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• W2079388718 abstract "Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.Background: LRP1 activation is neuroprotective in vitro. The role of LRP1 in axonal plasticity and regeneration is unknown.Results: LRP1-dependent cell signaling that includes TrkC activation promotes axonal growth in the CNS.Conclusion: LRP1 agonists promote regeneration after spinal cord injury.Significance: A significant role is established for LRP1 in axonal growth and regeneration after CNS injury, identifying a novel class of therapeutic targets for neurological disorders. Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders. Background: LRP1 activation is neuroprotective in vitro. The role of LRP1 in axonal plasticity and regeneration is unknown. Results: LRP1-dependent cell signaling that includes TrkC activation promotes axonal growth in the CNS. Conclusion: LRP1 agonists promote regeneration after spinal cord injury. Significance: A significant role is established for LRP1 in axonal growth and regeneration after CNS injury, identifying a novel class of therapeutic targets for neurological disorders. The LDL receptor-related protein 1 (LRP1) is a large (600-kDa) type I membrane receptor and is a member of the LDL receptor superfamily (1Strickland D.K. Gonias S.L. Argraves W.S. Diverse roles for the LDL receptor family.Trends Endocrinol. Metab. 2002; 13: 66-74Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Although originally known to function as an endocytic receptor for apolipoprotein E (2Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 5810-5814Crossref PubMed Scopus (456) Google Scholar) and amyloid Aβ (3Holtzman D.M. Herz J. Bu G. Apolipoprotein E and apolipoprotein E receptors. Normal biology and roles in Alzheimer disease.Cold Spring Harbor Perspect. Med. 2012; 2: a006312Crossref PubMed Scopus (534) Google Scholar), LRP1 is currently recognized as an endocytic and cell signaling receptor for diverse ligands, including tissue-type plasminogen activator (tPA), matrix metalloproteinase 9, and activated α2-macroglobulin (4Herz J. Strickland D.K. LRP. A multifunctional scavenger and signaling receptor.J. Clin. Invest. 2001; 108: 779-784Crossref PubMed Scopus (882) Google Scholar, 5Hu K. Yang J. Tanaka S. Gonias S.L. Mars W.M. Liu Y. Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression.J. Biol. Chem. 2006; 281: 2120-2127Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 6Hayashi H. Campenot R.B. Vance D.E. Vance J.E. Apolipoprotein E-containing lipoproteins protect neurons from apoptosis via a signaling pathway involving low-density lipoprotein receptor-related protein-1.J. 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Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein. Role of the intracellular adapter protein Fe65.J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar, 10Su H.P. Nakada-Tsukui K. Tosello-Trampont A.C. Li Y. Bu G. Henson P.M. Ravichandran K.S. Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).J. Biol. Chem. 2002; 277: 11772-11779Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In Schwann cells, LRP1 activates PI3K/Akt-mediated cell survival signaling pathways and counteracts endoplasmic reticulum stress (11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar, 12Mantuano E. Henry K. Yamauchi T. Hiramatsu N. Yamauchi K. Orita S. The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury.J. Neurosci. 2011; 31: 13376-13385Crossref PubMed Scopus (42) Google Scholar). LRP1 also interacts with other receptors, including integrins (13Salicioni A.M. Gaultier A. Brownlee C. Cheezum M.K. Gonias S.L. Low density lipoprotein receptor-related protein-1 promotes β1 integrin maturation and transport to the cell surface.J. Biol. Chem. 2004; 279: 10005-10012Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), platelet-derived growth factor receptors (14Muratoglu S.C. Mikhailenko I. Newton C. Migliorini M. Strickland D.K. Low density lipoprotein receptor-related protein 1 (LRP1) forms a signaling complex with platelet-derived growth factor receptor-β in endosomes and regulates activation of the MAPK pathway.J. Biol. Chem. 2010; 285: 14308-14317Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and urokinase-type plasminogen activator receptor (15Gonias S.L. Gaultier A. Jo M. Regulation of the urokinase receptor (uPAR) by LDL receptor-related protein-1 (LRP1).Curr. Pharm. Des. 2011; 17: 1962-1969Crossref PubMed Scopus (33) Google Scholar). As a result, LRP1 functions in a diversity of cellular activities, including cell signaling and