Abstract
Teneurins are type II transmembrane proteins comprised of four phylogenetically conserved homologs (Ten-1-4) that are highly expressed during neurogenesis. An additional bioactive peptide named teneurin C-terminal-associated peptide (TCAP-1-4) is present at the carboxyl terminal of teneurins. The possible correlation between the Ten/TCAP system and brain injuries has not been explored yet. Thus, this study examined the expression of these proteins in the cerebral cortex after mechanical brain injury. Adult rats were subjected to cerebral cortex injury by needle-insertion lesion and sacrificed at various time points. This was followed by analysis of the lesion area by immunohistochemistry and conventional RT-PCR techniques. Control animals (no brain injury) showed only discrete Ten-2-like immunoreactive pyramidal neurons in the cerebral cortex. In contrast, Ten-2 immunoreactivity was significantly up-regulated in the reactive astrocytes in all brain-injured groups (p < 0.0001) when compared to the control group. Interestingly, reactive astrocytes also showed intense immunoreactivity to LPHN-1, an endogenous receptor for the Ten-2 splice variant named Lasso. Semi-quantitative analysis of Ten-2 and TCAP-2 expression revealed significant increases of both at 48 h, 3 days and 5 days (p < 0.0001) after brain injury compared to the remaining groups. Immortalized cerebellar astrocytes were also evaluated for Ten/TCAP expression and intracellular calcium signaling by fluorescence microscopy after TCAP-1 treatment. Immortalized astrocytes expressed additional Ten/TCAP homologs and exhibited significant increases in intracellular calcium concentrations after TCAP-1 treatment. This study is the first to demonstrate that Ten-2/TCAP-2 and LPHN-1 are upregulated in reactive astrocytes after a mechanical brain injury. Immortalized cerebellar astrocytes expressed Ten/TCAP homologs and TCAP-1 treatment stimulated intracellular calcium signaling. These findings disclose a new functional role of the Ten/TCAP system in astrocytes during tissue repair of the CNS.
Highlights
Teneurins are type II transmembrane glycoproteins composed of four paralogues (Ten-1-4), mainly expressed during central nervous system (CNS) development (Baumgartner and ChiquetEhrismann, 1993; Baumgartner et al, 1994; Levine et al, 1994; Rubin et al, 1999; Tucker and Chiquet-Ehrismann, 2006)
Our in vivo immunohistochemistry data strongly suggest that reactive astrocytes are synthetizing Ten-2 and its up-regulation was confirmed by significant increase of Ten-2 messenger ribonucleic acid (RNA) (mRNA) in the cerebral cortex of animals with mechanical brain injury
Based on the limitations of the present study, a significant increase in Ten-2-LI reactive astrocytes was demonstrated for the first time after mechanical injury of the adult rat cerebral cortex
Summary
Teneurins are type II transmembrane glycoproteins composed of four paralogues (Ten-1-4), mainly expressed during central nervous system (CNS) development (Baumgartner and ChiquetEhrismann, 1993; Baumgartner et al, 1994; Levine et al, 1994; Rubin et al, 1999; Tucker and Chiquet-Ehrismann, 2006). The transmembrane and C-terminal extracellular domains comprise 34 and 2400 amino acids, respectively (Bagutti et al, 2003; Nunes et al, 2005; Tucker and Chiquet-Ehrismann, 2006). Latrophilins are constituted by three isoforms (LPHN-1-3), known as Abbreviations: Ab1, primary antibody; Ab1(LPHN-1), LPHN-1 primary antibody omission; Ab1(Ten-2) Ten-2 primary antibody omission; aCSF, artificial cerebrospinal fluid; ADS, adsorption; AM, fluo-4 acetomethyl; ANOVA, analysis of variance; BBB, blood-brain barrier; BDNF, brain-derived neurotrophic factor; Ca2+, unbound calcium; CaCl22H2O, calcium chloride dihydrate; CCD, camera-coupled device; cDNA, complementary DNA; CEUA, Institutional Committee of Animal Welfare; CNS, central nervous system; CRF, corticotrophin-releasing factor; Cy3, cyanine; DAB, diaminobenzidine; DAPI, 4’,6-diamidino-2-phenylindole; DGC, dystrophin-dystroglycan complex; ddH2O, double-distilled water; DEPC, diethyl pyrocarbonate; DMEM, Dulbecco’s modified eagle’s medium; DMSO,dimethyl sulfoxide; DNA, deoxyribonucleic acid; DNase, deoxyribonuclease; dNTP, deoxynucleotides; DOC2, double C2-like domain-containing protein; DTT, DL-Dithiothreitol; EtOH, ethanol; FBS, fetal bovine serum; FGF8, fibroblast growth factor; FITC, fluorescein isothiocyanate; GFAP-LI, glial fibrillary acidic protein-like immunoreactive; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; KCl, potassium chloride; Kg, kilograms; LPHN, latrophilin; -LPHN-1, absence of LPHN-1 antibody/ LPHN-1-LI, latrophilin-1-like immunoreactive; LPHN-2-LI, latrophilin-2-like immunoreactive; LPHN-3-LI, latrophilin-3-like immunoreactive; μg, microgram; μL, microliter; MAPK, mitogen-activated protein kinase; mL, milliliter; mg, milligram; MgCl2, magnesium chloride; MgCl26H2O, magnesium chloride hexahydrate; Mm, millimol; mRNA, messenger RNA; n, number; NaCl, sodium chloride; NFL, neurofilament light; nM, nanomol; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline and triton X-100; PCR, polymerase chain reaction; RCF, relative centrifugal force; RNA, ribonucleic acid; RNase, ribonuclease; rpm, revolutions per minute; RT-PCR, reverse transcription polymerase chain reaction; S100B, S100 calcium-binding protein B; SEM, standard error of the mean; TCAP, teneurin C-terminal-associated peptides; TCAP-1, teneurin C-terminal-associated peptide 1; TCAP-2, teneurin C-terminalassociated peptide 2; TCAP-3, teneurin C-terminal-associated peptide 3; TCAP-4, teneurin C-terminal-associated peptide 4;Ten-1, teneurin-1; Ten-2, teneurin-2; Ten-3, teneurin-3; Ten-4, teneurin-4; Ten-2-LI, teneurin-2-like immunoreactive; Uv, ultraviolet
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