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Biomedical subjects

Melitta Schachner

Publications and source records attributed to Melitta Schachner.

At least 109 records · Page 6Linked to original sources
PubMed2003

The proprotein convertase PC5A and a metalloprotease are involved in the proteolytic processing of the neural adhesion molecule L1.

The transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg(845) to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth.

Amino Acid Motifs↗
PubMed2003

The C264Y missense mutation in the extracellular domain of L1 impairs protein trafficking in vitro and in vivo.

The neural cell adhesion molecule L1, a member of the immunoglobulin superfamily, performs important functions in the developing and adult nervous system and is implicated in neuronal migration and survival, elongation, fasciculation and pathfinding of axons, and synaptic plasticity. This view is in line with the fact that mutations in the L1 gene result in severe neurological syndromes in humans. Patients with missense mutations in the extracellular domain of L1 often develop severe phenotypes. Here, we characterized in vitro and in vivo the missense mutation C264Y, which is located in the extracellular domain of L1 and causes a severe phenotype in humans. Transfection studies in vitro demonstrate that L1 carrying this missense mutation is not expressed at the cell surface but instead is located intracellularly, most likely within the endoplasmic reticulum. Lack of cell surface expression of L1 with a C264Y mutation was confirmed in a transgenic mouse line expressing the C264Y mutation under the control of the L1 promoter in an L1-deficient background. Analysis of these transgenic mice indicates that they represent functional null mutants, phenotypically indistinguishable from L1-deficient mice. These observations corroborate the view that impaired cell surface expression of mutated variants of L1 is a potential explanation for the high number of severe pathogenic mutations identified within the human L1 gene.

Animals↗
PubMed2003

Heterozygous null mutation of myelin P0 protein enhances susceptibility to autoimmune neuritis targeting P0 peptide.

Mice with a heterozygous null mutation in myelin protein zero (P0(+/-)) develop late-onset clinical paralysis associated with inflammatory pathology in the peripheral nerves. Although the development of this illness is known to require T cells and macrophages, little is understood regarding the immunological defect in the mice. Here we report that young P0(+/-) mice, free from clinical manifestations, have a defect in central tolerance to P0, and are more prone to induction of experimental autoimmune neuritis (EAN) by sensitization against P0(180-199 )peptide. Notably, we found that the P0 gene is transcribed in the thymus of wild-type and the P0(+/-) mice in an amount proportional to the gene dosage. We then replaced the thymus of wild-type mice with that of the P0-deficient mice and vice versa. Immunization of these mice with P0(180-199 )revealed that a lower thymic P0 transcript would be associated with the higher recall T cell response to P0(180-199), thus accounting for the higher susceptibility of the P0(+/-) mice to P0-induced EAN. These results imply that a heterozygous mutation in an autoantigen could cause defective central tolerance to the autoantigen. As such, autoimmune T cells may play some role in "genetic" diseases caused by a heterozygous gene defect.

Amino Acid Sequence↗
PubMed2003

Expression of protein zero is increased in lesioned axon pathways in the central nervous system of adult zebrafish.

The immunoglobulin superfamily molecule protein zero (P0) is important for myelin formation and may also play a role in adult axon regeneration, since it promotes neurite outgrowth in vitro. Moreover, it is expressed in the regenerating central nervous system (CNS) of fish, but not in the nonregenerating CNS of mammals. We identified a P0 homolog in zebrafish. Cell type-specific expression of P0 begins in the ventromedial hindbrain and the optic chiasm at 3-5 days of development. Later (at 4 weeks) expression has spread throughout the optic system and spinal cord. This is consistent with a role for P0 in CNS myelination during development. In the adult CNS, glial cells constitutively express P0 mRNA. After an optic nerve crush, expression is increased within 2 days in the entire optic pathway. Expression peaks at 1 to 2 months and remains elevated for at least 6 months postlesion. After enucleation, P0 mRNA expression is also upregulated but fails to reach the high levels observed in crush-lesioned animals at 4 weeks postlesion. Spinal cord transection leads to increased expression of P0 mRNA in the spinal cord caudal to the lesion site. The glial upregulation of P0 mRNA expression after a lesion of the adult zebrafish CNS suggests roles for P0 in promoting axon regeneration and remyelination after injury.