metabolism, migration, and blood-brain barrier integrity (16May P. Woldt E. Matz R.L. Boucher P. The LDL receptor-related protein (LRP) family. An old family of proteins with new physiological functions.Ann. Med. 2007; 39: 219-228Crossref PubMed Scopus (179) Google Scholar, 17Lillis A.P. Van Duyn L.B. Murphy-Ullrich J.E. Strickland D.K. LDL receptor-related protein 1. Unique tissue-specific functions revealed by selective gene knockout studies.Physiol. Rev. 2008; 88: 887-918Crossref PubMed Scopus (513) Google Scholar). LRP1 is widely expressed in uninjured central and peripheral neurons (18Wolf B.B. Lopes M.B. VandenBerg S.R. Gonias S.L. Characterization and immunohistochemical localization of α-2-macroglobulin receptor (low density lipoprotein receptor related protein) in human brain.Am. J. Pathol. 1992; 141: 37-42PubMed Google Scholar, 11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar). Recent reports have suggested a potential role for LRP1 in neural function and growth. LRP1 acts as a prosurvival and migratory receptor in Schwann cells after peripheral nerve injury (11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar, 19Mantuano E. Jo M. Gonias S.L. Campana W.M. Low density lipoprotein receptor-related protein (LRP1) regulates Rac1 and RhoA reciprocally to control Schwann cell adhesion and migration.J. Biol. Chem. 2010; 285: 14259-14266Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), transduces a signal for phagocytosis of degraded myelin in experimental models of multiple sclerosis (20Gaultier A. Wu X. Le Moan N. Takimoto S. Mukandala G. Akassoglou K. Campana W.M. Gonias S.L. Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis.J. Cell Sci. 2009; 122: 1155-1162Crossref PubMed Scopus (73) Google Scholar), and modulates processing of the amyloid precursor protein (20Gaultier A. Wu X. Le Moan N. Takimoto S. Mukandala G. Akassoglou K. Campana W.M. Gonias S.L. Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis.J. Cell Sci. 2009; 122: 1155-1162Crossref PubMed Scopus (73) Google Scholar, 21Goto J.J. Tanzi R.E. The role of the low-density lipoprotein receptor-related protein (LRP1) in Alzheimer's A β generation: development of a cell-based model system.J. Molec. Neurosci. 2002; 19: 37-41Crossref PubMed Scopus (33) Google Scholar, 22Liu Q. Zerbinatti C.V. Zhang J. Hoe H.S. Wang B. Cole S.L. Herz J. Muglia L. Bu G. Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1.Neuron. 2007; 56: 66-78Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3Holtzman D.M. Herz J. Bu G. Apolipoprotein E and apolipoprotein E receptors. Normal biology and roles in Alzheimer disease.Cold Spring Harbor Perspect. Med. 2012; 2: a006312Crossref PubMed Scopus (534) Google Scholar). Recently, LRP1 agonists were also reported to induce transactivation of TrkA in a Src family kinase (SFK) 3The abbreviations used are: SFK, Src family kinase; DRG, dorsal root ganglion; NF200, neurofilament 200; RBD, receptor binding domain; PEX, hemopexin domain; RAP, receptor-associated protein; pERK, phospho-ERK; pTrkC, phospho-TrkC; CTB, cholera toxin subunit B; NT-3, neurotrophin-3; PP2, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine; GFAP, glial fibrillary acidic protein. -dependent manner, promoting neurite outgrowth from PC12 cells (23Shi Y. Mantuano E. Inoue G. Campana W.M. Gonias S.L. Ligand binding to LRP1 transactivates Trk receptors by a Src family kinase-dependent pathway.Sci. Signal. 2009; 2: ra18Crossref PubMed Scopus (103) Google Scholar). The latter findings identify a candidate mechanism for potential actions of LRP1 on axonal function and suggest the hypothesis that LRP1 activation could promote axonal sprouting and regeneration in the adult CNS. Synthetic LRP1 ligands have been generated, including several fusion proteins that can be readily infused into the CNS to test potential activities in injury models. We now report potent effects of LRP1-binding ligands on axonal growth in vitro and after spinal cord injury in vivo, identifying a novel LRP1-dependent cell signaling mechanism involved in CNS plasticity and regeneration. These effects result from canonical LRP1 ligand transactivation of Trk. In vitro experiments determined whether LRP1 agonists promote neurite outgrowth from cultures of dissociated primary adult dorsal root ganglion (DRG) neurons and on signaling mechanisms activated by LRP1 receptor signaling. A second set of “ex vivo” experiments determined whether intrathecal infusion of LRP1 agonists for 3 days would also stimulate growth-related neuronal signaling and neurite outgrowth in DRG neurons that were