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					                                            Paris, December 1-2, 2009




            International Conference on Myasthenia




ok-7, MuSK, and the development of the neuromuscular junction




                                            Yuji Yamanashi
                                            Dept. Genetics
                                            Inst. Medical Science
                                            University of Tokyo
                                                            Paris, December 1-2, 2009




                International Conference on Myasthenia




ok-7, MuSK, and the development of the neuromuscular junction


   •   The receptor protein-tyrosine kinase MuSK controls where the NMJ
       develops.

   •   The cytoplasmic, adaptor-like protein Dok-7 is an essential activator of
       MuSK
The receptor protein-tyrosine kinase (PTK) MuSK


                  Ig-like domains
                  (Agrin response)

                  Frizzled-like cystein-rich domain
                  (Wnt response ?)


                   PTK domain
                   (catalytic domain)        (Jennings et al., PNAS. 90: 2895-2899, 1993)
                                             (Zhang et al., Develop. Cell. 16: 325-327, 1993)



•   MuSK is essential for the development of NMJs
                (DeChiara et al., Cell 85: 501-512, 1996)



•   MuSK controls where NMJs form in myotubes
                (Kim et al., Nat. Neurosci. 11: 19-27, 2008)
AChRs form postsynaptic clusters in the central region of the muscle

                                    Myotube
    Nerve        AChR
                                              Wild-type

                                              AChRs form postsynaptic clusters (red)
                                              in the central region of myotubes.


                               AChR cluster
    ( E18.5 Diaphragm )   (postsynaptic region)


                                                  MuSK is expressed
                                                  in the central region of myotubes.
            MuSK controls where the NMJs form

                                 Myotube
Nerve        AChR
                                              Wild-type

                                              AChRs form postsynaptic clusters
                                              in the central region of myotubes.


                                              MuSK Tg (overexpression in the muscle)

                                              Uniformly overexpressed MuSK induces
                                              scattered AChR cluster formation; and
                                              NMJs are also scattered.
( E18.5 Diaphragm )                             (Kim et al., Nat. Neurosci. 11: 19-27, 2008)
                           AChR cluster
                      (postsynaptic region)
           MuSK controls where the NMJs form

                             Myotube
Nerve      AChR
                                          Wild-type

                                          AChRs form postsynaptic clusters (red)
                                          in the central region of myotubes.


                                          MuSK Tg (overexpression in the muscle)

                                          Uniformly overexpressed MuSK induces
                                          scattered AChR cluster formation; and
                                          NMJs are also scattered.


                                          MuSK KO

                                          MuSK-KO mice form no AChR cluster


 ( Diaphragm )         AChR cluster
                  (postsynaptic region)
                                           (Lin et al., Nature 410: 1057-1064, 2001)
                                           (Kim et al., Nat. Neurosci. 11: 19-27, 2008)
Neural Agrin activates MuSK via LRP4 coreceptor.
                                       (Kim et al., Cell 135: 334-342, 2008)
                                       (Zhang et al., Neuron 60: 285-297, 2008)



      motor
                                               Motor nerve terminal
                                             motor nerve terminal
      nerve
 .. .. ..
                               Agrin    Agrin                              Agrin
              L Agrin      L                    L                      L
              R            R                    R                      R
              P            P                    P                      P
              4            4                    4                      4




                  Rapsyn                            AChR cluster
 AChR                      MuSK
                    Myotube                           Myotube
Nerve/Agrin-independent but MuSK-dependent AChR clustering
               Nerve      AChR                           Nerve       AChR

       WT                                 Agrin KO



 HB9 KO                                   MuSK KO

                                    (E14.5 Diaphragm)
                                                        ( Lin et al, Nature 410:1057-1064, 2001)

     motor
                                                              Motor nerve terminal
                                                            motor nerve terminal
     nerve
.. .. ..
                                Agrin                   Agrin                              Agrin
             L Agrin        L                                   L                      L
             R              R                                   R                      R
             P              P                                   P                      P
             4              4                                   4                      4




                 Rapsyn                                             AChR cluster
AChR                      MuSK
                   Myotube                                            Myotube
Nerve/Agrin-independent but MuSK-dependent AChR clustering
               Nerve      AChR                           Nerve       AChR

       WT                                   Agrin KO



 HB9 KO                                     MuSK KO

                                    (E14.5 Diaphragm)
                                                        ( Lin et al, Nature 410:1057-1064, 2001)

     motor
                                        Nerve/Agrin-independent, probably muscle-
     nerve
                                        intrinsic element(s) may exist to activate
.. .. ..                                MuSK
                                Agrin
             L Agrin        L                                L                         L
             R              R                                R                         R
             P              P                                P                         P
             4              4                                4                         4


                                  (   )   ?
                 Rapsyn                                            AChR cluster
AChR                      MuSK
                   Myotube                                            Myotube
Nerve/Agrin-independent but MuSK-dependent AChR clustering
               Nerve      AChR                           Nerve       AChR

       WT                                   Agrin KO



 HB9 KO                                     MuSK KO

                                    (E14.5 Diaphragm)
                                                        ( Lin et al, Nature 410:1057-1064, 2001)

     motor
                                        Nerve/Agrin-independent, probably muscle-
     nerve
                                        intrinsic element(s) may activate MuSK
.. .. ..
                                Agrin
             L Agrin        L                                L                         L
             R              R                                R                         R
             P              P                                P                         P
             4              4                                4                         4


                                          Dok-7
                 Rapsyn                                            AChR cluster
AChR                      MuSK
                   Myotube                                            Myotube
                                        Dok-family

                                   Y Y Y YY Y Y Y Y Y   Y: SH2 binding/target motif
Dok-1 (62kD)    PH        PTB
               (100%)     (100%)
                                   Y Y YY Y       Y
Dok-2 (56kD)    PH        PTB                           High expression
               ( 40%)     ( 50%)                        in hematopoietic cells
                                     Y Y      Y Y
Dok-3 (58kD)    PH        PTB
               ( 50%)     ( 40%)



                                   YY Y YY
Dok-4 (41kD)    PH        PTB
               ( - - -)   ( 30%)


Dok-5 (36kD)
                                      YY Y
                                                        High expression
                PH        PTB
               ( - - -)   ( 35%)                        in non-hematopoietic cells
                                     Y YYY
Dok-6 (36kD)    PH        PTB
               ( - - -)   ( 35%)



                                             YY
Dok-7 (55kD)    PH        PTB                            High expression in muscles
               ( - - -)   ( 20%)
  GM-CSFR                                SCFR
  IL-3R                                  M-CSFR
  TLR4
  BCR                Dok-1/2
                     Dok-1/2

                                            Receptor
                                               PTKs
Non-Receptor
       PTKs




                      Ras-Erk

         (Proliferation & Cytokine Production)

                                     Yamanashi & Baltimore, Cell, 1997
                                     Yamanashi et al., Genes Dev., 2000
                                     Yasuda et al., J. Exp. Med., 2004,
                                     Shinohara et al., J. Exp. Med., 2005
                          Dok-family proteins

                                   Y Y Y YY Y Y Y Y Y   Y: SH2 binding/target motif
Dok-1 (62kD)    PH        PTB
               (100%)     (100%)
                                   Y Y YY Y       Y
Dok-2 (56kD)    PH        PTB                           High expression
               ( 40%)     ( 50%)                        in hematopoietic cells
                                     Y Y      Y Y
Dok-3 (58kD)    PH        PTB
               ( 50%)     ( 40%)



                                   YY Y YY
Dok-4 (41kD)    PH        PTB
               ( - - -)   ( 30%)


Dok-5 (36kD)
                                      YY Y
                                                        High expression
                PH        PTB
               ( - - -)   ( 35%)                        in non-hematopoietic cells
                                     Y YYY
Dok-6 (36kD)    PH        PTB
               ( - - -)   ( 35%)



                                             YY
Dok-7 (55kD)    PH        PTB                            High expression in muscles
               ( - - -)   ( 20%)


                                                               (Okada et al., Science, 2006)
 Postsynaptic localization of Dok-7 at Neuromuscular Junction (NMJ)

         gastrocnemius
                                                                Presynaptic vesicle
                                                                (+ACh: acetylcholine)

