title: Silencing of tuberin enhances photoreceptor survival and function in a preclinical model of retinitis pigmentosa (an american ophthalmological society thesis).
abstract: OBJECTIVE
  To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis.
  
  
  METHODS
  In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival.
  
  
  RESULTS
  At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration.
  
  
  CONCLUSIONS
  Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.
authors: Institute of Human Nutrition, Department of Pathology and Cell Biology and the Department of Ophthalmology, Columbia University, New York, New York.
authors: Stephen H
authors: SH
authors: Tsang
authors: Department of Ophthalmology, Columbia University, New York, New York.
authors: Lawrence
authors: L
authors: Chan
authors: Department of Ophthalmology, Columbia University, New York, New York.
authors: Yi-Ting
authors: YT
authors: Tsai
authors: Department of Ophthalmology, Columbia University, New York, New York.
authors: Wen-Hsuan
authors: WH
authors: Wu
authors: Department of Ophthalmology, Columbia University, New York, New York.
authors: Chun-Wei
authors: CW
authors: Hsu
authors: Department of Ophthalmology, Columbia University, New York, New York; and Tianjin Medical University Eye Hospital, Tianjin, China.
authors: Jin
authors: J
authors: Yang
authors: Department of Ophthalmology, Columbia University, New York, New York; and Kresge Eye Institute, Wayne State University, Detroit, Michigan.
authors: Joaquin
authors: J
authors: Tosi
authors: Department of Ophthalmology, Columbia University, New York, New York; and Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors: Katherine J
authors: KJ
authors: Wert
authors: Department of Ophthalmology, Columbia University, New York, New York; and Neural Stem Cell Institute, Rensselaer, New York.
authors: Richard J
authors: RJ
authors: Davis
authors: Omics Lab, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa.
authors: Vinit B
authors: VB
authors: Mahajan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D004195:Disease Models, Animal; D020868:Gene Silencing; D051379:Mice; D008817:Mice, Mutant Strains; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000503:physiopathology*; D038601:Ribosomal Protein S6 / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D038601:Ribosomal Protein S6; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D010770:Phosphotransferases; D051057:Proto-Oncogene Proteins c-akt
references: Science. 1998 Oct 2;282(5386):117-21
references: 9756475
references: Cancer Cell. 2003 Aug;4(2):147-58
references: 12957289
references: J Biol Chem. 2004 Oct 1;279(40):41903-10
references: 15218024
references: Cell Signal. 2012 Jan;24(1):181-8
references: 21920434
references: Cancer Res. 2006 Dec 15;66(24):11605-12
references: 17178854
references: J Biol Chem. 2005 Apr 1;280(13):12585-92
references: 15668239
references: Exp Biol Med (Maywood). 2011 Oct;236(10):1211-7
references: 21885480
references: Nat Neurosci. 2009 Jan;12(1):44-52
references: 19060896
references: Mol Cell Biol. 2011 Jul;31(14):2787-801
references: 21576368
references: Oncogene. 2007 Jan 25;26(4):521-31
references: 16862180
references: Science. 2011 Jun 10;332(6035):1322-6
references: 21659605
references: J Biol Chem. 1991 Jun 15;266(17):10711-4
references: 1710212
references: Biochem Biophys Res Commun. 2004 Jan 23;313(4):1015-22
references: 14706644
references: Proc Natl Acad Sci U S A. 1981 Jan;78(1):152-6
references: 6264430
references: Am J Ophthalmol. 1995 May;119(5):543-62
references: 7733180
references: J Vis Exp. 2012;(69). pii: 4286. doi: 10.3791/4286
references: 23207897
references: Proc Natl Acad Sci U S A. 1924 Jul;10(7):329-33
references: 16576828
references: PLoS One. 2012;7(8):e43889
references: 22970106
references: Physiol Rev. 2001 Jan;81(1):117-151
references: 11152756
references: J Biol Chem. 1994 May 20;269(20):14329-32
references: 8182033
references: Mol Med. 2012;18:549-55
references: 22252712
references: Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3249-53
references: 7724547
