Cell adhesion and neurite outgrowth are promoted by neurofascin NF155 and inhibited by NF186

Neurofascin (NF) is a neural cell adhesion molecule in the L1-family containing six Ig domains and multiple fibronectin type III (FnIII) repeats in its extracellular region. NF has many splicing variants and two of these are exemplars that have different cellular patterns of expression during development. NF186, which is expressed on neurons, contains an unusual mucin-like region and NF155, which is expressed on glia, contains a unique FnIII repeat with an RGD motif. Analysis of Fc fusion proteins representing different extracellular regions of NF indicate that NF186 inhibits cell adhesion and neurite outgrowth, and the inhibition is associated with the region containing the mucin-like domain. NF155 promotes neural cell adhesion and neurite outgrowth, and the RGD motif in its third FnIII repeat is critical for cell spreading and neurite outgrowth. The results suggest that different splicing variants of NF expressed on neurons and glia play distinct roles during neural development.     

Distinct effects of recombinant tenascin-C domains on neuronal cell adhesion,growth cone guidance,and neuronal polarity

Using a set of recombinantly expressed proteins, distinct domains of the mouse extracellular matrix glycoprotein tenascin-C, hereafter called tenascin, have been identified to confer adhesion, anti-adhesion, and changes in morphology of neuronal cells. In short-term adhesion assays (1 hr), cerebellar and hippocampal neurons adhered to several domains, encompassing the fibronectin type III-like (FN III) repeats 1–2 and 6–8, as well as to the alternatively spliced FN III repeats and to tenascin itself. Although no short-term adhesion to the EGF repeats containing fragment could be detected under the conditions used, it was anti-adhesive for neuronal cell bodies and repellent for growth cone advance and neuritogenesis. FN III repeats 3–5 were repellent only for growth cones but not for neuronal cell bodies. Neurite outgrowth promoting activities at early stages and induction of a polarized neuronal morphology at later stages of differentiation were associated with the EGF repeats and the FN III repeats 6–8. These observations suggest differential effects of particular domains of the tenascin molecule on distinct cellular compartments, i.e., cell body, axon and dendrite, and existence of multiple neuronal receptors with distinct intracellular signaling features. © 1996 Wiley-Liss, Inc.     

Characterization of the murine splice variant Mobp155: developmental CNS expression pattern and subcellular localization of epitope-tagged protein

Members of the myelin-associated oligodendrocytic basic protein (MOBP) family constitute the third most abundant protein in CNS myelin. Although MOBP localizes to the major dense line (MDL) of CNS myelin, the function of the individual isoforms is unknown. Alternative splicing of pre-Mobp mRNA gives rise to six characterized splice variants in both the mouse and the rat. These splice variants share a common N-terminal encoded in Mobp exon 3 comprising 68 amino acids. The predicted protein isoforms differ in their C-termini. Sequence analysis of intron 3 revealed the presence of a putative initiation codon followed by an open reading frame (ORF) encoding 53 amino acids that extends in frame into Mobp exon 4 yielding a predicted MOBP isoform comprising 155 amino acids, designated MOBP155. This newly characterized isoform possessing a novel N-terminus shares a common C-terminus with MOBP170. Mobp170 message is detectable at low abundance throughout myelinogenesis. In contrast, the novel splice variant encoding MOBP155 is expressed at modest levels late in CNS development, coincident with the expression of the abundant splice variant, Mobp81A. Immunostaining of Cos7 cells transiently expressing an epitope-tagged MOBP155 suggested that most of the product was translocated to mitochondria. Although Mobp155 and Mobp170 encode a common predicted C-terminus they have different expression profiles and their products are targeted to mitochondria and the nucleus, respectively, in transiently transfected Cos7 cells.     

zfNLRR, a novel leucine-rich repeat protein is preferentially expressed during regeneration in zebrafish.

zfNLRR is a novel transmembrane protein that is most prominently expressed during regeneration of the zebrafish central nervous system. Retinal ganglion cells and descending spinal cord neurons strongly increase zfNLRR mRNA levels after axotomy in the adult. In contrast, during development expression is hardly detectable and is restricted to a few sensory systems. In the adult brain, zfNLRR mRNA is found at low levels in several motor and premotor systems. Sequence analysis reveals that zfNLRR contains in its extracellular region 12 leucine-rich repeats, 1 immunoglobulin-like domain and 1 fibronectin type III-like domain. The same protein binding motifs were identified in transmembrane proteins from frog, mouse, and human. Together, they constitute a novel family of vertebrate neuronal leucine-rich repeat proteins. Three distinct isoforms are identified so far. On the basis of its structural features and expression pattern, we propose that zfNLRR functions as a neuronal-specific adhesion molecule or soluble ligand binding receptor, primarily during restoration of the nervous system after injury.     

