Udo Bartsch

Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin

Using homologous recombination in embryonic stem cells, we have generated mice with a null mutation in the gene encoding the myelin-associated glycoprotein (MAG), a recognition molecule implicated in myelin formation. MAG-deficient mice appeared normal in motor coordination and spatial learning tasks. Normal myelin structure and nerve conduction in the PNS, with N-CAM overexpression at sites normally expressing MAG, suggested compensatory mechanisms. In the CNS, the onset of myelination was delayed, and subtle morphological abnormalities were detected in that the content of oligodendrocyte cytoplasm at the inner aspect of most myelin sheaths was reduced and that some axons were surrounded by two or more myelin sheaths. These observations suggest that MAG participates in the formation of the periaxonal cytoplasmic collar of oligodendrocytes and in the recognition between oligodendrocyte processes and axons.

Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS

The MAG-deficient mouse was used to test whether MAG acts as a significant inhibitor of axonal regeneration in the adult mammalian CNS, as suggested by cell culture experiments. Cell spreading, neurite elongation, or growth cone collapse of different cell types in vitro was not significantly different when myelin preparations or optic nerve cryosections from either MAG-deficient or wild-type mice were used as a substrate. More importantly, the extent of axonal regrowth in lesioned optic nerve and corticospinal tract in vivo was similarly poor in MAG-deficient and wild-type mice. However, axonal regrowth increased significantly and to a similar extent in both genotypes after application of the IN-1 antibody directed against the neurite growth inhibitors NI-35 and NI-250. These observations do not support the view that MAG is a significant inhibitor of axonal regeneration in the adult CNS.

The Disintegrin/Metalloproteinase ADAM10 Is Essential for the Establishment of the Brain Cortex

The metalloproteinase and major amyloid precursor protein (APP) α-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10 −/− mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that α-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP α-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

Multiple functions of the myelin-associated glycoprotein MAG (siglec-4a) in formation and maintenance of myelin.

The myelin‐associated glycoprotein, a minor component of myelin in the central and peripheral nervous system, has been implicated in the formation and maintenance of myelin. Although the analysis of MAG null mutants confirms this view, the phenotype of this mutant is surprisingly subtle. In the CNS of MAG‐deficient mice, initiation of myelination, formation of morphologically intact myelin sheaths and to a minor extent, integrity of myelin is affected. In the PNS, in comparison, only maintenance of myelin is impaired. Recently, the large isoform of MAG has been identified as the functionally important isoform in the CNS, whereas the small MAG isoform is sufficient to maintain the integrity of myelinated fibers in the PNS. Remarkably, none of the different defects in the MAG mutant is consistently associated with each myelinated fiber. These observations suggest that other molecules performing similar functions as MAG might compensate, at least partially, for the absence of MAG in the null mutant. GLIA 29:154–165, 2000.

Reduced Perisomatic Inhibition, Increased Excitatory Transmission, and Impaired Long-Term Potentiation in Mice Deficient for the Extracellular Matrix Glycoprotein Tenascin-R

The role of the extracellular matrix molecule tenascin-R (TN-R) in regulation of synaptic transmission and plasticity in the CA1 region of the hippocampus was studied using mice deficient in expression of this molecule. The mutant mice showed normal NMDA-receptor-mediated currents but an impaired NMDA-receptor-dependent form of long-term potentiation (LTP) as compared to wild-type littermates. Reduced LTP in mutants was accompanied by increased basal excitatory synaptic transmission in synapses formed on CA1 pyramidal neurons. A possible mechanism for increased excitatory synaptic transmission in mutants could involve modulation of inhibition, since TN-R and its associated carbohydrate HNK-1 decorate perisomatic interneurons. Indeed, the amplitudes of unitary perisomatic inhibitory currents were smaller in mutants compared to wild-type mice. Thus, our data show that a deficit in TN-R results in reduction of perisomatic inhibition and, as a consequence, in an increase of excitatory synaptic transmission in CA1 to the levels close to saturation, impeding further expression of LTP.

Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice

Vision impairment caused by degeneration of photoreceptors, termed retinitis pigmentosa, is a debilitating condition with no cure presently available. Cell-based therapeutic approaches represent one treatment option by replacing degenerating or lost photoreceptors. In this study the potential of transplanted primary retinal cells isolated from neonatal mice to integrate into the outer nuclear layer (ONL) of adult mice and to differentiate into mature photoreceptors was evaluated. Retinal cells were isolated from retinas of transgenic mice ubiquitously expressing enhanced green fluorescence protein (EGFP) at either postnatal day (P) 0, P1 or P4 and transplanted into the subretinal space of adult wild-type mice. One week to 11 months post-transplantation experimental retinas were analyzed for integration and differentiation of donor cells. Subsequent to transplantation some postnatal retinal cells integrated into the ONL of the host and differentiated into mature photoreceptors containing inner and outer segments as confirmed by immunohistochemistry and electron microscopy. Notably, the appearance of EGFP-positive photoreceptors was not the result of fusion between donor cells and endogenous photoreceptors. Retinal cells isolated at P4 showed a significant increase in their capacity to integrate into the ONL and to differentiate into mature photoreceptors when compared with cells isolated at P0 or P1. As cell suspensions isolated at P4 are enriched in cells committed towards a rod photoreceptor cell fate it is tempting to speculate that immature photoreceptors may have the highest integration and differentiation potential and thus may present a promising cell type to develop cell replacement strategies for diseases involving rod photoreceptor loss.

Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules

We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N‐terminal signal sequence, six immunoglobulin (Ig)‐like domains, 4.5 fibronectin type III (FN)‐like repeats, a transmembrane domain and a C‐terminal, most likely intracellular domain of ˜100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng‐CAM (˜40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr‐CAM, Drosophila neuroglian and zebrafish L1.l (37‐28% amino acid identity), and mouse F3, rat TAG‐1 and rat BIG‐1 (˜27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N‐CAM), DCC, HLAR, rse] is 16‐11%. The intracellular domain is most similar to mouse and chicken Nr‐CAM, mouse and rat neurofascin (˜60% amino acid identity) followed by chicken neurofascin and Ng‐CAM, Drosophila neuroglian and zebrafish L1.l and L1.2 (˜40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng‐CAM; chicken/mouse Nr‐CAM; Drosphila neuroglian; zebrafish L1.l and L1.2; chicken/mouse neurofascin/rat ankyrin‐binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig‐like domains and FN‐like repeats. These show a collinearity in the six Ig‐like domains and four adjacent FN‐like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI‐linked forms of the F3 subgroup (mouse F3/chicken F1l/human CNTN1; rat BIG‐l/mouse PANG; rat TAG‐l/mouse TAX‐l/chicken axonin‐1). The colorectal cancer molecule (DCC), previously introduced as an N‐CAM‐like molecule, conforms to the L1 family cassette. Other structural features of CHL l shared between members of the L1 family are a high degree of N‐glycosidically linked carbohydrates (˜20% of its molecular mass), which include the HNK‐1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kDa band and smaller fragments of 165 and 125 kDa. As for the other L1 family members, predominant expression of CHL l is observed in the nervous system and at later developmental stages. In the central nervous system CHL l is expressed by neurons, but, in contrast to L1, also by glial cells. Our findings suggest a common ancestral L1‐like molecule which evolved via gene duplication to generate a diversity of structurally and functionally distinct yet similar molecules.