Age Factors↗
PubMed2003

Region and lamina-specific distribution of extracellular matrix proteoglycans, hyaluronan and tenascin-R in the mouse hippocampal formation.

The extracellular matrix is known to show region-specific characteristics in the adult brain. Our comparative cytochemical study is focused on the laminar organisation of major extracellular matrix constituents in the murine hippocampal formation, including the regions CA1, CA2 and CA3 of the hippocampus proper, the dentate gyrus, the subiculum and the presubiculum. Components related to chondroitin sulphate proteoglycans were detected by N-acetylgalactosamine-binding Wisteria floribunda agglutinin, colloidal iron staining, and antibodies to different proteoglycan domains, including the Cat-301 and Cat-315 epitopes of aggrecan, as well as neurocan, brevican and phosphacan. The distribution patterns of these components were correlated with the patterns revealed for hyaluronan and the brain-specific extracellular matrix glycoprotein, tenascin-R, known to be ligands of extracellular matrix proteoglycans. Lectin binding clearly labelled perineuronal nets of the extracellular matrix around interneurons, which were preferentially located within or near the principal cell layers in all regions. In the hippocampus proper, the CA2 subfield showed an intense labelling of the neuropil around pyramidal cell bodies and the neuropil zones in the strata oriens and radiatum. These patterns were also seen after immunoreaction for chondroitin proteoglycan domains, brevican and phosphacan, as well as after detection of hyaluronan and tenascin-R. Characteristic laminar and intralaminar patterns were additionally expressed in the neuropil in all regions. In the dentate gyrus, the staining intensity for brevican, phosphacan and tenascin-R was predominant in the middle molecular layer, and for Cat-315 in the inner molecular layer, whereas immunoreactivity for neurocan increased within the outer molecular layer towards the hippocampal fissure. Our findings indicate that proteoglycans, hyaluronan and tenascin-R show differential patterns of co-expression in the individual regions and laminae of the hippocampal formation. The inhomogeneous composition of these major components suggests that the extracellular matrix is specifically adapted to the functional domains of intrahippocampal connections and afferent fibre systems.

Animals↗
PubMed2003

Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival.

Many uncertainties remain regarding the physiological function of the prion protein PrP and the consequences of its conversion into the pathological scrapie isoform in prion diseases. Here, we show for the first time that different signal transduction pathways are involved in neurite outgrowth and neuronal survival elicited by PrP in cell culture of primary neurons. These pathways include the nonreceptor Src-related family member p59(Fyn), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase. Regulation of Bcl-2 and Bax expression also correlates with the survival effect elicited by PrP. The combined results, along with our observation that PrP carries the recognition molecule-related HNK-1 carbohydrate, argue strongly for a role of the molecule in neural recognition by interacting with yet unknown heterophilic neuronal receptors, as shown by comparison of neurite outgrowth from neurons of PrP-deficient and wild-type mice.

Animals↗
PubMed2003

Single chain Fv antibodies against neural cell adhesion molecule L1 trigger L1 functions in cultured neurons.

