Abstract
Several eye diseases are associated with axonal injury in the optic nerve, which normally leads to degeneration of retinal ganglion cells (RGCs) and subsequently to loss of vision. There is experimental evidence that some members of the small heat shock protein family (HspBs) are upregulated upon optic nerve injury (ONI) in the retina and sufficient to promote RGC survival. These data raise the question as to whether other family members may play a similar role in this context. Here, we performed a comprehensive comparative study comprising all HspBs in an experimental model of ONI. We found that five HspBs were expressed in the adult rat retina at control conditions but only HspB1 and HspB5 were upregulated in response to ONI. Furthermore, HspB1 and HspB5 were constitutively phosphorylated in Müller cells at serine 15 and serine 59, respectively. In RGCs, phosphorylation was stimulated by ONI and occurred at serine 86 of HspB1 and at serine 19 and 45 of HspB5. These data suggest that of all small heat shock proteins, only HspB1 and HspB5 might be of protective value for RGCs after ONI and that this process might be regulated by phosphorylation at serine 86 of HspB1 and serine 19 and serine 45 of HspB5. The molecular targets of phosphoHspB1 and phosphoHspB5 remain to be identified.
Similar content being viewed by others
References
Arac A, Brownell SE, Rothbard JB, Chen C, Ko RM, Pereira MP, Albers GW, Steinman L, Steinberg GK (2011) Systemic augmentation of alphaB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation. Proc Natl Acad Sci U S A 108:13287–13292
Arrigo AP, Welch WJ (1987) Characterization and purification of the small 28,000-dalton mammalian heat shock protein. J Biol Chem 262:15359–15369
Bartelt-Kirbach B, Golenhofen N (2011) Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons. J Neurosci Res 89:162–175
Bartelt-Kirbach B, Golenhofen N (2014) Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons. Cell Stress Chaperones 19:145–153
Bellyei S, Szigeti A, Pozsgai E, Boronkai A, Gomori E, Hocsak E, Farkas R, Sumegi B, Gallyas F Jr (2007) Preventing apoptotic cell death by a novel small heat shock protein. Eur J Cell Biol 86:161–171
Benn SC, Perrelet D, Kato AC, Scholz J, Decosterd I, Mannion RJ, Bakowska JC, Woolf CJ (2002) Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron 36:45–56
Berkelaar M, Clarke DB, Wang YC, Bray GM, Aguayo AJ (1994) Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats. J Neurosci 14:4368–4374
Brown IR (1990) Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective. J Neurosci Res 27:247–255
Brownell SE, Becker RA, Steinman L (2012) The protective and therapeutic function of small heat shock proteins in neurological diseases. Front Immunol 3:74
Cheng G, Basha E, Wysocki VH, Vierling E (2008) Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry. J Biol Chem 283:26634–26642
Chiesa R, Gawinowicz-Kolks MA, Kleiman NJ, Spector A (1987) The phosphorylation sites of the B2 chain of bovine alpha-crystallin. Biochem Biophys Res Commun 144:1340–1347
Dieckmann-Schuppert A, Schnittler HJ (1997) A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces. Cell Tissue Res 288:119–126
Diekmann H, Leibinger M, Fischer D (2013) Do growth-stimulated retinal ganglion cell axons find their central targets after optic nerve injury? New insights by three-dimensional imaging of the visual pathway. Exp Neurol 248:254–257
Fischer D, Leibinger M (2012) Promoting optic nerve regeneration. Prog Retin Eye Res 31:688–701
Fischer D, Pavlidis M, Thanos S (2000) Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture. Invest Ophthalmol Vis Sci 41:3943–3954
Fischer D, Heiduschka P, Thanos S (2001) Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats. Exp Neurol 172:257–272
Gaestel M, Schroder W, Benndorf R, Lippmann C, Buchner K, Hucho F, Erdmann VA, Bielka H (1991) Identification of the phosphorylation sites of the murine small heat shock protein hsp25. J Biol Chem 266:14721–14724
Garrido C, Gurbuxani S, Ravagnan L, Kroemer G (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286:433–442
