Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.

Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

The structure and function of tenascins in the nervous system.

The tenascins are a family of large extracellular matrix glycoproteins that comprise five known members. Three of these, tenascin-C (TN-C) tenascin-R (TN-R) and tenascin-Y (TN-Y) are expressed in specific patterns during nervous system development and are down-regulated after maturation. The expression of TN-C, the best studied member of the family, persists in restricted areas of the nervous system that exhibit neuronal plasticity and is reexpressed after lesion. Numerous studies in vitro suggest specific roles for tenascins in the nervous system involving precursor cell migration, axon growth and guidance. TN-C has been shown to occur in a large number of isoform variants generated by combinatorial variation of alternatively spliced fibronectin type III (FNIII) repeats. This finding indicates that TN-C might specify neural microenvironments, a hypothesis supported by recent analysis of TN-C knockout animals, which has begun to reveal subtle nervous system dysfunctions.

Evidence for combinatorial variability of tenascin-C isoforms and developmental regulation in the mouse central nervous system.

The extracellular matrix glycoprotein tenascin-C (TN-C) displays a restricted and developmentally regulated distribution in the mouse central nervous system. Defined modules of the molecule have been shown to mediate specific functions, such as neuron migration, neurite outgrowth, cell adhesion, and cell proliferation. The smallest TN-C form contains a stretch of eight fibronectin type III (FNIII) domains, which are common to all TN-C isoforms. Unrestricted and independent alternative splicing of six consecutive FNIII cassettes between the fifth and sixth constitutive FNIII domain bears the potential to generate 64 different combinations that might code for TN-C proteins with subtly different functions. To explore TN-C isoform variability in mouse brain, the alternatively spliced region of TN-C mRNAs was examined by the reverse transcription-polymerase chain reaction technique. Polymerase chain reaction products of uniform size were subcloned and analyzed using domain-specific probes to reveal the expression of particular combinations of alternatively spliced FNIII domains. 27 TN-C isoforms were identified to be expressed in mouse central nervous system, of which 22 are novel. Furthermore, during development, specific TN-C isoforms were found to occur in distinct relative frequencies, as demonstrated for isoforms containing two alternatively spliced FNIII domains. We conclude that TN-C is expressed in a complex and regulated pattern in mouse central nervous system. These findings highlight the potential role of TN-C in mediating specific neuron glia interactions.

Up-regulation of astrocyte-derived tenascin-C correlates with neurite outgrowth in the rat dentate gyrus after unilateral entorhinal cortex lesion.

The extracellular matrix protein tenascin-C has been implicated in the regulation of axonal growth. Using unilateral entorhinal cortex lesions, which induce a massive sprouting response in the denervated outer molecular layer of the rat fascia dentata, the role of tenascin-C for axonal growth was investigated in vivo. Monoclonal antibodies against the neurite outgrowth and anti-adhesive domains of the molecule were employed. Immunostaining was increased throughout the denervated outer molecular layer by day 2, reached a maximum around day 10, and was back to control levels by four weeks post lesion. Growth cone deflecting as well as neurite outgrowth promoting isoforms of tenascin-C were up-regulated after the lesion. Using electron microscopy, single intensely tenascin-C immunoreactive cells were identified as reactive astrocytes that phagocytose degenerated terminals. In situ hybridization histochemistry for tenascin-C messenger RNA revealed numerous cellular profiles in the denervated outer molecular layer of the ipsilateral and contralateral dentate gyrus two days post lesion. Tenascin-C messenger RNA-positive cells in the outer molecular layer were identified as astrocytes using double-labelling for tenascin-C messenger RNA and glial fibrillary acidic protein immunohistochemistry. Thus, a tenascin-C-rich substrate is present in the outer molecular layer during the time of sprouting and a sharp boundary is formed against the inner molecular layer. This pattern may contribute to the layer-specific sprouting response of surviving afferents after entorhinal lesion. Neurite outgrowth may be promoted within the denervated zone, whereas axons trying to grow into the denervated outer molecular layer, for example from the inner molecular layer, would be deflected by a tenascin-C-rich barrier.

Tenascin-C synthesis and influence on axonal growth during rat cortical development.

Several putative guidance molecules are restricted to the marginal and subplate zones, the major fibre tracts in the developing cortex. It is presently unknown how their distribution is achieved and how these molecules affect neurite extension. Tenascin-C is of particular interest in this context, because it may either promote or deflect growing axons depending on its mode of presentation. Therefore, the cellular origin of tenascin-C in the developing rat cortex and its effects on the extension of cortical afferents and efferents were examined. Tenascin-C protein is first restricted to the marginal and subplate zones and spreads later into the developing grey matter, in close correlation with afferent innervation. In situ hybridization showed that tenascin-C mRNA is first confined to the ventricular zone, at some distance from the location of the protein, while at later stages tenascin-C-synthesizing cells become scattered throughout the cortical thickness, concomitant with the spread of the protein. In order to assess its function, monoclonal antibodies directed against different domains of tenascin-C were used in a quantitative axonal outgrowth assay. These perturbation experiments suggested that distinct tenascin-C fibronectin type III repeats sustain the growth of thalamic and cortical axons on cortical membrane carpets, whereas the EGF-type repeats are not involved. The combination of different antibodies revealed that separate fibronectin-type III repeats exert cooperative effects. These results suggest that ventricular zone cells regulate the establishment of thalamic and cortical axonal projections through locally restricted deposition of tenascin-C.

Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons.

The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Götz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell-binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.