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In Vivo to In Vitro A Predictive Math Model

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					Undergraduate Research in
   Computer Science
       Program Director:
      Dr. Fatma Mili Ph.D.
Application of a Coherence
    Network to Retinal
Ganglion Cell Regeneration
Investigators:
   Marianne Mara (M2)
            University of Michigan-Dearborn
   Noelle Nelson (N2)
            Oakland University
Advisors:
    Dr. M. Zohdy PhD
      Electrical and Computer Engineering Department Oakland University
    Dr. S. Chintala PhD
      Eye Research Institute Oakland University
         Research Objectives
• Understand the basic mechanisms of retinal
  ganglion cell regeneration
• Apply a new extended coherence network
  simulation to retinal ganglion cell interactions
• Study the important protein kinases involved in
  regeneration of retinal ganglion cells
• Compare computer simulation (in silico) results
  to actual laboratory experiments performed at
  the Eye Research Institute
          Coherence Network
• A coherence network is made up of nodes
  (concepts) connected by arcs, that possibly
  carry real or complex weights (relationships), to
  arrive at coherent (consistent) solutions.
• We extended the basic coherence network to
  include hierarchical classes of nodes and arcs in
  order to better suit retinal ganglion cell protein
  interaction.
• Our additional contribution is to consider the
  Sigmoidal function for input/output relationships
  again to suit retinal ganglion cell neuro-biology
 Applying the Extended Coherence
    Network to Protein Kinase
             Interaction
• Network: Nodes represent the proteins and
  associated kinases involved in retinal ganglion
  cell regeneration
• Coherence represents consistent relationships
  that will show the qualitative and quantitative
  effects of protein interactions
• The extended coherence network model that we
  are developing describes the significance of
  protein kinase pathways that may promote
  retinal ganglion cell regeneration
     Extended Coherent Network


                    Class 2   Class 4   Class 1
Class 3
                                                                Legend
                                                  Active
          Class 5                                 Node
Class 5             Class 4             Class 5
                                                           Class 1
                                                           (Protein
                                                           Kinase)         Class 4
                                                                         Up Regulates
Class 1             Class 2
                                                            Class 2
                                        Class 3                            Class 5
                                                           (Inhibitor)
                                                                            Down
                                                                          Regulates
                    Class 4
                                                             Class 3
                                                           (Protease)


                    Class 1             Class 5
A Protein Kinase and Inhibitor Interaction in
       a Retinal Ganglion Cell Line

                    Staurosporine
                                                               Up
                                                            Regulates

         Down
        Regulates               Down
                               Regulates




                                                            Up
                          Up                            Regulates            tPA           Neurite
           PKC                             PKA                                            Outgrowth
                       Regulates                     In the presence
                                                         Of cAMP



                                                                        Forms Complexes
                                                                          To Inactivate
                            Legend

       Protease                     Protease
       Inhibitors
                                                                          Plaminogen
                                                                           Activator
        Protein                  Biochemical Path                           Inhibitor
        Kinase                  Functional Outcome
            Extended Coherent Network of the
                Protein Kinase Interaction
                                                      Legend
                              Active
                              Node                                                                   Staurosporine
                                                 Class 1                                                                                            Up
                                                 (Protein                                                                                        Regulates
                                                 Kinase)                   Class 4
                                                                         Up Regulates    Down
                                                  Class 2
                                                                                        Regulates             Down
                                                 (Inhibitor)               Class 5                           Regulates
                                                                            Down
                                                                          Regulates
Staurosporin
  Class 2                                          Class 3
                                                 (Protease)
                                                                                                                                                 Up
                                                                                                           Up                                Regulates
                                                                                          PKC                                 PKA                                tPA
                                                                                                        Regulates                         In the presence
                                                                                                                                              Of cAMP

                                            Class 4
                                              (1)
  Class 5
    (-1)

                                                                                                                                                        Forms Complexes
               Class 5                                                                                                                                    To Inactivate
                 (-1)