Differential activities in adhesion and neurite growth of fibronectin type III repeats in the PTP-δ extracellular domain

The full-length extracellular domain (ECD) of protein tyrosine phosphatase delta (PTP-delta) functions as a ligand to promote cell adhesion and neurite outgrowth; this ECD contains three immunoglobulin (Ig) repeats and eight fibronectin type III (FN III) repeats. However, it is not known which regions of the ECD regulate its ligand functions. Therefore, we constructed and expressed a fusion protein of the PTP-delta ECD lacking FN III repeats 4-8, and tested this protein for neuronal adhesion and neurite-promoting ability. Compared to the full-length isoform, the truncated ECD was poorer at promoting adhesion, but a more potent promoter of neurite growth. The results suggest that distal FN III repeats of PTP-delta are important in adhesive functions, but dispensable for neurite outgrowth promotion. As the predominant isoform of PTP-delta during neural development (type D) also lacks distal FN III repeats, the functional properties we observe may be relevant to periods of axon extension, suggesting that splice variants of receptor PTPs play distinct roles in neural development.     

Different Domains of the F3 Neuronal Adhesion Molecule are Involved in Adhesion and Neurite Outgrowth Promotion

The mouse F3 cell surface protein is preferentially expressed on axons of subpopulations of neurons and is anchored to the membrane by a glycosyl-phosphatidylinositol group. It consists of six immunoglobulin-like domains and four fibronectin type III homologous repeats, and can be found both in membrane-anchored and soluble forms. We have previously established that F3 fulfils the operational criteria of a cell adhesion molecule when anchored to the plasma membrane and that its soluble form stimulates neurite initiation and neurite outgrowth. To further characterize F3-mediated adhesion and to investigate whether adhesion and neurite outgrowth promoting activities are displayed by different parts of the molecule, we (i) selected F3 transfected CHO cells expressing increasing levels of F3 at their surface and (ii) prepared transfectants expressing an F3 molecule with its fibronectin type III repeats deleted. We show that the F3 molecule mediates divalent-cation-independent, temperature-dependent binding. The levels of aggregation of F3 transfectants are proportional to the level of F3 expression. Transfectants expressing F3 deleted of the fibronectin type Ill repeats lose their adhesive properties; conversely, cells expressing wild-type F3 and treated with collagenase, specifically removing the immunoglobulin-like domains, are still able to aggregate. Therefore, in this model adhesion site(s) mapped to the fibronectin type III repeats. By contrast, transfectants expressing deleted F3, as well as the soluble forms of this F3 deleted molecule, were able to stimulate neurite outgrowth of sensory neurons similarly to wild-type F3. Our data indicate that F3 is a multifunctional molecule and that adhesion and neurite outgrowth promoting properties are expressed by distinct and independent domains.     

PSF suppresses tau exon 10 inclusion by interacting with a stem-loop structure downstream of exon 10

Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA–protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5′ splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.     

Retinal lesions induce differential changes in the expression of flip and flop isoforms of the glutamate receptor subunit GluR1 in the chick optic tectum

A sensitive RNase protection assay was employed to determine the levels of mRNA encoding the GluR1 subunit flip and flop isoforms in the chick optic tectum and forebrain. We found that the flip GluR1 mRNA predominates in the forebrain, whereas the flop variant is more strongly expressed in the optic tectum. A temporal analysis of GluR1 variants in the embryonic and adult chick brain revealed that the flip isoform is more highly expressed at E12 than at P15-21, whereas mRNA levels of the flop isoform are higher at P15-21 than at E12. To study the effect of deafferentation on GluR1 expression, unilateral retinal lesions were performed. Two days later the mRNA levels of GluR1 flip and flop variants were decreased in the deafferented tectum, especially for the flop isoform. However, 7 days after the lesion, the mRNA levels of both GluR1 isoforms were increased, especially for the flip isoform. These results reveal an important control of the retinal input upon the expression of the different GluR1 isoforms. Furthermore, they indicate a differential spatial and temporal regulation of the flip and flop splice variants, suggesting the existence of a mechanism regulating differential splicing or possibly differential RNA stability.     