                                                                AChR (ACh receptor)
                                        myotube
                                                                cluster
   Dok-7      AChR            Merge
                                        NMJ (Neuromuscular Junction)
(a-Dok-7 Ab) (bungarotoxin)




                                              motor
                                              nerve
                                                           NMJ


                                                      myotube


          MuSK        AChR
          (Apel et al., Neuron, 1997)
 Postsynaptic localization of Dok-7 at Neuromuscular Junction (NMJ)

          gastrocnemius
                                                                                 Presynaptic vesicle
                                                                                 (+ACh: acetylcholine)

                                                                                 AChR (ACh receptor)
                                                    myotube
                                                                                 cluster
   Dok-7      AChR            Merge
                                                    NMJ (Neuromuscular Junction)
(a-Dok-7 Ab) (bungarotoxin)

                               Agrin-MuSK -Dok-7 signaling ?
        motor
                                                                          Motor nerve terminal
                                                                        motor nerve terminal
        nerve
   .. .. ..
                                      Agrin                         Agrin                           Agrin
                L Agrin          L                                          L                   L
                R                R                                          R                   R
                P                P        Dok-7           Dok-7             P                   P
                4                4                                          4                   4
                                                              PH
                                              PH              PH



                                         ?    PTB             PTB
                                                                    ?
                    Rapsyn
                                              Y               Y
                                                                                AChR cluster
   AChR                      MuSK
                                              Y               Y
                      Myotube                                                    Myotube
Dok-7 induces MuSK activation and AChR clustering in myotubes

                 / Dok-7          AChR          AChR
  IP:             IB: a pY
                    apY
  aMuSK           IB: a MuSK
                  IB:apY
  AChR-PD         apY-AChRb1

                  IB: a Dok-7
    WCL                                                      myotubes
                  IB: a Tubulin
                                    (    )        Dok-7     (C2C12)
            myotubes              ( AChR : ACh Receptor )


 IP : Immunoprecipitation
 IB : Immunoblotting
 PD: Pull-down
WCL: Whole cell lysates
apY: anti-phosphotyrosine
Dok-7 induces MuSK activation and AChR clustering in myotubes

                 / Dok-7                     AChR                AChR
  IP:             IB: a pY
                    apY
  aMuSK           IB: a MuSK
                  IB:apY
  AChR-PD         apY-AChRb1

                  IB: a Dok-7
    WCL                                                                           myotubes
                  IB: a Tubulin
                                               (    )             Dok-7          (C2C12)


                Agrin           Dok-7 can activate MuSK independently of
                                Agrin
            L                     L                               L                    L
            R                     R                               R                    R
            P                     P   Dok-7                       P                    P
            4                     4                               4                    4
                                       PH                  PH
                                                           PH                              PH

                                       PTB                 PTB                             PTB
                                                   Dok-7
                Rapsyn
                                       Y
                                                                        AChR cluster       Y
  AChR                   MuSK                              Y
                                       Y                   Y                               Y
                  Myotube                                                Myotube
Nerve/Agrin-independent but MuSK-dependent AChR clustering
               Nerve      AChR                           Nerve       AChR

       WT                                   Agrin KO



 HB9 KO                                     MuSK KO

                                    (E14.5 Diaphragm)
                                                        ( Lin et al, Nature 410:1057-1064, 2001)

     motor
                                        Nerve/Agrin-independent, probably muscle-
     nerve
                                        intrinsic element(s) may activate MuSK
.. .. ..
                                Agrin
             L Agrin        L                                L                         L
             R              R                                R                         R
             P              P                                P                         P
             4              4                                4                         4


                                          Dok-7
                 Rapsyn                                            AChR cluster
AChR                      MuSK
                   Myotube                                            Myotube
  Is Dok-7 a muscle-intrinsic activator of MuSK in vivo ?