                                                                                                              Legend
  Protein
  Kinase            Class 4             PKA                    Class 4
     C                (1)              Class 1                   (1)          tPA
  Class 1                                                                   Class 3                                      Protease                            Plaminogen
                                                                                        Protease
                                                                                        Inhibitors                                                            Activator
                                                                                                                                                               Inhibitor
                                                                            Class 5
                                                                              (-1)
                                                                                         Protein                      Biochemical Path
                                                                                         Kinase                      Functional Outcome
                                                                          Plasminogen
                                                                            Activator
                                                                            Inhibitor
                                                                             Class 2
Sigmoidal Functions
Anatomy of the Human Eye
                              Bipolar Cells
             Amacrine Cells               Horizontal Cells




           Retinal Ganglion Cells                Rod and Cone Cells
        Importance of Retinal Ganglion Cells
•   Major eye diseases like glaucoma are prevalent in the US and the world
    population.
•   Irreversible loss of retinal ganglion cells occurs in glaucoma



                                                                Vision




                                                     Glaucoma
          Cross section of retinas
     Laboratory Analysis Model for Retinal
                Ganglion Cells
• Retinal ganglion cells in adult retina are terminally differentiated, do
  not divide, survive for 1-3 generations in laboratory conditions, and
  cannot be used for long-term experiments.
• RGC-5 cells are transformed cells, they take on many of the
  characteristics of adult retinal ganglion cells, divide indefinitely under
  laboratory conditions, and therefore can be used to study the
  mechanisms involved in regeneration of retinal ganglion cells.
• Unlike adult retinal ganglion cells, RGC-5 cells divide in culture and
  appear as fibroblasts, and not like ganglion cells.
• However, RGC-5 cells can be programmed to behave like adult and
  differentiated ganglion cells after treatment with staurosporin (a
  chemical agent) that inhibits a number of intracellular protein
  kinases.
RGC-5 cells treated with staurosporine to induce differentiation



        Undifferentiated                  Differentiated
              Known Protein Kinases
               Staurosporine Inhibits
As staurosporine inhibits a number of protein kinases, it is important to
know which protein kinase (s) staurosporine inhibits, and which kinases
       are responsible for inducing RGC-5 cells to differentiate.

                                  Protein Kinase
                                     Inhibitor


                                     Stauro-
                                     sporine




                                                                                                    JAK-5


                                                                                           EGFR
      AKT                   MLCK
             MEK     PKC
                                                                                PDGFR
                                                                CDK2/
                                                               Cyclin A
                                   CAM             CDK5                   PKA



                                                          CDK1/
              cGMP                         Rho-           Cyclin B                      Protein Kinases
               PDE         cAMP           Kinase
                            PDE
             Research Tools

1. Matlab, and Matlab Toolboxes for developing an
   extended coherence network
2. Microsoft Office Visio
3. Pathway Studio software
4. Designed experiments from Dr. Chintala’s Eye
   Research Institute laboratory
5. NCBI Protein databanks with gene sequences,
   and related scientific journal articles
                                                                                                           CDK2
                                                                                            CDK1                              AKT


    Biochemical
                                                                                                              CDK2
                                                                                             CDK1                             AKT
                                                                                                                                                 JAK-5
                                                                                                                                                 JAK-5



       Paths                                                                               Down
                                                                                          RegulatesDown
                                                                                            Down
                                                                                                 Regulates
                                                                                                             Down
                                                                                          RegulatesDown Regulates
                                                                                                             Down Down
                                                                                                                                          EGFR
                                                                                                                                          EGFR
                                                                                                  Regulates
                                                                                                            Regulates
                                                                                                                    Down
                                                                                                                Regulates
                                                                                                                  Regulates

                                                                                                                  Down                    PDGFR
                                                                                                                 Regulates
              Staurosporine                                                                                        Down                   PDGFR
                                                                                                                 Regulates
                                                                                                                    Down
                                                        Up
                                                                                                                     Regulates
                                                     Regulates                                                         Down
                                                                                                                     Regulates                    MEK
   Down                                                                                                                Down
  Regulates            Down                                                                                          Regulates                     MEK
                                                                                              Stauro-                  Down
                      Regulates                                                               sporine
                                                                                              Staurospo               Regulates
                                                                                Up
                                                                                                 rine                  Down
                                                                             Regulates
                                                                                                                     Regulates
                                                                                                                       Down                MLCK
                                                                                                                  Regulates
                                                                                                                     Down
                                                     Up                                                                                     MLCK
                                                                                                                   Regulates
                                                                                                                      Down
                    Up                           Regulates
     PKC                             PKA                               tPA                                          Regulates
                 Regulates                    In the presence
                                                  Of cAMP                                                                 Down
                                                                                tPA                             Down Regulates                           CAM
                                                                                                                          Down
                                                                                                              Regulates Regulates
                                                                                                                                                         CAM
                                                                                                                 Down
                                                                                                               Regulates
                                                                                                           Down
                                                                            Inactivates
                                                                Forms Complexes                                                                           Rho
                                                                                                         Regulates
                                                                                                          Down
                                                                  To Inactivate                                                                          Kinase
                                                                                                    Down Regulates                                         Rho
                                                                                                  Regulates                                              Kinase
                                                                                                   Down
                     Legend                                                                       Regulates                         PKC
                                                                         Plasminogen                                                                Down
                                                                                                                                                             cAMP
                                                                           Activator                                                PKC           Regulates
                                                                                                                                                     Down     PDE
Protease                      Protease                                     Inhibitor                                                                         cAMP
                                                                                                                                                  Regulates
Inhibitors                                                                                                                                                    PDE
                                                               Plaminogen                                                                   Down
                                                                 Activator                                                                 Regulates
                                                          Up                                                             CDK5                Down
                                                                 Inhibitor
                                                       Regulates                                                                           Regulates    cGMP
Protein                   Biochemical Path                                                                                CDK5                           PDE
Kinase                   Functional Outcome                                                                                                             cGMP
                                                                                                                                                         PDE
                                                                                                          PKA
                                                                                                          PKA         Down
                                                                                                                     Regulates   Up
                                                                                                                       Down
                                                                                                                     RegulatesRegulates
Qualitative Analysis of a Coherent
             Network


                                                                                                     Legend

                                                                      Active                   Inactive
                                                                      Node                      Node

                                                                                 Class 1
                                                                                 (Protein                            Class 4
     Staurosporin                                                                Kinase)                           Up Regulates
       Class 2
                                                                                                                     Class 5
                                                                                  Class 2
                                                                                                                      Down
                                                                                 (Inhibitor)
                                                                                                                    Regulates


                                                                                   Class 3
                                                                                 (Protease)


                                        Class 4
                                          (1)
       Class 5
         (-1)


                    Class 5
                      (-1)




       Protein                                                                                       Plasminogen
       Kinase            Class 4    PKA           Class 4                      Class 5                 Activator
          C                (1)     Class 1          (1)       tPA                (-1)                  Inhibitor
       Class 1                                              Class 3                                     Class 2
                          Qualitative Evaluation with an
                          Extended Coherent Network



                                                                                                             Alpha-2
                                      Plasminogen                 Class 5    Urokinase                     Anti-plasmin
                                        Activator                   (-1)    Plasminogen                      Class 2
                                        Inhibitor                             Activator
                                         Class 2                               Class 3



                                                                                                                                      Matrix
                                                                              Class 4                        Class 5
                                                                                                                                    Metallorotein
                                                                                (1)                            (-1)
                                                                                                                                     Inhibitor
                                                                                          Active Matrix                               Class2
                                                                                          MetalloProtein
                                                                                            Class 3


                                                                                            Class 4
                                                                                              (1)
                                                            Class 5         Plasminogen                      Plasmin
                                                              (-1)             Class 3                       Class 3
                             Legend                                                                                                    Class 5
                                                                                                                                         (-1)
Active                 Inactive
Node                    Node                                                  Class 4
         Class 1                                                                (1)
         (Protein                              Class 4                                                                    Class 4
         Kinase)                             Up Regulates                                                                   (1)

                                               Class 5
          Class 2
                                                Down
         (Inhibitor)
                                              Regulates
                                                                               Tissue
                                                                            Plasminogen                      Class 4                 Active Matrix
           Class 3                                                            Activator                        (1)                   MetalloProtein
         (Protease)                                                            Class 3                                                 Class 3
       Qualitative Analysis of Staurosporine
        and the Protein Kinases it Inhibits



                                  CAM                                                                    JAK-5                               PDGF
                                 Class 1                                                                 Class 1                             Class 1




 MEK        AKT
Class 1    Class 1     Rho                  CDK1                                             CDK2
                                                                                             Class 1                     EGFR                               MLCK
                      Kinase                Class1                                                                       Class 1                            Class 1
                      Class 1




                                                                                                          Class 5




                                                                                                                                              Class 5
                                                                                                            (-1)
            Class 5
 Class 5




                                                                                                                                                (-1)
                                             Class 5
              (-1)
   (-1)




                                               (-1)
                                  Class 5
                                    (-1)




                                                                                               Class 5
                       Class 5




                                                                                                                          Class 5
                                                                                                 (-1)




                                                                                                                                                             Class 5
                         (-1)




                                                                                                                            (-1)




                                                                                                                                                               (-1)
                                                           Staurosporine
                                                              Class 2


                                                                                                                                                          Legend
                                                                                         cGMP
                                                                            Class 5




                                                                                          PDE
                                                                              (-1)




                                                                                         Class 1
                                                                                                                                    Active                 Inactive
                                                 Class 5




                                                                                                                                    Node                    Node
                                                   (-1)




                                                                                                               Class 5
                                                                                                                 (-1)
                                                                                                                                              Class 1                  Class 4
                                                                                                                                                                         Up
                                                                                          Class 5




                                                                                                                                              (Protei
                                                                                            (-1)



                                                                                                                                                 n                     Regulate
                                                                            PKC                                                               Kinase)
                                                                           Class 1
                                                                                          Class 5
                                                                                            (-1)




                                                                                                                                               Class 2
                                                                                                                                               (Inhibit                Class 5
                                                                                                                                                  or)                   Down
                                                PKA
                                                                                                                                                                       Regulate
                                               Class 1
                                                                                         cAMP
                                                                                          PDE                CDK5
                                                                                         Class 1             Class 1                           Class 3
                                                                                                                                              (Proteas
                                                                               Class 5
                                                                                 (-1)




                                                                                                                                                  e)
            Qualitative Analysis of RGC-5 Cell Line
                   Exposed to Staurosporine




                                           CAM                                                                                       JAK-5                               PDGF
                                          Class 1                                                                                    Class 1                             Class 1




 MEK        AKT
Class 1    Class 1      Rho                                       CDK1                                                   CDK2
                                                                                                                         Class 1                     EGFR                               MLCK
                       Kinase                                     Class1                                                                             Class 1                            Class 1
                       Class 1




                                                                                                                                      Class 5




                                                                                                                                                                          Class 5
                                                                                                                                        (-1)
            Class 5
 Class 5




                                                                                                                                                                            (-1)
                                                                   Class 5
              (-1)
   (-1)




                                                                     (-1)
                                           Class 5
                                             (-1)




                                                                                                                           Class 5
                         Class 5




                                                                                                                                                      Class 5
                                                                                                                             (-1)




                                                                                                                                                                                         Class 5
                           (-1)




                                                                                                                                                        (-1)




                                                                                                                                                                                           (-1)
                                                                                 Staurosporine
                                                                                    Class 2


                                                                                                                                                                                      Legend
                                                                                                                     cGMP
                                                                                                     Class 5




                                                                                                                      PDE
                                                                                                       (-1)




                                                                                                                     Class 1
                                   Plasminogen
                                     Activator                                                                                                                  Active                 Inactive
                                                                       Class 5




                                     Inhibitor                                                                                                                  Node                    Node
                                                        Class 4




                                                                         (-1)




                                                                                                                                           Class 5
                                                                                                                                             (-1)
                                                          (1)




                                      Class 3                                                                                                                                                      Class 4
                                                                                                                                                                          Class 1
                                                                                                                                                                                                     Up
                                                                                                                      Class 5




                                                                                                                                                                          (Protei
                                                                                                                        (-1)




                                                                                                                                                                             n                     Regulate
                      Class 5




                                                                                                    PKC                                                                   Kinase)
                        (-1)




                                                                                                   Class 1
                                                                                                                      Class 5
                                                                                                                        (-1)




                                                                                                                                                                           Class 2
                                                                                                                                                                           (Inhibit                Class 5
                                       tPA
                                                                                                                                                                              or)                   Down
                                                     Class 4




                                     Class 3                          PKA
                                                                                                 Class 4




                                                                                                                                                                                                   Regulate
                                                       (1)




                                                                                                   (1)




                                                                     Class 1
                                                                                                                     cAMP
                                                                                                                      PDE                CDK5
                                                                                                                     Class 1             Class 1                           Class 3
                                                                                                                                                                          (Proteas
                                                                                                           Class 5
                                                                                                             (-1)




                                                                                                                                                                              e)
 Results from a Quantitative
Extended Coherence Network
        Simulation I
 Results from a Quantitative
Extended Coherence Network
        Simulation II
            Conclusion
We have gained a much better
understanding of RGC differentiation,
regeneration, and neurite formation.
Although it needs more work, our
coherence network was able to achieve
some similarity to the laboratory generated
data.
At this point the in silico results seem to
agree with cell line laboratory research
        Further Research
As new data is discovered in relation to
the interaction of staurosporine and
protein kinases, the extended coherent
network can be further explored.
The extended coherent network may be
tested with well researched biochemical
paths such as glycolysis and the Kreb’s
cycle.
   Conference Opportunities

American Chemical Society – Division of Biological
Chemistry, XXVI Midwest Enzyme Chemistry
Conference, September 30, 2006 Northwestern
University, Evanston, IL. www.midwestenzyme.org

2006 Huntsville Simulation Conference, October 18 -
19, 2006 Huntsville, Alabama. www.scs.org.hsc
                                       References
2006
S. Chintala. Data from axon lengthening experiments with staurosporine, tissue plasminogen activator,and urokinase
     plasminogen activator. Received June 29, 2006.
    The advisor provided data related to varying concentrations of staurosporine and the generation
     of tissue plasminogen activator and urokinase plasminogen activator.

S. Chintala. The emerging role of proteases in retinal ganglion cell death. Experimental Eye Research. Vol. 82, No.1,
     pp. 5-12. , 2006.
    The article is a review of the known and proposed interplay of protein kinases, proteases, and inhibitors which
     results in the destruction of retinal ganglion cells.

L. Frassetto, C. Schlieve, C. Lievenen, A. Utter, M. Jones, N. Agarwal, & L. Levin. Kinase-dependent differentiation of a
     retinal ganglion cell precursor. Inv. Ophth. & Vis. Sci. Vol. 47, No. 1, pp. 427-438, Jan. 2006.
     The authors report the effects of staurosporine induced differentiation in the RGC-5 cell line.

K. Hu, J. Yang, S. Tanaka, S. Gonias, W. Mars, & Y. Liu. Tissue-type plasminogen activator acts as a cytokine that
     triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression. Jour. Of Bio.
     Chem. Vol. 281, No. 4, pp. 2120-2127, Jan. 27, 2006.
    The articale describes the activation of matrix metalloproteins in light of the protein kinases inhibited.