Alternative splicing of amino-terminal Tau mRNA in rat spinal cord during development and following axonal injury

Tau is a family of microtubule-associated phosphoproteins in which isoform variation is produced by alternative splicing of a single gene and posttranslational modifications. Tau isoforms that include exon 10 are overexpressed in frontotemporal dementia and progressive supranuclear palsy. Therefore, we examined the expression of tau mRNA splice variants during axonal regeneration and abortive regeneration. Previous work in our laboratory demonstrated that expression of exon 10 tau isoforms during regeneration and abortive regeneration was altered and partially recapitulated the developmental patterns of tau isoform expression. Using RT-PCR, we examined the alternative splicing of exons 2 and 3 in tau during early postnatal development and regeneration in the rat spinal cord. The levels of tau lacking exons 2 and 3 were high on the day of birth and rapidly declined. Conversely, tau isoforms containing exon 2 or exons 2 and 3 first appeared at low levels and steadily increased. During axonal regeneration, the levels of all three tau mRNA isoforms were significantly lower 7 days after injury. In a model of abortive regeneration, all of the tau isoforms were elevated 14 and 42 days postinjury. The relative levels of exon 2 and 3 tau splice variants were not altered during regeneration or abortive regeneration as occurred during development. These results suggest that tau isoform expression following neuronal injury does not recapitulate the developmental pattern and is not independently regulated as in development. Our previous results together with these data suggest that alterations in tau mRNA isoform expression that occur in neurodegeneration are not secondary to axonal injury but may be a more primary event underlying cytoskeletal derangement.     

Molecular characterization of microtubule-associated proteins tau and MAP2.

Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule-binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms.     

Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR

N-methyl-D-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943-954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306-313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180-3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1-16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31-108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies.     

Influence of janusin and tenascin on growth cone behavior in vitro

Janusin and tenascin are glia-derived, structurally related, extracellular matrix glycoproteins of the J1 family that are expressed in vivo at times and in locations where active neurite outgrowth occurs, but also when the formation or stabilization of cytoarchitectonic boundaries appears to be in operation. To resolve this apparent functional dichotomy, we have studied the behavioral response of growth cones, growing in culture on the permissive substrate laminin, to janusin and tenascin, by video time lapse microscopy. When janusin and tenascin were offered as sharp substrate boundaries, dorsal root ganglion (DRG) and retinal ganglion neuron growth cones avoided growing on these molecules, but were not induced to collapse. On the other hand, when janusin and tenascin were offered, in a mixture with laminin, as uniform substrates, DRG growth cones displayed a collapsed morphology and were able to advance at a faster rate than on laminin alone. In contrast, the outgrowth of retinal ganglion neuron growth cones was completely inhibited under these conditions, underscoring a cell type specificity in the response of growth cones to these molecules. Using several monoclonal antibodies binding to distinct epitopes on the tenascin molecule, we have identified two domains responsible for growth cone repulsion, on epidermal growth factor (EGF)-like repeats 3-5 and fibronectin type III homologous repeats 4 and 5. These domains are different from the one previously recognized to be involved in neurite outgrowth on a uniform tenascin substrate. We conclude that both molecules may promote or retard growth cone advance, depending on the spatial expression pattern and the neuronal cell type. © 1993 Wiley-Liss, Inc.     

Isolating and characterizing three alternatively spliced mu opioid receptor variants: mMOR‐1A,mMOR‐1O,and mMOR‐1P

Extensive alternative pre‐mRNA splicing of the mu opioid receptor gene, OPRM1, has demonstrated an array of splice variants in mice, rats and humans. Three classes of splice variants have been identified: full‐length seven transmembrane (TM) domain variants with C‐terminal splicing, truncated 6TM variants and single TM variants. The current studies isolates and characterizes an additional three full‐length C‐terminal splice variants generated from the mouse OPRM1 gene: mMOR‐1A, mMOR‐1O, and mMOR‐1P. Using RT‐qPCR, we demonstrated differential expression of these variants' mRNAs among selected brain regions, supporting region‐specific alternative splicing. When expressed in Chinese Hamster Ovary cells, all the variants displayed high mu binding affinity and selectivity with subtle differences in the affinities toward some agonists. [³⁵S]γGTP binding assays revealed marked differences in agonist‐induced G protein activation in both potency and efficacy among the variants. Together with the previous studies of mu agonist‐induced phosphorylation and internalization in several carboxyl terminal splice variants, the current studies further suggest the existence of biased signaling of various agonists within each individual variant and/or among different variants. Synapse 68:144–152, 2014 . © 2013 Wiley Periodicals, Inc.