           Agrin    Dok-7 can activate MuSK independently of
                    Agrin
       L            L                          L                  L
       R            R                          R                  R
       P            P    Dok-7                 P                  P
       4            4                          4                  4
                           PH            PH
                                         PH                           PH

                           PTB           PTB                          PTB
                                 Dok-7
           Rapsyn
                           Y
                                                   AChR cluster       Y
AChR                MuSK                 Y
                           Y             Y                            Y
             Myotube                                Myotube
            Nerve        AChR       Merge                     Nerve     AChR     Merge


 WT                                                WT
                E14.5




Dok-7                                             Dok-7
 KO                                                KO
                E14.5


              (E14.5 diaphragm)                                     (E18.5 diaphragm)

      motor
      nerve
                                     Dok-7 is essential for AChR clustering
                                                        and NMJ formation in vivo
 .. .. ..
                                    Agrin
              L Agrin           L                               L                    L
              R                 R                               R                    R
              P                 P       Dok-7                   P                    P
              4                 4                               4                    4
                                            PH            PH
                                                          PH                             PH

                                            PTB           PTB                            PTB
                                                  Dok-7
                    Rapsyn
                                            Y
                                                                      AChR cluster       Y
 AChR                          MuSK                       Y
                                            Y             Y                              Y
                        Myotube                                        Myotube
             E18.5
             WT Tg
                       100
                             Dok-7-
                       75
                             EGFP

Dok-7                  50

           IP: Dok-7
           IB: Dok-7


     motor
     nerve
                                  Overexpression of Dok-7 in vivo

.. .. ..
                                 Agrin
             L Agrin         L                               L                  L
             R               R                               R                  R
             P               P       Dok-7                   P                  P
             4               4                               4                  4
                                         PH            PH
                                                       PH                           PH

                                         PTB           PTB                          PTB
                                               Dok-7
                 Rapsyn
                                         Y
                                                                 AChR cluster       Y
AChR                        MuSK                       Y
                                         Y             Y                            Y
                     Myotube                                      Myotube
                                                  Nerve          AChR     Merge
             E18.5
             WT Tg
                                                                                       WT
                       100
                             Dok-7-
                       75
                             EGFP
                                                                                      Dok-7
Dok-7                  50
                                                                                       Tg
           IP: Dok-7
           IB: Dok-7                                    E18.5 Diaphragm
                                                       ( Inoue et al. Science Signaling, 2009 )

     motor
     nerve
                                  Overexpression of Dok-7 in vivo
                                            facilitates AChR cluster formation
.. .. ..
                                 Agrin
             L Agrin         L                               L                        L
             R               R                               R                        R
             P               P       Dok-7                   P                        P
             4               4                               4                        4
                                         PH            PH
                                                       PH                                   PH

                                         PTB           PTB                                  PTB
                                               Dok-7
                 Rapsyn
                                         Y
                                                                   AChR cluster             Y
AChR                        MuSK                       Y
                                         Y             Y                                    Y
                     Myotube                                        Myotube
                                                                           Nerve          AChR     Merge
                   100
    Myofibers(%)                                      WT
                   80
                                                      Dok-7 Tg                                                  WT
                   60

                   40

                   20

                    0
                                                                                                               Dok-7
                         1   2    3   4   5   6       7   8   9                                                 Tg
                     AChR clusters per myofiber
                                                                                 E18.5 Diaphragm
                                                                                ( Inoue et al. Science Signaling, 2009 )

     motor
     nerve
                                                          Overexpression of Dok-7 in vivo
                                                                    facilitates AChR cluster formation
.. .. ..
                                                      Agrin
                         L Agrin                  L                                   L                        L
                         R                        R                                   R                        R
                         P                        P           Dok-7                   P                        P
                         4                        4                                   4                        4
                                                                  PH            PH
                                                                                PH                                   PH

                                                                  PTB           PTB                                  PTB
                                                                        Dok-7
                                 Rapsyn
                                                                  Y
                                                                                            AChR cluster             Y
AChR                                      MuSK                                  Y
                                                                  Y             Y                                    Y
                                   Myotube                                                   Myotube
                                               Nerve          AChR     Merge
AChR cluster(s) on a myotube
                                                                                    WT
                               WT

                                                                                   Dok-7
                              Dok-7
                                                                                    Tg
                               Tg
                                                     E18.5 Diaphragm
                                                    ( Inoue et al. Science Signaling, 2009 )

     motor
     nerve
                               Overexpression of Dok-7 in vivo
                                         facilitates AChR cluster formation
.. .. ..
                              Agrin
             L Agrin      L                               L                        L
             R            R                               R                        R
             P            P       Dok-7                   P                        P
             4            4                               4                        4
                                      PH            PH
                                                    PH                                   PH

                                      PTB           PTB                                  PTB
                                            Dok-7
                 Rapsyn
                                      Y
                                                                AChR cluster             Y
AChR                      MuSK                      Y
                                      Y             Y                                    Y
                   Myotube                                       Myotube
                       Dok-7 induces NMJ formation in vivo
AChR cluster(s) on a myotube                                               E18.5




                                                   Number of NMJs
                                                                    200
                                                                               **
                                  WT                                150             **P < 0.001




                                                   (% of WT)
                                                                    100

                                                                    50
                                 Dok-7
                                                                     0
                                  Tg
                                                                          WT Dok-7 Tg

                                                           ( Inoue et al. Science Signaling, 2009 )

     motor
     nerve
                                  Overexpression of Dok-7 in vivo
                                            facilitates NMJ formation
.. .. ..
                                 Agrin
             L Agrin         L                                        L                       L
             R               R                                        R                       R
             P               P       Dok-7                            P                       P
             4               4                                        4                       4
                                         PH                 PH
                                                            PH                                    PH

                                         PTB               PTB                                    PTB
                                               Dok-7
                 Rapsyn
                                         Y
                                                                            AChR cluster          Y
AChR                      MuSK                              Y
                                         Y                  Y                                     Y
                   Myotube                                                    Myotube
                                                      Dok-7 activates MuSK in vivo
                                                             pY-MuSK                        AChR-PD (Btx)                                                 pY-AChR
       IP: MuSK                                       160                                                                                          160
                                                      140
                                                            *P < 0.05   *            Tg     -     -      -    +                                    140   **P < 0.003
        WT       Tg




                                                                                                                              Relative intensity
                                 Relative intensity
                                                      120                          Dok-7 +/+      -/-   -/-   +/+                                  120
100                   pY-MuSK                         100                                                                                          100
                                                                                                                                                                   **
                                                                                      50
                                                       80                                                           pY-AChR                         80
        IB: PY
                                                       60                                                                                           60
                                                                                                   IB: PY
                                                       40                                                                                           40
100                   MuSK                                                            50                            AChR
                                                       20                                                                                           20

       IB: MuSK                                         0                                       IB: AChR (b1)                                        0
                                                             WT     Dok-7 Tg                                                                             WT       Dok-7 Tg

                                                                                                        ( Inoue et al. Science Signaling, 2009 )

           motor
           nerve                                                             Dok-7 is an essential activator of MuSK
      .. .. ..
                                                                   Agrin
                       L Agrin                                 L                                              L                                               L
                       R                                       R                                              R                                               R
                       P                                       P            Dok-7                             P                                               P
                       4                                       4                                              4                                               4
                                                                             PH                         PH
                                                                                                        PH                                                              PH

                                                                             PTB                        PTB                                                             PTB
                                                                                           Dok-7
                             Rapsyn
                                                                             Y
                                                                                                                    AChR cluster                                        Y
      AChR                             MuSK                                                             Y
                                                                             Y                          Y                                                               Y
                                Myotube                                                                               Myotube
                 Nerve     AChR                       Nerve       AChR

       WT                                Agrin KO



 HB9 KO                                  MuSK KO


                                  (E14.5 diaphragm)
                                                      ( Lin et al, Nature 410:1057-1064, 2001)

     motor
     nerve        Agrin            Dok-7 is an essential activator of MuSK
.. .. ..
             L               L                              L                        L
             R               R                              R                        R
             P               P    Dok-7                     P                        P
             4               4                              4                        4
                                   PH                 PH
                                                      PH                                  PH

                                   PTB                PTB                                 PTB
                                             Dok-7
                  Rapsyn
                                   Y
                                                                 AChR cluster             Y
AChR                       MuSK                       Y
                                   Y                  Y                                   Y
                    Myotube                                        Myotube
            Nerve        AChR       Merge                      Nerve     AChR     Merge


 WT                                                  WT
                E14.5




Dok-7                                              Dok-7
 KO                                                 KO
                E14.5


              (E14.5 diaphragm)                                      (E18.5 diaphragm)

      motor
      nerve
                                            Why Agrin cannot induce AChR
                                                      clustering in Dok-7 KO mice?
 .. .. ..
                                    Agrin
              L Agrin           L                                L                    L
              R                 R                                R                    R
              P                 P       Dok-7                    P                    P
              4                 4                                4                    4
                                            PH             PH
                                                           PH                             PH

                                            PTB            PTB                            PTB
                                                   Dok-7
                    Rapsyn
                                            Y
                                                                       AChR cluster       Y
 AChR                          MuSK                        Y
                                            Y              Y                              Y
                        Myotube                                         Myotube
            Nerve        AChR       Merge                      Nerve     AChR     Merge


 WT                                                 WT
                E14.5




Dok-7                                             Dok-7
 KO                                                KO
                E14.5


              (E14.5 diaphragm)                                      (E18.5 diaphragm)

      motor
      nerve
                                            Dok-7 may be required for
                                                   Agrin-mediated MuSK activation
 .. .. ..
                                    Agrin
              L Agrin           L                                L                    L
              R                 R                                R                    R
              P                 P       Dok-7                    P                    P
              4                 4                                4                    4
                                            PH             PH
                                                           PH                             PH

                                            PTB            PTB                            PTB
                                                   Dok-7
                    Rapsyn
                                            Y
                                                                       AChR cluster       Y
 AChR                          MuSK                        Y
                                            Y              Y                              Y
                        Myotube                                         Myotube
                WT      Dok-7 -/-
    Agrin     0 10 30 0 10 30 (min)
        150

        100
 IP:
                   IB: pY
 MuSK
        150

        100
                 IB: MuSK


                            ( myotube )


                                         Dok-7 is required for
                                                    Agrin-mediated MuSK activation
                                        Agrin
              L Agrin               L                               L                  L
              R                     R                               R                  R
              P                     P       Dok-7                   P                  P
              4                     4                               4                  4
                                                PH            PH
                                                              PH                           PH

                                                PTB           PTB                          PTB
                                                      Dok-7
                  Rapsyn
                                                Y
                                                                        AChR cluster       Y
AChR                        MuSK                              Y
                                                Y             Y                            Y
                     Myotube                                             Myotube
                WT      Dok-7 -/-
    Agrin     0 10 30 0 10 30 (min)                                Agrin (-)           Agrin (+)
        150
                                                         WT
        100
 IP:
                   IB: pY                             Dok-7-/-
 MuSK
        150                                           Dok-7-/-
        100                                           + Dok-7
                 IB: MuSK
                                                                               AChR


                            ( myotube )                          ( Inoue et al. Science Signaling, 2009 )


                                         Dok-7 is required for
                                                    Agrin-mediated MuSK activation
                                        Agrin
              L Agrin               L                                   L                          L
              R                     R                                   R                          R
              P                     P       Dok-7                       P                          P
              4                     4                                   4                          4
                                                PH                 PH
                                                                   PH                                   PH

                                                PTB               PTB                                  PTB
                                                         Dok-7
                  Rapsyn
                                                Y
                                                                               AChR cluster             Y
AChR                        MuSK                                  Y
                                                Y                 Y                                     Y
                     Myotube                                                    Myotube
                Nerve       AChR                            Nerve      AChR     Merge


Agrin KO                                            WT



MuSK KO                                            Dok-7
                                                    KO

  ( Lin et al, Nature 410:1057-1064, 2001)                       (E18.5 diaphragm)

     motor
     nerve                          Dok-7 is a key muscle-intrinsic activator of MuSK

.. .. ..
                                   Agrin
             L Agrin           L                                 L                   L
             R                 R                                 R                   R
             P                 P       Dok-7                     P                   P
             4                 4                                 4                   4
                                             PH            PH
                                                           PH                            PH

                                             PTB           PTB                           PTB
                                                   Dok-7
                 Rapsyn
                                             Y
                                                                     AChR cluster        Y
AChR                       MuSK                            Y
                                             Y             Y                             Y
                    Myotube                                           Myotube
How does Dok-7 activate MuSK from the cytoplasmic compartment ?


     motor
     nerve                    Dok-7 is a key muscle-intrinsic activator of MuSK

.. .. ..
             L            L                             L                  L
             R            R                             R                  R
             P            P     Dok-7                   P                  P
             4            4                             4                  4
                                   PH             PH
                                                  PH                           PH

                                  PTB             PTB                          PTB
                                          Dok-7
                 Rapsyn
                                   Y
                                                            AChR cluster       Y
AChR                      MuSK                    Y
                                   Y              Y                            Y
                   Myotube                                   Myotube
How does Dok-7 activate MuSK from the cytoplasmic compartment ?


     motor
     nerve                    Dok-7 is a key muscle-intrinsic activator of MuSK

.. .. ..
             L            L                              L                  L
             R            R      ?                       R                  R
             P            P                              P                  P
             4            4                              4                  4
                                     PH            PH
                                                   PH                           PH

                                     PTB           PTB                          PTB
                                           Dok-7
                 Rapsyn
                                     Y
                                                             AChR cluster       Y
AChR                      MuSK                     Y
                                     Y             Y                            Y
                   Myotube                                    Myotube
The ectodmain of MuSK is dispensble for Dok-7-mediated activation



     MuSK      WT DIg
     Dok-7       +     +
                              pY-MuSK                              PH
                              pY-MuSK-DIg
 IB: apY                                                           PTB


                                                                   Y
                                                                   Y
                                                    MuSK-DIg
                              WT                                  Dok-7
                              MuSK-DIg
 IB: amyc                                    Dok-7 may target the cytoplasmic
   (MuSK)                                    portion of MuSK



IB: aDok-7                    DOK-7


              (293T cells)
  ( Inoue et al. Science Signaling, 2009 )
The ectodmain and transmembrane region of MuSK are dispensble
                 for Dok-7-mediated activation

     MuSK      WT DIg      Cyt
                                                             PH
     Dok-7       +     +      +
                                                             PTB
                                    pY-MuSK
                                    pY-MuSK-DIg               Y
 IB: apY                                                      Y

                                    pY-MuSK-Cyt   MuSK-Cyt   Dok-7

                                    WT
                                    MuSK-DIg
 IB: amyc
   (MuSK)
                                    MuSK-Cyt
                                                             PH

IB: aDok-7                          DOK-7                    PTB


                                                              Y
                                                              Y
              (293T cells)                        MuSK-DIg
                                                             Dok-7
  ( Inoue et al. Science Signaling, 2009 )
The ectodmain and transmembrane region of MuSK are dispensble
                 for Dok-7-mediated activation

    MuSK    WT DExt Cyt
                                                                          PH
    Dok-7       +    +   +
                                                                          PTB
                                 pY-MuSK
                                 pY-MuSK-DIg                               Y
 IB: apY                                                                   Y

                                 pY-MuSK-Cyt                 MuSK-Cyt    Dok-7


                         Dok-7 targets the cytoplasmic portion of MuSK


            L                L                                L                  L
            R                R                                R                  R
            P                P      Dok-7                     P                  P
            4                4                                4                  4
                                      PH               PH
                                                       PH                            PH

                                      PTB              PTB                           PTB
                                               Dok-7
                Rapsyn
                                      Y
                                                                  AChR cluster       Y
 AChR                      MuSK                        Y
                                      Y                Y                             Y
                    Myotube                                        Myotube
 Does Dok-7 directly activate the cytoplasmic portion of MuSK?



                     Dok-7 targets the cytoplasmic portion of MuSK


        L              L                           L                  L
        R              R                           R                  R
        P              P    Dok-7                  P                  P
        4              4                           4                  4
                              PH             PH
                                             PH                           PH

                              PTB            PTB                          PTB
                                     Dok-7
            Rapsyn
                              Y
                                                       AChR cluster       Y
AChR                 MuSK                    Y
                              Y              Y                            Y
              Myotube                                   Myotube
        MuSK-cyt
                   Dok-7
                                  PH

                                  PTB
 250
 150
 100
  75                              Y
                                  Y     Dok-7
  50

  37


  25
                                 MuSK-Cyt
(kDa)
               (CBB)



                                        Dok-7 targets the cytoplasmic portion of MuSK


                           L              L                           L                  L
                           R              R                           R                  R
                           P              P     Dok-7                 P                  P
                           4              4                           4                  4
                                                 PH             PH
                                                                PH                           PH