Golenhofen N, Bartelt-Kirbach B (2015) “HspB5/alpha-B-crystallin in the brain”. The Big Book of Small Heat Shock Proteins. R. M. Tanguay and L. E. Hightower, Springer International Publishing AG, pp 365-381
Golenhofen N, Ness W, Koob R, Htun P, Schaper W, Drenckhahn D (1998) Ischemia-induced phosphorylation and translocation of stress protein alpha B-crystallin to Z lines of myocardium. Am J Physiol 274:H1457–H1464
Golenhofen N, Arbeiter A, Koob R, Drenckhahn D (2002) Ischemia-induced association of the stress protein alpha B-crystallin with I-band portion of cardiac titin. J Mol Cell Cardiol 34:309–319
Golenhofen N, Redel A, Wawrousek EF, Drenckhahn D (2006) Ischemia-induced increase of stiffness of alphaB-crystallin/HSPB2-deficient myocardium. Pflugers Arch 451:518–525
Gusev NB, Bogatcheva NV, Marston SB (2002) Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins. Biochemistry (Mosc) 67:511–519
Haslbeck M, Franzmann T, Weinfurtner D, Buchner J (2005) Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Mol Biol 12:842–846
Hauk TG, Leibinger M, Muller A, Andreadaki A, Knippschild U, Fischer D (2009) Stimulation of axon regeneration in the mature optic nerve by intravitreal application of the toll-like receptor 2 agonist Pam3Cys. Invest Ophthalmol Vis Sci 51:459–464
Hauk TG, Leibinger M, Muller A, Andreadaki A, Knippschild U, Fischer D (2010) Stimulation of axon regeneration in the mature optic nerve by intravitreal application of the toll-like receptor 2 agonist Pam3Cys. Invest Ophthalmol Vis Sci 51:459–464
Heinzel W, Vogt A, Kallee E, Faller W (1965) A new method for the quantitative determination of antibody and antigen protein, with a sensitivity to five micrograms. J Lab Clin Med 66:334–340
Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA (2006) Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites. Proc Natl Acad Sci U S A 103:7159–7164
Huang L, Min JN, Masters S, Mivechi NF, Moskophidis D (2007a) Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption. Genesis 45:487–501
Huang W, Fileta JB, Filippopoulos T, Ray A, Dobberfuhl A, Grosskreutz CL (2007b) Hsp27 phosphorylation in experimental glaucoma. Invest Ophthalmol Vis Sci 48:4129–4135
Ito H, Okamoto K, Nakayama H, Isobe T, Kato K (1997) Phosphorylation of alphaB-crystallin in response to various types of stress. J Biol Chem 272:29934–29941
Jaya N, Garcia V, Vierling E (2009) Substrate binding site flexibility of the small heat shock protein molecular chaperones. Proc Natl Acad Sci U S A 106:15604–15609
Kampinga HH, Hageman J, Vos MJ, Kubota H, Tanguay RM, Bruford EA, Cheetham ME, Chen B, Hightower LE (2009) Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 14:105–111
Kappe G, Franck E, Verschuure P, Boelens WC, Leunissen JA, de Jong WW (2003) The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10. Cell Stress Chaperones 8:53–61
Kappe G, Boelens WC, de Jong WW (2010) Why proteins without an alpha-crystallin domain should not be included in the human small heat shock protein family HSPB. Cell Stress Chaperones 15:457–461
Kiang JG, Tsokos GC (1998) Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther 80:183–201
Kim YH, Choi MY, Kim YS, Han JM, Lee JH, Park CH, Kang SS, Choi WS, Cho GJ (2007) Protein kinase C delta regulates anti-apoptotic alphaB-crystallin in the retina of type 2 diabetes. Neurobiol Dis 28:293–303
Kirbach BB, Golenhofen N (2011) Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons. J Neurosci Res 89:162–175
Krueger-Naug AM, Emsley JG, Myers TL, Currie RW, Clarke DB (2002) Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system. Neuroscience 110:653–665
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Landry J, Chretien P, Laszlo A, Lambert H (1991) Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells. J Cell Physiol 147:93–101
Landry J, Lambert H, Zhou M, Lavoie JN, Hickey E, Weber LA, Anderson CW (1992) Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. J Biol Chem 267:794–803
Lee J, Kim H, Lee JM, Shin T (2006) Immunohistochemical localization of heat shock protein 27 in the retina of pigs. Neurosci Lett 406:227–231