subsequently removed and cultured in vitro. Finally, in vivo experiments assessed the ability of intrathecal infusions of LRP1 agonists to promote sprouting or regeneration of the central processes of DRG neurons projecting through the dorsal columns after C4 dorsal spinal cord lesions. All experiments examined the same population of large-diameter, neurofilament 200 (NF200)-expressing, NT-3-sensitive DRG neurons. GST fusion proteins that directly bind LRP1 and are LRP1 agonists were prepared as described previously (24Mettenburg J.M. Webb D.J. Gonias S.L. Distinct binding sites in the structure of α 2-macroglobulin mediate the interaction with β-amyloid peptide and growth factors.J. Biol. Chem. 2002; 277: 13338-13345Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 25Mantuano E. Mukandala G. Li X. Campana W.M. Gonias S.L. Molecular dissection of the human α2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling.J. Biol. Chem. 2008; 283: 19904-19911Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Purified GST and GST fusion proteins were subjected to chromatography on Detoxi-Gel endotoxin-removing columns (Pierce) and endotoxin dialysis decontamination (8Mantuano E. Inoue G. Li X. Takahashi K. Gaultier A. Gonias S.L. The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of Schwann cells by binding to low-density lipoprotein receptor-related protein.J. Neurosci. 2008; 28: 11571-11582Crossref PubMed Scopus (135) Google Scholar). LRP1 agonists include an 18-kDa receptor binding domain of α2-macroglobulin (α2M), henceforth referred to as RBD (25Mantuano E. Mukandala G. Li X. Campana W.M. Gonias S.L. Molecular dissection of the human α2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling.J. Biol. Chem. 2008; 283: 19904-19911Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. Selective mutations in cloned and expressed α-macroglobulin receptor binding fragment alter binding to either the α2-macroglobulin signaling receptor or the low density lipoprotein receptor-related protein/α2-macroglobulin receptor.J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. Identification of residues in alpha-macroglobulins important for binding to the α2-macroglobulin receptor/low density lipoprotein receptor-related protein.J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and the 18-kDa LRP1 binding domain of matrix metalloproteinase 9, the hemopexin domain, henceforth referred to as PEX (28Van den Steen P.E. Van Aelst I. Hvidberg V. Piccard H. Fiten P. Jacobsen C. Moestrup S.K. Fry S. Royle L. Wormald M.R. Wallis R. Rudd P.M. Dwek R.A. Opdenakker G. The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors.J. Biol. Chem. 2006; 281: 18626-18637Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 8Mantuano E. Inoue G. Li X. Takahashi K. Gaultier A. Gonias S.L. The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of Schwann cells by binding to low-density lipoprotein receptor-related protein.J. Neurosci. 2008; 28: 11571-11582Crossref PubMed Scopus (135) Google Scholar). The LRP1 antagonist, receptor-associated protein (RAP), and GST controls were prepared as described previously (27Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. Identification of residues in alpha-macroglobulins important for binding to the α2-macroglobulin receptor/low density lipoprotein receptor-related protein.J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 29Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/α 2-macroglobulin receptor.J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). The resulting protein preparations yielded clearly defined bands with the correct molecular masses when assessed by Coomassie Blue staining of SDS gels or immunoblot analysis with GST-specific antibody. LRP1-dependent cell signaling was confirmed by inhibition of pERK with RAP. The stability of fusion proteins over time at 37 °C (for subsequent in vivo infusions) was assessed by incubating 100 nm RBD or GST in PBS at 37 °C for 0, 24, 48, 72, and 96 h, adding to PC12 cells for 10 min, and examining ERK activation, as described below. Primary cultures of adult F344 L4-L6 DRG neurons were cultured on poly-L-lysine-coated (16.6 μg/ml) 12-well plates as described previously (30Blesch A. Lu P. Tsukada S. Alto L.T. Roet K. Coppola G. Geschwind D. Tuszynski M.H. Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects.Exp. Neurol. 