Despite the identification of over 160 different mutations in the Cx32 coding sequence, it is not known whether the mutations cause the disease manifestations through a loss of Cx32 function or through toxic effects on peripheral nerve. We created transgenic mice with a frameshift mutation at codon 175 (175fs), identified in a large CMTX pedigree. Light microscopic examination of the peripheral nerves from adult transgenic animals showed no pathological features. Western blotting did not show transgenic Cx32 protein in any of the 26 lines, although expression of transgenic messenger RNA was detected by reverse-transcriptase polymerase chain reaction and by ribonuclease protection assay. Our findings indicate that the 175fs mutation results in a loss of Cx32 function, without additional toxic effects.

Animals↗
PubMed1998

Maturation-dependent apoptotic cell death of oligodendrocytes in myelin-deficient rats.

Mutations in the proteolipid protein gene (PLP/plp), which encodes the major intrinsic membrane protein in central nervous system (CNS) myelin, cause inherited dysmyelination in mammals. One of these mutants, the myelin-deficient (md) rat, has severe dysmyelination that is associated with oligodendrocyte cell death. Using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay, which labels apoptotic cells, we find that cell death is increased in multiple white matter tracts of md rats. The tracts that myelinate the earliest show the earliest increase in cell death, and cell death persists for at least 22 days, the lifespan of these mutant animals. In all tracts, and at all developmental ages examined, apoptotic cells expressed the markers of mature oligodendrocytes, such as myelin basic protein, myelin-associated glycoprotein, and the Rip antigen, but not chondroitin sulfate proteoglycan, a marker of oligodendrocyte precursors. Mature oligodendrocytes fail to accumulate in md brain because they die before they fully mature.

Age Factors↗
PubMed1998

Accumulation of F-spondin in injured peripheral nerve promotes the outgrowth of sensory axons.

F-spondin, an extracellular matrix protein, is present in peripheral nerve during embryonic development, but its amount diminishes by birth. Axotomy of adult rat sciatic nerve, however, causes a massive upregulation of both F-spondin mRNA and protein distal to the lesion. F-spondin in the distal stump of axotomized nerve promotes neurite outgrowth of sensory neurons, as revealed by protein neutralization with F-spondin-specific antibodies. Thus, F-spondin is likely to play a role in promoting axonal regeneration after nerve injury.

Animals↗
PubMed1998

Promyelinating Schwann cells express Tst-1/SCIP/Oct-6.

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are transiently arrested at the "promyelinating" stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined beta-galactosidase (beta-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. beta-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenous tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-beta-D-galactopyranoside- (X-gal) and halogenated indolyl-beta-D-galactoside- (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of beta-gal, both in developing and in regenerating nerves. Thus, the expression of beta-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested in tst-1/scip/oct-6-null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.

Age Factors↗
PubMed1998

Functional gap junctions in the schwann cell myelin sheath.

The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32-null mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Connexin32-null mice develop demyelinating peripheral neuropathy.

Mutations in the gene encoding the gap junction protein connexin32 (Cx32) cause X-linked Charcot-Marie-Tooth disease (CMTX), a common form of inherited demyelinating peripheral neuropathy. To learn more about the pathogenesis of CMTX, we examined the PNS and CNS of cx32-null mice (cx32-/Y males and cx32-/-females) by light and electron microscopy. These mice develop a progressive demyelinating peripheral neuropathy beginning by 3 months of age, and at all ages, motor fibers are more affected than sensory fibers. Like other genes of the X chromosome, the cx32 gene appears to be randomly inactivated, since only some myelinating Schwann cells express Cx32 in heterozygous cx32 +/- females. Heterozygous cx32 +/- females have fewer demyelinated and remyelinated axons than age-matched homozygous cx32-/- females and cx32-/Y males. Although oligodendrocytes also express Cx32, no abnormalities in CNS myelin were found. These findings indicate that a null cx32 allele in myelinating Schwann cells is sufficient to cause an inherited demyelinating neuropathy, so that Cx32 has an essential role in myelinating Schwann cells both in mice and in humans.