The neural cell adhesion molecule L1 plays important roles in cell adhesion, neuronal migration, neurite outgrowth, fasciculation and pathfinding, neuronal survival, and synaptic plasticity. Many of these functions have been identified and characterized by using antibodies. Because of the need for reproducible and functionally active antibodies, we have generated two single-chain variable fragment antibodies against mouse L1 from a human synthetic phage display library. The complementarity determining region 3 of the variable heavy chains of the two antibodies differed in length and sequence. Both antibodies recognized mouse, but not human L1, by enzyme-linked immunosorbent assay, Western blot, and immunofluorescence staining of cultured neurons. Epitope mapping showed reactivity with the fibronectin type III repeats 1-2 of mouse L1. The antibodies stimulated neurite outgrowth from cerebellar dorsal, root ganglion and motor neurons when offered in substrate-coated form in a dose-dependent manner with maximal effects at approximately 32 nM. Furthermore, substrate-coated antibodies enhanced survival of cerebellar neurons. Peptides comprising 8 and 11 amino acids derived from the complementarity determining region 3 of the variable heavy chains of the two single-chain variable fragment antibodies also promoted neurite outgrowth. The combined observations indicate that single-chain variable fragment antibodies against L1 and peptides derived from their binding domains can mimic some beneficial functions of homophilically binding L1 in vitro and may thus serve to trigger these functions in vivo.

Amino Acid Sequence↗
PubMed2003

Tenascin-N: characterization of a novel member of the tenascin family that mediates neurite repulsion from hippocampal explants.

Tenascin-N, a novel member of the tenascin family, was identified and shown to encode characteristic structural motifs of a cysteine-rich stretch, 3.5 epidermal growth factor-like repeats, 12 fibronectin type III homologous domains, and a fibrinogen-like domain. The third fibronectin type III homologous domain is altered by RNA splicing. Characterization of the expression of tenascin-N by in situ hybridization analysis assigned transcripts to many types of neurons in the central nervous system, to the medullary region in the kidney, and to resident macrophages of the T-cell zone in the splenic white pulp. By immunohistochemistry, tenascin-N expression is detectable in all brain regions, with a characteristic staining pattern in the hippocampus demarcating the CA3 region. Recombinantly expressed protein fragments of the alternatively spliced isoforms were presented in choice assays on patterned substrates to neurites and migrating neurons from hippocampal CA3 region explant cultures. The smaller splice variant inhibited neurite outgrowth or cell migration, whereas the longer splice form did not inhibit these functions. These observations suggest that the novel tenascin family member mediates specific repulsive properties on neurites and neurons by generating splice isoforms.

Alternative Splicing↗
PubMed2003

Recognition molecule associated carbohydrate inhibits postsynaptic GABA(B) receptors: a mechanism for homeostatic regulation of GABA release in perisomatic synapses.

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA(B) receptor-mediated activation of K(+) currents in CA1 pyramidal cells, which elevates extracellular K(+) concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA(B) receptors and inhibits GABA(B) receptor-activated K(+) currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA(A) receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA(B) receptor activity.

Animals↗
PubMed2003

Potentiation of amygdaloid and hippocampal auditory-evoked potentials in a discriminatory fear-conditioning task in mice as a function of tone pattern and context.

According to the local memory storage hypothesis, information about the tone-shock association in an auditory fear-conditioning paradigm is stored in synapses within the lateral amygdala. Thus, fear-conditioning-induced potentiation of auditory-evoked potentials in response to a conditioned stimulus (CS+, a series of short lasting tones; patterned tone) has been interpreted as an in vivo correlate of amygdaloid synaptic plasticity. Here, we re-examine the specificity of potentiation of auditory-evoked potentials in terms of (i) local confinement to the lateral amygdala, (ii) parameters of CS+ and (iii) influence of context, using a discriminatory fear-conditioning paradigm. Adult male C57BL/6J mice were implanted with recording electrodes aimed at the lateral amygdala, the CA1 region of the hippocampus and the neck muscles for simultaneous recordings of auditory-evoked potentials and startle responses. In a neutral context, auditory-evoked potentials within lateral amygdala and CA1 as well as startle and freezing responses to the CS+ were significantly potentiated following conditioning, as compared with pre-conditioning values and responses to a neutral stimulus (CSn; tone of different frequency). Potentiation was only evident if CS+ was presented as a uniform series but not if presented mixed with CSn. Accordingly, mice failed to show intensified freezing to a patterned tone if a single lasting tone of the same frequency served as CS+. Both CA1 and lateral amygdala auditory-evoked potentials were potentiated in response to CSn if presented in the conditioning context. These findings demonstrate that (i) potentiation of auditory-evoked potentials is not restricted to the lateral amygdala, (ii) both tone frequency and pattern of tone presentation are essential for proper CS+ recognition and (iii) contextual memory leads to a general potentiation of auditory-evoked potentials.