Y. Yin, M. Henzl, B. Lorber, T. Nakazawa, T. Thomas, F. Jiang, R. Langer, & L. Benowitz. Oncomodulin is a
     macrophage-derived signal for axon regeneration in retinal ganglion cells. Nature Neuroscience. Vol. 9, No. 6, pp.
     June 2006.
     Tissue macrophages release oncomodulin which induces axon lengthening.
                     References Continued
2005
A. Davy, & P. Soriano. Ephrin signaling in vivo: Look both ways. Dev. Dynamics. Vol. 232, pp. 1-10, 2005.
     The authors describe embronic extension of the gangalion into the developing brain.

F. Mann, W. Harris, & C. Holt. New views on retinal axon development: a navigation guide. Int. Jour. Dev. Bio. Vol. 48, pp.
     957-964, 2004.
    The authors describe the cell marker which assist in the “direction” of axon lengthening into the central nervous system.

X. Wang, S. Lee, K. Arai, S. Lee, K. Tsuji, G. Rebeck, & E. Lo. Lipoprotein receptor-mediated induction of matrix
    metalloproteinase by tissue plasminogen activator. Nat. Med. Vol. 9, No. 10, pp. 1313-1317, Oct. 2003.
    The article describes tPA as a signaling agent in combination with a lipoprotein receptor in retinal ganglion cell
    degradation by matrix metalloproteins.

2002
M. Aon, & S. Cortassa. Coherent and robust modulation of a metabolic network by cytosketal organization and dynamics.
     Biophysical Chem. Vol. 97, Issue 2-3, pp. 213-231, June 2002.

B. Patel & D. Vactor. Axon guidance: the cytoplasmic tail. Current Opinion in Cell Biology. Vol. 14, pp. 221-229, Jan. 30,
     2002.
     The Rho-kinase is investigated in light of the signaling necessary for axon lengthening .
                 References Continued
R. Krishnamoorthy, P. Agarwal, G. Prasanna, K. Vopat, W. Lambert, H. Sheedlo, I. Pang, D. Shade, R. Wordinger, T.
         Yorio, A. Clark & N. Agarwal. Characterization of a transformed rat ganglion cell line. Mol. Brain Res. Vol. 86,
         pp. 1-12, 2001.
         The authors describe a cell line, RGC-5, which can be transformed for use in place of harvesting rat retinal
         ganglion cells.

P. Thagard. Coherent and creative conceptual combinations. Creative
      thought: An investigation of conceptual thought and structures.
      Washington, DC. American Psychological Association. pp. 29-141, 1997.

F. Meggio, A. Donella Deana, M. Ruzzene, A. Brunati, L. Cesaro, B. Guerra, T. Meyer, H. Mett, D. Fabbro, P. Furet, G.
        Dobrowolska, & L. Pinna. Different susceptibility of protein kinases to staurosporine inhibition kinetic studies
        and molecular bases for the resistance of protein kinase CK2. Eur. Jour. Biochem. Vol. 234, pp. 3317-322,
        1995.
          The author describe the relative sensitivities of protein kinases to the inhibitory effects of staurosporine.

P. Leprince, C. Bonvoisin, B. Rogister, C. Mazy-Servais, & . Moonen. Protein kinase- and staurosporine-dependent
         induction of neurite outgrowth and plasminogen activator activity in PC12 cells. Vol. 52, pp. 1399-1405, 1996.
         Protein kinase A and Protein kinase C interactions are studied to understand the effect of staurosporine on
         neurite lengthening in PC12 cell line.

N. Yanagihara, E. Tachikawa, F. Izumi, S. Yasugawa, H. Yammamoto, & E. Miyamoto. Staurosporine: An effictive
       Inhibitor for Ca+2/calmodulin-dependent protein kinase 2. Jour. Of Neurochem. Vol. 56, No. 1, pp. 294-298,
       1991.
       The authors investigated the inhibitory role of staurosporine on the protein kinase Ca +2/calmodulin-dependent
       protein kinase II.

				
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