2012; 235: 162-173Crossref PubMed Scopus (79) Google Scholar). Cells were treated with 100 nm RBD, PEX, or GST for 18 or 24 h. In some assays, cells were pretreated with GST-RAP (200 nm) 30 min prior to the addition of LRP1 agonists. Some conditions included plating of cells on inhibitory myelin substrates, as described below. Myelin-coated plates were prepared by extracting myelin from rat spinal cord, diluting in water at 10 μg/well, drying overnight at room temperature, and washing with DMEM/F-12 the next day before use. Cells were fixed and labeled for NF200, a specific marker of neurites emerging from the NT-3-responsive subpopulation of DRG neurons (31Lawson S.N. Waddell P.J. Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.J. Physiol. 1991; 435: 41-63Crossref PubMed Scopus (358) Google Scholar, 32McMahon S.B. Armanini M.P. Ling L.H. Phillips H.S. Expression and coexpression of Trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets.Neuron. 1994; 12: 1161-1171Abstract Full Text PDF PubMed Scopus (495) Google Scholar) The in vitro portion of our studies focused on the NT-3-responsive population of DRG neurons because they are the subject of the subsequent spinal cord injury model in vivo. Images were acquired manually at ×100 magnification, and longest neurite length per cell was measured in a minimum of 80–100 neurons/well using ImageJ. Quantification was performed in a blinded manner. Experiments were replicated twice with internal triplicates. Immunolabeling was performed using anti-NF200 (1:4000, Millipore). Dual labeling of NF200 and LRP1 was performed in frozen DRG sections using a polyclonal LRP1 antibody (1:1000, Sigma) (11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar). Adult F344 female rats weighing 150–200 g (n = 28 rats) were anesthetized with a mixture (2 ml/kg) of ketamine (25 mg/ml), rompun (1.8 mg/ml), and acepromazine (0.25 mg/ml). National Institutes of Health and Institutional Animal Use and Safety Committee guidelines for laboratory animal care and safety were strictly followed for all animal use and post-operation care. Alzet osmotic minipumps (model 1003) were implanted in the spinal cord at the L4–5 level for intrathecal delivery of GST or RBD (5 μm, 1 μl/h) for 3 days. After infusions, DRGs were rapidly isolated, lysed in radioimmune precipitation assay buffer for subsequent immunoblot analysis, or cultured for 18 h, fixed with 4% PFA, stained for NF200, imaged, and quantitated as described above. L4-L6 rat DRGs were harvested and plated on poly-L-lysine-coated 6-well plates. The cells were treated with 200 nm RBD, PEX, GST, or NT-3 (positive control, Abcam, 20 nm) or vehicle (0.01% Brij35 in PBS) for 30 min in neural basal medium. In some cases, cells were pretreated with the Trk activation inhibitor k252a (10 nm) or with an SFK inhibitor, PP2 (1 μm), 30 min prior to the addition of LRP1 agonists. Cell extracts were prepared using a total protein extraction kit (Millipore). An equivalent amount of cellular protein (20–50 μg/lane) was subjected to SDS-PAGE, immunoblotting, and densitometry, as described previously (11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar, 12Mantuano E. Henry K. Yamauchi T. Hiramatsu N. Yamauchi K. Orita S. The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury.J. Neurosci. 2011; 31: 13376-13385Crossref PubMed Scopus (42) Google Scholar). Primary antibodies used included pTrkC (1:1000, Syd Laboratories), tTrkC (Abcam), pERK or total ERK (1:1000, Cell Signaling Technology). Experiments were replicated five to 11 times. Blots were scanned (Cannoscan), and densitometry was performed using Image J, as previously described (8Mantuano E. Inoue G. Li X. Takahashi K. Gaultier A. Gonias S.L. The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of Schwann cells by binding to low-density lipoprotein receptor-related protein.J. Neurosci. 2008; 28: 11571-11582Crossref PubMed Scopus (135) Google Scholar, 11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar). Dissociated DRG neurons were plated on poly-L-lysine-coated coverslips and treated with 100 nm PEX, GST, NT-3 (positive control, 20 nm), or vehicle (0.01% Brij35 in PBS) for 30 min in neural basal medium. In some cases, cells were pretreated with an SFK inhibitor, PP2 (1 μm), 30 min prior to the addition of the LRP1 agonist and then fixed with 4% paraformaldehyde. Dual-label immunofluorescence was performed as described (11Campana W.M. Li X. Dragojlovic N. Janes J. Gaultier A. Gonias S.L. The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells. Possible implications in peripheral nerve injury.J. Neurosci. 