title: Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells.
abstract: BACKGROUND
  Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.
  
  
  RESULTS
  Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.
  
  
  CONCLUSIONS
  The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.
authors.affiliation: Institute for Biochemistry and Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Congcong
authors.initials: C
authors.lastname: Zhang
authors.forename: Saskia
authors.initials: S
authors.lastname: Helmsing
authors.forename: Marta
authors.initials: M
authors.lastname: Zagrebelsky
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
authors.forename: Andrea L J
authors.initials: AL
authors.lastname: Marschall
authors.forename: Manuela
authors.initials: M
authors.lastname: Schüngel
authors.forename: Martin
authors.initials: M
authors.lastname: Korte
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D016716:PC12 Cells; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020800:Receptor, Nerve Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D051020:bcl-X Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C494813:Bcl2l1 protein, rat; D012333:RNA, Messenger; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein