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Rat Retina Expresses Beta Subunit 2 of CaMKIb2 During Its Development

This research investigates the expression of CaMKIb2 in the rat retina from mid-gestational until adulthood, using immunohistochemistry. Results show that CaMKIb2 is expressed in the retina during the prenatal period.

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Rat Retina Expresses Beta Subunit 2 of CaMKIb2 During Its Development

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  1. Rat retina expresses the beta subunit 2 of Ca2+/calmodulin-dependent protein kinase I (CaMKIb2) during its development Ahmad Aulia Jusuf 1 Toshio Terashima 2 Hiroyuki Sakagami 3 1Depart of Histology Faculty of Medicine University of Indonesia 2Depart of Anatomy Kobe University School of Medicine 3Division of Histology, Department of Cell Biology, Graduate School of Medical Science, Tohoku University, Sendai Japan

  2. AGENDA • Introduction • Purpose of the Research • Material and methods • Results • Discussion • Summary AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  3. Introduction • Intracellular Ca2+ • regulate cellular functions1 • Neurotransmitter release • Excitation-contraction coupling in muscle • Ion channel permeability • Active state of a variety of intracellular enzyme • Mediated by Ca2+ receptor, Calmodulin (CaM)2 • Complex Ca2+/Calmodulin (CaM) 1R.P Rubin, The role of calcium in the release of neurotransmitter substances and hormone, Pharmacol. Rev 22 (1972) 389-428 2 AS Manalan and CB Klee, In advances in cyclic nucleotide and protein phosphorylase research (P. Geengard and G.A. Robinson, eds) Vol 18 Raven press, NY, 1984 227-278 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  4. Introduction • Ca2+/Calmodulin-dependent protein kinase (CaMKs)3,4 • A family of structurally related protein kinases • Phosphorylase kinase3 • Myosin light chain kinase • Death associated protein kinase4 • CaMKI-IV • Depends on the complex of Ca2+/Calmodulin 3A.P Braun and H. Schulman. The Multifunctional calcium/calmodulin-dependent protein kinase from form to function. Annu.Rev.Physiol.57 (1995) 417-445 4 P.I.Hanson and H. Schulman. Neuronal Ca2+/calmodulin-dependent protein kinase. Annu.Rev. Biochem. 6 (1992) 559-601 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  5. Introduction • CaMKI b2 • One form of CaMKI isoforms5 • alfa, beta, gamma • 37-43 KDa • In the adult mainly in the central nervous system (CNS)6 • During embryogenesis • Brain • Spinal cord • Trigeminal nerve 5 Y. Fukuda. A Three groups classification of rat retinal ganglion cells histological and functional studies. Brain Res.119 (1977) 327-344 6 T. Ueda, H.Sakagami, K. Abe, I.Oishi, A. Maruo, H.Kondo, T.Terashima, M.Ichihashi, H.Yamamura and Y. Minami. Distribution and intracellular localization of a mouse homologue of ca2+/calmodulin-dependent protein kinase Ib2 in the nervous system. J. Neurochem. 73 (1999) 2119-2129 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  6. Introduction • Retina • embriologically arises from an evaginated portion of the brain7 • expressed a variety of neurotransmitter and protein kinase7 • expressed the other CaMKs8 7 J.M Bronstein, C.G. Wasterland and D.B. Farber, A retinal calmodulin-dependent protein kinase: Calcium /calmodulin-stimulated and inhibited states, J. Neurochem. 50 (1998) 8 T. Ochiishi, T. Terashima, H. Sugiura, T. Yamauchi, Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in the rat retina Brain Res 634 (1994) 257-265 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  7. Purpose of the research • The purpose of research • To investigate the expression of CaMKIb2 in the rat retina • From mid gestational until the adulthood AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  8. Material and Methods • Animal • Rat fetuses at a variety of embryonic age, which are obtained from time pregnant Wistar rats • E10, E12, E13, E15, E18 • Rat pups at a variety of age • P0, P2, P7, P12, P18 • Adult rat • Tissue processing • Supra vital fixation with paraformaldehyde 4% dan 7% saturated picric acid • Cryoprotected with 15% sucrose • Embedding with OCT compound • Cut with cryostat at a thickness of 10 mm • Mounted on the gelatin-coated slides AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  9. Material and Methods • Immunohistochemistry • Avidin-biotin peroxidase complex (ABC) method • Blocking solution : bovine serum albumin (BSA) 0.5% • Primary antibody : rabbit polyclonal antibody against murine CaMKIb2 at a dilution of 1: 200, overnight, at 40C • Secondary Antibody : biotinylated goat anti rabbit IgG at a dilution of 1: 200, 1 hr, at room temperature • Visualization with 3.3% 3,3’-diaminobenzidine tetrahydrochloride (DAB) AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  10. Results • CaMKIb2 expression in the retina during prenatal period • Appeared with the low level in the earliest development of eye • At