부산대학교 도서관

Byline: Gonzalo S Tejeda, Sara Ayuso-Dolado, Raquel Arbeteta, Gema M Esteban-Ortega, Gema M Esteban-Ortega, Margarita Diaz-Guerra Keywords: TrkB; excitotoxicity; ischaemia; metalloproteinases; [gamma]-secretases; stroke Abstract Stroke remains a leading cause of death and disability in the world with limited therapies available to restrict brain damage or improve functional recovery after cerebral ischaemia. A promising strategy currently under investigation is the promotion of brain-derived neurotrophic factor (BDNF) signalling through tropomyosin-related kinase B (TrkB) receptors, a pathway essential for neuronal survival and function. However, TrkB and BDNF-signalling are impaired by excitotoxicity, a primary pathological process in stroke also associated with neurodegenerative diseases. Pathological imbalance of TrkB isoforms is critical in neurodegeneration and is caused by calpain processing of BDNF high affinity full-length receptor (TrkB-FL) and an inversion of the transcriptional pattern of the Ntrk2 gene, to favour expression of the truncated isoform TrkB-T1 over TrkB-FL. We report here that both TrkB-FL and neuronal TrkB-T1 also undergo ectodomain shedding by metalloproteinases activated after ischaemic injury or excitotoxic damage of cortical neurons. Subsequently, the remaining membrane-bound C-terminal fragments (CTFs) are cleaved by [gamma]-secretases within the transmembrane region, releasing their intracellular domains (ICDs) into the cytosol. Therefore, we identify TrkB-FL and TrkB-T1 as new substrates of regulated intramembrane proteolysis (RIP), a mechanism that highly contributes to TrkB-T1 regulation in ischaemia but is minor for TrkB-FL which is mainly processed by calpain. However, since the secreted TrkB ectodomain acts as a BDNF scavenger and significantly alters BDNF/TrkB signalling, the mechanism of RIP could contribute to neuronal death in excitotoxicity. These results are highly relevant since they reveal new targets for the rational design of therapies to treat stroke and other pathologies with an excitotoxic component. Copyright [c] 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. CAPTION(S): Supplementary materials and methods Figure S1. Histochemical characterization of photothrombotic focal cerebral ischaemia in mice. Five (A) and 24 h (C) after photothrombotic brain injury, vital-dye TTC staining of coronal sections (1 mm) reliably delineate the infarct (pale areas versus non-injured deep red coloured tissue). Cresyl violet (Nissl) staining of cryosections (30 A[micro]m) prepared from 5 h (B) or 24 h (D) post-injury mice brain revealed an evolving hypochromatic area in the ipsilateral neocortex (shown in detail in panels 3 and 6) indicative of neuronal injury, compared with the equivalent regions of the contralateral hemisphere (panels 1 and 4). Transition zones between damaged and non-ischaemic tissue (panels 2 and 5) highlight cell morphology alterations due to ischaemic injury. Figure S2. Time course of TrkB processing depends on the severity of focal cerebral ischaemia. (A) Immunoblot analysis of TrkB-FL processing in infarcted (I) and equivalent contralateral (C) regions of mice showing lower susceptibility to brain ischaemia (CD-1 strain) subjected to photothrombosis as before and sacrificed after the indicated times. (B) Quantification of normalized TrkB-FL levels. Results are represented relative to the data obtained for the contralateral region in sham-operated animals, arbitrarily assigned a 100% value (n = 3, mean [+ or -] SEM). Statistical significance was evaluated using Student's t-test (*p < 0.05). Figure S3. BDNF does not prevent TrkB-FL inactivation and metalloproteinase or calpain processing induced by NMDAR overactivation. Cortical cultures incubated with BDNF (100 ng/ml) and/or NMDA for the indicated periods of time were analysed by immunoblotting. Figure S4. Identification of GFP-fused CTF and ICD fragments resulting from HA-TrkB-T1-GFP RIP. Immunoblot analysis of cortical cultures infected with LV-HA-TrkB-T1-GFP (moi 1) and treated with NMDA for 6 h compared with cells infected with the same multiplicity of control virus LV-GFP. Recombinant TrkB-T1 C-terminal fragment (CTF-GFP) and intracellular domain (ICD-GFP) were identified with an anti-GFP antibody and different exposure times of the original blot are shown for each lentivirus. Figure S5. Purification of a recombinant HA-TrkB-ECD from a heterologous system. HEK293T cells transfected with plasmid HA-TrkB-T1 were treated with APMA (100 A[micro]m) for 6 h before media collection. Released HA-ECD fragment was then purified by using anti-HA conjugated agarose beads. Immunoblot with HA antibodies of collected medium and first three eluted fractions is shown.