Localization of Janusin mRNA in the Central Nervous System of the Developing and Adult Mouse

Janusin (formerly termed J1‐160/180) is an oligodendrocyte‐derived extracellular matrix molecule which is restricted to the central nervous system and which is expressed late during development (Pesheva et al., J. Cell Biol., 1765 – 1778, 1989). To gain insights into the molecules morphogenetic functions and to identify its cellular source in vivo, we have studied the localization of janusin messenger RNA in the optic nerve, retina and spinal cord and the expression of janusin protein in the spinal cord of developing and adult mice. Moreover, we have analysed optic nerve cell cultures and retinal cell suspensions in double‐labelling experiments using a janusin‐specific anti‐sense complementary RNA probe and cell type‐specific antibodies to identify the cell types containing janusin transcripts. In developing animals, oligodendrocytes were strongly labelled with the janusin anti‐sense cRNA probe during the period of myelination. The number of labelled cells and intensity of the hybridization signal decreased significantly with increasing age. Interestingly, expression of janusin was not confined to oligodendrocytes. Some neuronal cell types and type‐2 astrocytes present in optic nerve cell cultures also contained janusin transcripts. In contrast to oligodendrocytes, the number and labelling intensity of neurons containing janusin transcripts remained constant during postnatal development and into adulthood. Expression of janusin protein in the spinal cord was developmentally regulated, with a peak of expression in 2‐or 3‐week‐old animals. The molecule was visible in the white and grey matter. In myelinated regions, it was associated with myelinated fibres and accumulated at nodes of Ranvier. These observations suggest that janusin may be of functional relevance for myelination.

The raft-associated protein MAL is required for maintenance of proper axon–glia interactions in the central nervous system

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode–axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Immunohistological Localization of Tenascin in the Developing and Lesioned Adult Mouse Optic Nerve

To gain insight into the morphogenetic functions of the recognition molecule tenascin in the central nervous system, we have studied its localization in the developing and lesioned adult mouse optic nerve using light and electron microscopic immunocytochemistry. Since tenascin is a secreted molecule, we have analysed the tenascin‐synthesizing cells in tissue sections of retinae and optic nerves by in situ hybridization. A weak and homogeneous tenascin immunoreactivity was detectable in the developing retinal nerve fibre layer and optic nerve of 14‐day‐old mouse embryos, the earliest developmental age investigated. In the optic nerve of neonatal and 1‐week‐old animals, a high number of tenascin messenger RNA (mRNA)‐containing cells were present, and antibodies to tenascin labelled the surfaces of astrocytes and unmyelinated retinal ganglion cell axons. With increasing age, expression of tenascin in the optic nerve was down‐regulated at the mRNA and protein levels. At the fourth postnatal week, blood vessels in the optic nerve and collagen fibrils in the vicinity of meningeal fibroblast‐like cells still showed significant immunoreactivity, but the optic nerve tissue proper no longer did so. In adult animals, tenascin was no longer detectable in association with blood vessels located in the myelinated part of the optic nerve, and meninges were only weakly immunoreactive. Also, tenascin mRNA‐containing cells were no longer detectable in the myelinated part of the adult mouse optic nerve and few labelled cells were found in the meninges. In the retina, ganglion cells contained no detectable levels of tenascin mRNA at any of the developmental ages analysed. No significant up‐regulation of tenascin expression was seen in the nerve tissue proper of transected proximal (i.e. retinal) and distal (i.e. cranial) optic nerve stumps of adult mice during the first 4 weeks after lesioning, the time period studied. However, collagen fibrils associated with meningeal fibroblast‐like cells and located near the lesion site became strongly tenascin‐immunoreactive 2 days after lesioning. Also, some blood vessels at the lesion site became immunoreactive. We conclude that tenascin in the optic nerve is synthesized by glial cells and not by retinal ganglion cells. The detectability of tenascin at embryonic ages suggests that it may mediate neurite growth in vivo. The absence of a strong, lesion‐induced up‐regulation of tenascin expression in the regeneration‐prohibitive mouse optic nerve contrasts with the lesion‐induced pronounced up‐regulation in the regeneration‐permissive peripheral nervous system, and may indicate a functional involvement of tenascin in regenerative processes. The high tenascin positivity of collagen fibrils at early postnatal ages and after lesioning suggests that tenascin expression may be correlated with mitotic activity of the associated meningeal fibroblast‐like cells. Finally, tenascin may be involved in the process of vascularization, since the molecule is associated with blood vessels in developing and adult lesioned, but not intact adult, optic nerves.