2006; 26: 11197-11207Crossref PubMed Scopus (77) Google Scholar). Briefly, cells were incubated with primary antibodies to pTrk (1:500 Tyr-490, Santa Cruz Biotechnology) or pERK (1:1000, Cell Signaling Technology) and with the appropriate fluorescent antibodies (Alexa Fluor 488- or 594-conjugated antibodies). A second primary antibody, NF200 (1:4000; Millipore) and secondary antibody were added. Preparations were mounted on slides using Pro-Long Gold with DAPI for nuclear labeling (Invitrogen). Images were captured on an Olympus Fluoview1000 confocal microscope and quantified using Veloctiy three-dimensional image analysis software (PerkinElmer Life Sciences). A total of 80–110 neurons were counted from four independent experiments. 17 adult female Fisher 344 rats weighing 150–200 g were deeply anesthetized as described above under animal protocols approved by the Institutional Animal Welfare Committee. Alzet osmotic minipumps were implanted in the spinal cord at the L4–5 level for intrathecal delivery of either 5 μm GST (n = 8 animals) or RBD (n = 9 animals). Three days later, C4 dorsal column spinal cord lesions were placed using a Scouten tungsten wire knife (33Weidner N. Ner A. Salimi N. Tuszynski M.H. Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 3513-3518Crossref PubMed Scopus (448) Google Scholar). The lesion site was filled with syngeneic bone marrow stromal cells to provide a matrix in the lesion site to which injured axons could attach and grow (34Alto L.T. Havton L.A. Conner J.M. Hollis 2nd, E.R. Blesch A. Tuszynski M.H. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.Nat. Neurosci. 2009; 12: 1106-1113Crossref PubMed Scopus (176) Google Scholar). Rat marrow stromal cells were prepared as described previously (35Hofstetter C.P. Schwarz E.J. Hess D. Widenfalk J. El Manira A. Prockop D.J. Olson L. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2199-2204Crossref PubMed Scopus (891) Google Scholar, 36Kadoya K. Tsukada S. Lu P. Coppola G. Geschwind D. Filbin M.T. Blesch A. Tuszynski M.H. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.Neuron. 2009; 64: 165-172Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and transplanted into the lesion site using glass pipettes and a picospritzer. Pumps were replaced at the same time with fresh RBD. Dorsal column sensory axons were labeled transganglionically by cholera toxin B subunit (CTB) injection into the sciatic nerve (2 μl of 1% solution/sciatic nerve) 3 days before perfusion (36Kadoya K. Tsukada S. Lu P. Coppola G. Geschwind D. Filbin M.T. Blesch A. Tuszynski M.H. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.Neuron. 2009; 64: 165-172Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 37Lu P. Yang H. Jones L.L. Filbin M.T. Tuszynski M.H. Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury.J. Neurosci. 2004; 24: 6402-6409Crossref PubMed Scopus (324) Google Scholar, 38Taylor L. Jones L. Tuszynski M.H. Blesch A. Neurotrophin-3 gradients established by lentiviral gene delivery promote short-distance axonal bridging beyond cellular grafts in the injured spinal cord.J. Neurosci. 2006; 26: 9713-9721Crossref PubMed Scopus (154) Google Scholar). Four weeks after spinal cord lesions, animals were transcardially perfused with 4% paraformaldehyde, post-fixed overnight, and cryoprotected in 30% sucrose at 4 °C. Spinal cords were sectioned sagittally on a cryostat set to 30-μm intervals. All sections were processed free-floating. CTB-labeled sensory axons were visualized as described previously (36Kadoya K. Tsukada S. Lu P. Coppola G. Geschwind D. Filbin M.T. Blesch A. Tuszynski M.H. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.Neuron. 2009; 64: 165-172Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). GFAP was detected subsequently in the same sections by fluorescence labeling. We quantified the mean number of dorsal column sensory axons penetrating the lesion site, the total length of axons penetrating the lesion site, and the mean number of axons reaching the midportion of the lesion site using serial 30-μm-thick sections (a series of one-in-seven) labeled for CTB and GFAP. The number of CTB-labeled axons encountered at a virtual line drawn in the midportion of the graft, identified by GFAP labeling (Fig. 4A), was counted using a ×10 ocular with a calibrated" @default.
• W2079388718 created "2016-06-24" @default.
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• W2079388718 creator A5019172846 @default.
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• W2079388718 date "2013-09-01" @default.
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• W2079388718 title "Low-density Lipoprotein Receptor-related Protein 1 (LRP1)-dependent Cell Signaling Promotes Axonal Regeneration" @default.
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