Acoustic Stimulation↗
PubMed2003

The immunoglobulin-superfamily molecule basigin is a binding protein for oligomannosidic carbohydrates: an anti-idiotypic approach.

Recognition molecules that carry carbohydrate structures regulate cell interactions during development and play important roles in synaptic plasticity and regeneration in the adult. Glycans appear to be involved in these interactions. We have searched for binding proteins for oligomannosidic structures using the L3 antibody directed against high mannose-type glycans in an anti-idiotypic approach. A selected monoclonal anti-idiotype antibody was used for affinity chromatography and identified basigin as a binding protein from mouse brain detergent lysates. Basigin was found to bind to high mannose-carrying cell recognition molecules, such as myelin-associated glycoprotein, L1, the beta2-subunit of Na+/K+-ATPase and an oligomannosidic neoglycolipid. Furthermore, basigin was involved in outgrowth of astrocytic processes in vitro. A striking homology between the first immunoglobulin (Ig)-like domain of basigin and the fourth Ig-like domain of NCAM, previously shown to bind to oligomannosidic glycans, and the lectin domain of the mannose receptor confirms that basigin is an oligomannose binding lectin. To our knowledge this is the first report that anti-idiotypic antibodies can be used to identify binding partners for carbohydrates.

Amino Acid Sequence↗
PubMed2003

Antibodies to myelin-associated glycoprotein accelerate preferential motor reinnervation.

Predegeneration of nerve enhances its ability to support axon regeneration. Trophic factors are upregulated by reactive Schwann cells while potentially inhibitory molecules are removed. These experiments isolate the effects of one such inhibitory molecule, the myelin-associated glycoprotein (MAG), to determine its role in modifying regeneration after nerve repair. Suture of the mouse femoral nerve was followed by daily intraperitoneal injection of antibodies to MAG, antibodies to HNK-1, a specific muscle pathway marker, or no further treatment. Regeneration was assayed by double-labeling the femoral cutaneous and muscle branches with horseradish peroxidase and fluoro-gold after 4 weeks or 6 weeks of regeneration. Four weeks after nerve repair, selective reinnervation of the muscle branch by motoneurons, or preferential motor reinnervation (PMR), was not seen in either controls or L2-antibody-treated animals. In contrast, treatment with MAG antibodies resulted in dramatic PMR. By 6 weeks, the controls had achieved borderline specificity, substantial PMR developed in the L2 antibody group and the MAG group changed little. Blocking access to MAG in the distal nerve stump thus accelerated and enhanced PMR. Sensory regeneration was depressed by both antibody treatments at 4 weeks but recovered by 6 weeks. Antibody administration has a generalized effect on sensory regeneration that is unrelated to the behavior of motoneurons in the same nerve.

Animals↗
PubMed2003

Soluble cell adhesion molecule L1-Fc promotes locomotor recovery in rats after spinal cord injury.