E10 • In the optic vesicle (data not shown) AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  11. Results E12 E13 E15 E18 • CaMKIb2 expression in the retina during prenatal period Lns = Lens PL = Pigmented layer of retina NRL = Neural layer of Retina IBL= inner neuroblastic layer OBL = outer neuroblastic layer AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  12. Results • CaMKIb2 expression in the retina during prenatal period • low in the primary retina at E12 and gradually increased during further development • stronger in the inner neuroblastic layer (IBL) than that in the outer (OBL) at E15 until E18 • Located in the nuclei of developing retinal cells • Play the role in the process within the nucleus AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  13. Results P0 P7 P12 P18 • CaMKIb2 expression in the retina during post natal period GCL = Ganglionic Cell IPL = inner plexiform INL = inner nuclear OPL = outer plexiform ONL = outer nucelar RCL = rod-cone OBL= outer neuroblastic AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  14. Results • CaMKIb2 expression in the retina during retina development AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  15. Results • CaMKIb2 expression in the adult retina GCL = Ganglionic Cell IPL = inner plexiform INL = inner nuclear OPL = outer plexiform ONL = outer nucelar RCL = rod-cone OBL= outer neuroblastic D : GCL E : INL F : RCL AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  16. Results • CaMKIb2 expression in the retina during retina development AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  17. Discussion • From this study • CaMKIb2 is expressed at the rat retina from earliest development stage until adulthood • may be involved in the proliferation and differentiation process of precursor cells into specific cells in the retina • formation of functional layers AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  18. Discussion • From this study • CaMKIb2 is more widely expressed in the retina (GCL, INL,RCL) during development period compared to other CaMKs • Two other isoforms of CaMKs are also expressed in the retina • CaMKII 9,10 • ganglion cells and displaced amacrine cells in GCL • amacrine cells in the INL • CamKIV11 • Bipolar cells in INL • Ganglion cell and displaced amacrine cells in GCL (weak) • CaMKIb2 plays a more crucial role in the retina during developmental period and adulthood than two other isoforms of CaMKs 9T.Ochiisi, T. Terashima, H. Sugiura, T. Yamauchi, Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in the rat retina. Brain Res. 634 (1994) 257-265 10T. Terashima, T. Ochiishi, T. Yamauchi, Immunohistochemical localization of calcium/calmodulin-dependent protein kinase II isoform in the ganglion cells of the rat retina : immunofluorescence histochemistry combined with a fluorescent retrograde tracer, Brain Res 650 (1994) 133-139 11H. Sakagami, M. Watanabe and H. Kondo, Gene expression of Ca2+/calmodulin-dependent protein kinase type IV in the mature and developing rat retina, Brain Res. 719 (1996) 154-160 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  19. Discussion • From other study cAMP-responsive binding protein (CREB) CaMKIb212 Phosphorylated CREB light stimulation13 c-fos and somatostatin in GCL and INL activated c-fos and somatostatin genes 13 M.Sheng, M.A. Thompson and M.E. Greenberg, CREB : a Ca2+ regulated transcription factor phosphorylated by calmodulin-dependent kinases, Science. 252 (1991) 1427-1430 13K. Yoshida, J. Imaki, H. Matsuda and M. Hiaragawa, light induced CREB phosphorylation and gene expression in rat retinal cells, J. Neurochem. 65 (1995) 1499-1504 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  20. Discussion • From this study • Since CaMKIb2 is expressed in the rod-cone cell suggesting that CaMKIb2 may play the important role for visualization process • light alters CaMKs activity in synaptic membrane of retina14 • First electroretinographic responses are recorded around P1315 14K.F. Jensen, C.A. Ohmstede, R.S. Fisher, J.K. Olin and N. Sahyoun. Acquistion and loss of a neuronal Ca2+/calmodulin-dependent protein kinase during neuronal differentiation, Proc. Natl. Acad. Sci. USA. 88 (1991) 4050-4055 15T.A. Weidman and T. Kuwabata. Postnatal development of the rat retina. Arch Opthal.79 (1968) 470-484 AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  21. Summary • From this study • CaMKIb2 is expressed at the rat retina from earliest development stage until adulthood • CaMKIb2 may be involved in the proliferation and differentiation process of precursor cells into specific cells in the retina • CaMKIb2 is expressed in the rod-cone cell suggesting that CaMKIb2 may play the important role for visualization process AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

  22. thank you AAJ/rat retina expresses beta subunit 2 of CAMKI during its development

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