references: Invest Ophthalmol Vis Sci. 1995 Feb;36(2):263-75
references: 7843898
references: Invest Ophthalmol Vis Sci. 2005 Sep;46(9):3443-50
references: 16123450
references: Toxicol Sci. 2005 Dec;88(2):331-9
references: 16150885
references: Front Biosci. 2005;10:1191-204
references: 15769618
references: J Gene Med. 2003 Sep;5(9):757-64
references: 12950066
references: Hum Gene Ther. 2013 Mar;24(3):242-4
references: 23458444
references: J Neurosci. 2006 Apr 26;26(17):4472-80
references: 16641226
references: Hum Mol Genet. 2013 Feb 1;22(3):558-67
references: 23108158
references: Biochemistry. 1990 Mar 20;29(11):2657-64
references: 2161252
references: J Urol. 2008 Jun;179(6):2187-91; discussion 2191
references: 18423725
references: Hum Gene Ther. 2005 Jun;16(6):649-63
references: 15960597
references: Annu Rev Physiol. 2002;64:153-87
references: 11826267
references: J Biol Chem. 1979 Nov 25;254(22):11669-77
references: 227876
references: J Cyclic Nucleotide Res. 1976;2(3):139-48
references: 6493
references: N Engl J Med. 2008 May 22;358(21):2231-9
references: 18441371
references: Cell Tissue Res. 2005 May;320(2):213-22
references: 15789220
references: Hum Mol Genet. 2014 Jan 15;23(2):514-23
references: 24101599
references: Nat Rev Mol Cell Biol. 2011 Jan;12(1):21-35
references: 21157483
references: Amino Acids. 2006 Jun;30(4):391-6
references: 16773241
references: Proc Natl Acad Sci U S A. 2013 Feb 5;110(6):E517-25
references: 23341635
references: J Physiol. 2007 Mar 1;579(Pt 2):303-12
references: 17138607
references: Lancet. 2006 Nov 18;368(9549):1795-809
references: 17113430
references: Ophthalmology. 2009 May;116(5):971-80
references: 19410955
references: Vision Res. 2002 Feb;42(4):439-45
references: 11853759
references: Genes Dev. 2005 Sep 15;19(18):2199-211
references: 16166381
references: Cell Signal. 2011 Oct;23(10):1584-9
references: 21616145
references: Exp Eye Res. 2006 Aug;83(2):380-9
references: 16626700
references: Am J Ophthalmol. 2008 Nov;146(5):780-8
references: 18723146
references: J Vis Exp. 2011;(57). pii: 3184. doi: 10.3791/3184
references: 22126835
references: Invest Ophthalmol Vis Sci. 2008 Nov;49(11):5067-76
references: 18658088
references: Science. 1977 May 6;196(4290):664-6
references: 193183
references: Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1620-9
references: 16565401
references: Hum Mutat. 2007 Mar;28(3):243-54
references: 17044014
references: J Biol Chem. 2006 Jun 2;281(22):15412-22
references: 16595671
references: Neuron. 2005 Nov 3;48(3):387-401
references: 16269358
references: Science. 1974 Nov 1;186(4162):449-51
references: 4369896
references: Curr Opin Cell Biol. 2005 Dec;17(6):596-603
references: 16226444
references: Invest Ophthalmol Vis Sci. 1999 Nov;40(12):2770-2
references: 10549634
references: Arch Ophthalmol. 2007 Feb;125(2):151-8
references: 17296890
references: Invest Ophthalmol Vis Sci. 2008 Oct;49(10):4573-7
references: 18539930
references: Annu Rev Neurosci. 2001;24:779-805
references: 11520918
references: Mol Imaging Biol. 2010 Aug;12(4):386-93
references: 19937134
references: Cold Spring Harb Perspect Biol. 2012 Nov;4(11). pii: a012252. doi: 10.1101/cshperspect.a012252
references: 22888049
references: J Biol Chem. 2004 Nov 19;279(47):49206-13
references: 15355981
references: Biochem Biophys Res Commun. 2009 Dec 25;390(4):1149-53
references: 19878658
references: Hum Mutat. 2001;17(1):42-51
references: 11139241
references: J Neurosci. 2013 Aug 14;33(33):13475-83
references: 23946405
references: N Engl J Med. 2008 May 22;358(21):2240-8
references: 18441370
references: Invest Ophthalmol Vis Sci. 1993 Apr;34(5):1659-76
references: 8473105
references: Transplantation. 2010 Apr 27;89(8):911-9
references: 20164818
references: J Hered. 1966 Mar-Apr;57(2):47-50
references: 5916892
references: Brain Res. 2007 Jan 19;1129(1):116-29
references: 17156753
references: J Cell Mol Med. 2011 Aug;15(8):1778-87
references: 20950332
references: Retina. 2005 Dec;25(8 Suppl):S21-S24
references: 16374320
references: J Neurosci. 2008 Feb 27;28(9):2064-74
references: 18305241
references: Mol Cell Proteomics. 2006 Feb;5(2):324-36
references: 16253986