Morrison LE, Hoover HE, Thuerauf DJ, Glembotski CC (2003) Mimicking phosphorylation of alphaB-crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis. Circ Res 92:203–211
Morrison LE, Whittaker RJ, Klepper RE, Wawrousek EF, Glembotski CC (2004) Roles for alphaB-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion-induced damage in a KO mouse model. Am J Physiol Heart Circ Physiol 286:H847–H855
Munemasa Y, Kwong JM, Caprioli J, Piri N (2009) The role of alphaA- and alphaB-crystallins in the survival of retinal ganglion cells after optic nerve axotomy. Invest Ophthalmol Vis Sci 50:3869–3875
O’Reilly AM, Currie, RW, Clarke, DB (2010) HspB1 (Hsp 27) Expression and Neuroprotection in the Retina. Mol Neurobiol
Ousman SS, Tomooka BH, van Noort JM, Wawrousek EF, O'Connor KC, Hafler DA, Sobel RA, Robinson WH, Steinman L (2007) Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination. Nature 448:474–479
Peschek J, Braun N, Rohrberg J, Back KC, Kriehuber T, Kastenmuller A, Weinkauf S, Buchner J (2013) Regulated structural transitions unleash the chaperone activity of alphaB-crystallin. Proc Natl Acad Sci U S A 110:E3780–E3789
Plumier JC, Hopkins DA, Robertson HA, Currie RW (1997) Constitutive expression of the 27-kDa heat shock protein (Hsp27) in sensory and motor neurons of the rat nervous system. J Comp Neurol 384:409–428
Quraishe S, Asuni A, Boelens WC, O'Connor V, Wyttenbach A (2008) Expression of the small heat shock protein family in the mouse CNS: differential anatomical and biochemical compartmentalization. Neuroscience 153:483–491
Ray PS, Martin JL, Swanson EA, Otani H, Dillmann WH, Das DK (2001) Transgene overexpression of aB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J 15:393–402
Reddy VS, Reddy GB (2015) Emerging role for alphaB-crystallin as a therapeutic agent: pros and cons. Curr Mol Med 15:47–61
Schmidt T, Bartelt-Kirbach B, Golenhofen N (2012) Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/alphaB-crystallin in cultured hippocampal neurons. Histochem Cell Biol 138:407–418
Shao W, Zhang SZ, Tang M, Zhang XH, Zhou Z, Yin YQ, Zhou QB, Huang YY, Liu YJ, Wawrousek E et al (2013) Suppression of neuroinflammation by astrocytic dopamine D2 receptors via alphaB-crystallin. Nature 494:90–94
Sun Y, MacRae TH (2005) The small heat shock proteins and their role in human disease. FEBS J 272:2613–2627
Thanos S, Bohm MR, Meyer zu Horste M, Prokosch-Willing V, Hennig M, Bauer D, Heiligenhaus A (2014) Role of crystallins in ocular neuroprotection and axonal regeneration. Prog Retin Eye Res 42:145–161
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
Verschuure P, Croes Y, van den IJssel PR, Quinlan RA, de Jong WW, Boelens WC (2002) Translocation of small heat shock proteins to the actin cytoskeleton upon proteasomal inhibition. J Mol Cell Cardiol 34:117–128
Welch WJ (1992) Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev 72:1063–1081
Wu N, Yu J, Chen S, Xu J, Ying X, Ye M, Li Y, Wang Y (2014) alpha-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush. Life Sci 94:17–23
Xi J, Farjo R, Yoshida S, Kern TS, Swaroop A, Andley UP (2003) A comprehensive analysis of the expression of crystallins in mouse retina. Mol Vis 9:410–419
Ying X, Zhang J, Wang Y, Wu N, Wang Y, Yew DT (2008) Alpha-crystallin protected axons from optic nerve degeneration after crushing in rats. J Mol Neurosci 35:253–258
Acknowledgments
We thank Diana Reinhardt for her excellent technical assistance and the International Graduate School in Molecular Medicine (University of Ulm, Germany) of which Thomas Schmidt was a member.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(GIF 380 kb)
Rights and permissions
About this article
Cite this article
Schmidt, T., Fischer, D., Andreadaki, A. et al. Induction and phosphorylation of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in the rat retina upon optic nerve injury. Cell Stress and Chaperones 21, 167–178 (2016). https://doi.org/10.1007/s12192-015-0650-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12192-015-0650-8