Previous studies suggest that the cell adhesion molecule L1 promotes neurite growth by neutralizing white matter associated inhibitors of axonal growth. We made a soluble chimeric dimer by linking mouse L1 to human Fc. This L1-Fc construct (40 microg/mL) markedly facilitated neurite outgrowth, as well as neuronal adhesion to white matter on frozen sections of spinal cord. We applied L1-Fc intrathecally (200 microg/mL at 0.5 microL/h) to rat spinal cords for 2 weeks after a 25-mm weight drop contusion of the T13 spinal cord. Initial experiments indicated that L1-Fc is present in the spinal cord after 2 weeks of intrathecal infusion and significantly improved locomotor recovery by 6-12 weeks after injury. We then randomized 45 rats to intrathecal infusion of L1-Fc (L1), phosphate-buffered saline controls (PBS), and a mouse monoclonal IgM antibody (M1). By 12 weeks after injury, L1-treated rats recovered significantly (p < 0.005) better locomotor function (BBB score 10.57 +/- 0.25, n = 14) than PBS-treated rats (BBB score 9.00 +/- 0.33, n = 14) or M1-treated (BBB score 8.71 +/- 0.16, n = 14). Only two rats of 22 treated with saline recovered weight-supported ambulation. Of 20 L1-Fc-treated rats, however, 18 recovered weight-supported walking by 12 weeks. The L1-Fc-treated rats also showed more consistent hindlimb contact placing than saline controls. We injected biotinylated dextran amine (BDA) into the motor cortices of 14 rats treated with L1-Fc to label corticospinal axons, comparing these with 13 rats treated with saline. In saline-treated rats, BDA-labeled corticospinal axons often grew up to the impact edge and occasionally into the impact site. L1-treated rats showed longer corticospinal tract growth at the injury site. Three rats had BDA-labeled axons that extended beyond the impact center. One L1-Fc-treated rat showed axonal extension and synapse formation in cord distal to the injury. These results indicate that soluble L1-Fc promotes axonal growth and functional recovery after spinal cord injury. However, the limited corticospinal tract growth across the injury site cannot account for the observed locomotor recovery. Thus, L1 may be stimulating growth of other motor tracts or protecting axons and neurons. More studies are required to elucidate the mechanisms of L1-Fc-induced locomotor recovery.

Animals↗
PubMed2002

Exercise increases mRNA levels for adhesion molecules N-CAM and L1 correlating with BDNF response.

In situ hybridization was used to evaluate whether long-term moderate locomotor exercise, which up-regulates BDNF and TrkB levels in the spinal gray matter of the adult rat, similarly influences the expression of the cell adhesion molecules N-CAM and L1. Exercise doubled the level of N-CAM mRNA hybridization signal in the lumbar spinal gray. The increase in L1 mRNA was less consistent. N-CAM mRNA levels slightly increased in the white matter. BDNF mRNA levels also increased in cells of the ventral horn and the white matter due to the exercise. These results suggest that exercise-induced rearrangements of the spinal network involve N-CAM, L1 and BDNF, crucial in different aspects of synaptic plasticity and synapse formation.

Animals↗
PubMed2002

Tracing myelin protein zero (P0) in vivo by construction of P0-GFP fusion proteins.

BACKGROUND: Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein. RESULTS: In contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed. CONCLUSIONS: The present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation.

Afferent Pathways↗
PubMed2002

Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts.

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses.

Animals↗
PubMed2002

Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons.

We investigated the hypothesis that neural stem cells (NSCs) possess an intrinsic capacity to "rescue" dysfunctional neurons in the brains of aged mice. The study focused on a neuronal cell type with stereotypical projections that is commonly compromised in the aged brain-the dopaminergic (DA) neuron. Unilateral implantation of murine NSCs into the midbrains of aged mice, in which the presence of stably impaired but nonapoptotic DA neurons was increased by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was associated with bilateral reconstitution of the mesostriatal system. Functional assays paralleled the spatiotemporal recovery of tyrosine hydroxylase (TH) and dopamine transporter (DAT) activity, which, in turn, mirrored the spatiotemporal distribution of donor-derived cells. Although spontaneous conversion of donor NSCs to TH(+) cells contributed to nigral reconstitution in DA-depleted areas, the majority of DA neurons in the mesostriatal system were "rescued" host cells. Undifferentiated donor progenitors spontaneously expressing neuroprotective substances provided a plausible molecular basis for this finding. These observations suggest that host structures may benefit not only from NSC-derived replacement of lost neurons but also from the "chaperone" effect of some NSC-derived progeny.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