Aging↗
PubMed1997

Altered trafficking of mutant connexin32.

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot-Marie-Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting in a potentially toxic cytoplasmic accumulation of Cx32 in these cells. These results and evidence of cytoplasmic accumulation of other mutated myelin proteins suggest that diseases affecting myelinating cells may share a common pathophysiology.

Amino Acid Sequence↗
PubMed1997

Transient expression of the neurofilament proteins NF-L and NF-M by Schwann cells is regulated by axonal contact.

Expression of the genes that encode neurofilament proteins is considered to be confined normally to neurons. However, in demyelinating peripheral nerves Schwann cells upregulate the mRNA for the medium-sized neurofilament protein (NF-M), and cultured Schwann cells of the myelin-forming phenotype can also synthesize and incorporate NF-M protein into their intermediate filament (IF) cytoskeleton. The purpose of this study was to establish how axonal contact might influence glial neurofilament gene expression and regulate the synthesis of neurofilament proteins. We show that the gene encoding NF-M is expressed at early stages of differentiation in myelin-forming Schwann cells in vivo; nevertheless, little NF-M protein can be detected in these cells. The transient induction of NF-M mRNA is also apparent in dedifferentiating Schwann cells during Wallerian degeneration. In these Schwann cells the mRNAs for NF-M and NF-L (the smallest polypeptide), but not NF-H (the largest neurofilament subunit), are coordinately expressed. In contrast to differentiating myelin-forming Schwann cells, the cells of degenerating nerves express both NF-M and NF-L polypeptides. Restoration of axonal contact in the growing nerve stimulates the recapitulation of Schwann cell differentiation including the elevation of NF-M and NF-L mRNA expression. These results demonstrate that the transient induction of neurofilament mRNAs in Schwann cells is a feature of both differentiation and dedifferentiation. However translation of these mRNAs is confined to Schwann cells deprived of axonal contact either by nerve injury or by culture in the absence of axons. These findings suggest that the expression of the NF-M and NF-L polypeptides is an important characteristic of those Schwann cells that will contribute to the repair of damaged peripheral nerves.

Animals↗
PubMed1997

Cloning and sequence of the cDNA encoding the beta 4 integrin subunit in rat peripheral nerve.

beta 4 and alpha 6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. beta 4 is also expressed in Schwann cell (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for beta 4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding beta 4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in beta 4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of beta 4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived beta 4 integrin are highly similar to those cloned from epithelia.

Amino Acid Sequence↗
PubMed1997

Neuregulin expression in PNS neurons: isoforms and regulation by target interactions.

Neuregulins have several important functions in the development of the peripheral nervous system, acting on both developing Schwann cells and muscle fibers. To determine whether these factors are also important for peripheral nerve regeneration, we have analyzed neuregulin expression in motor and sensory neurons by Northern blots and in situ hybridization. The results of this analysis show that the predominant neuregulin isoform expressed in these neurons is a novel transmembrane splice variant. After axotomy, there is a rapid decline in neuregulin expression in both motor and sensory neurons, but following reinnervation of target tissues, neuregulin expression returns to near normal levels. These results indicate that the normal expression of neuregulins in these neurons is maintained by the interactions with target tissues.

Amino Acid Sequence↗
PubMed1997

Connexin32 and X-linked Charcot-Marie-Tooth disease.

Mutations in the gap junction gene connexin32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. More than 130 different mutations have been described, affecting all portions of the Cx32 protein. In transfected cells, the mutant Cx32 proteins encoded by some Cx32 mutations fall to reach the cell surface; other mutant proteins reach the cell surface, but only one of these forms functional gap junctions. In peripheral nerve, Cx32 is localized to incisures and paranodes, regions of noncompact myelin within the myelin sheath. This localization suggests that Cx32 forms "reflexive" gap junctions that allow ions and small molecules to diffuse directly across the myelin sheath, which is a thousandfold shorter distance than the circumferential pathway through the Schwann cell cytoplasm. Cx32 mutations may interrupt this shorter pathway or have other toxic effects, thereby injuring myelinating Schwann cells and their axons.

Animals↗