                                                 PTB            PTB                          PTB
                                                        Dok-7
                               Rapsyn
                                                 Y
                                                                          AChR cluster       Y
AChR                                    MuSK                    Y
                                                 Y              Y                            Y
                                 Myotube                                   Myotube
Dok-7              MuSK-cyt
                                       MuSK-cyt
                                       + Dok-7
                 0 5 15 30 60 120   0 5 15 30 60 120 (min)
 PH
            75
 PTB                                                 pY-Dok-7-His
            50

 Y          37                                       pY-MuSK-cyt
 Y
            25

                     IB: pY            IB: pY

 MuSK-Cyt         pY: phosphotyrosine
                                                                    ( Inoue et al. Science Signaling, 2009 )



        Dok-7 directly activates the cytoplasmic portion of MuSK


        L                     L                                     L                             L
        R                     R                                     R                             R
        P                     P     Dok-7                           P                             P
        4                     4                                     4                             4
                                        PH                   PH
                                                             PH                                         PH

                                       PTB                   PTB                                        PTB
                                                   Dok-7
            Rapsyn
                                        Y
                                                                          AChR cluster                  Y
 AChR                   MuSK                                 Y
                                        Y                    Y                                          Y
                 Myotube                                                     Myotube
Dok-7              MuSK-cyt
                                       MuSK-cyt                            MuSK-cyt
                                       + Dok-7                             + Dok-7DN
                 0 5 15 30 60 120   0 5 15 30 60 120 (min)               0 5 15 30 60 120 (min)
 PH
            75
 PTB                                                 pY-Dok-7-His
            50

            37                                                                              pY-MuSK-cyt
 Y                                                   pY-MuSK-cyt
 Y
            25

                     IB: pY            IB: pY                                IB: pY

 MuSK-Cyt         pY: phosphotyrosine
                                                                    ( Inoue et al. Science Signaling, 2009 )



        Dok-7 directly activates the cytoplasmic portion of MuSK


        L                     L                                     L                             L
        R                     R                                     R                             R
        P                     P     Dok-7                           P                             P
        4                     4                                     4                             4
                                        PH                   PH
                                                             PH                                         PH

                                       PTB                   PTB                                        PTB
                                                   Dok-7
            Rapsyn
                                        Y
                                                                          AChR cluster                  Y
 AChR                   MuSK                                 Y
                                        Y                    Y                                          Y
                 Myotube                                                     Myotube
                                                                                               *
                                    Myotube
                                                    Nerve          AChR     Merge

             WT                                                                          WT



           Dok-7                                                                        Dok-7
            Tg                                                                           Tg
Overexpression              AChR cluster                  E18.5 Diaphragm
       of Dok7         (postsynaptic region)
                                                         ( Inoue et al. Science Signaling, 2009 )

     motor
     nerve
                                   Why uniform overexpression of Dok-7
                                     facilitates centrally formation of the NMJ ?
.. .. ..
                                   Agrin
             L Agrin           L                               L                        L
             R                 R                               R                        R
             P                 P       Dok-7                   P                        P
             4                 4                               4                        4
                                           PH            PH
                                                         PH                                   PH

                                           PTB           PTB                                  PTB
                                                 Dok-7
                  Rapsyn
                                           Y
                                                                    AChR cluster              Y
AChR                       MuSK                          Y
                                           Y             Y                                    Y
                    Myotube                                           Myotube
                                                                                  *
                            Myotube
                                         Nerve     AChR        Merge

          WT                                                                WT



        Dok-7                                                              Dok-7
         Tg                                                                 Tg
Overexpression        AChR cluster           E18.5 Diaphragm
       of Dok7   (postsynaptic region)
                                            ( Inoue et al. Science Signaling, 2009 )


    Is MuSK expressed in the central region of Dok-7 Tg muscle?
   Dok-7 is essential for mid-muscle expression of MuSK and AChR
                          WT   Dok-7Tg Dok-7 -/-           WT   Dok-7Tg Dok-7 -/-




                  E14.5




                                                   E14.5
                  E18.5




                                                   E18.5
in situ hybrid.
  (Diaphragm)                   MuSK                            AChRa




        MuSK is expressed in the central region of Dok-7 Tg muscle, and

  Dok-7 is required for the centrally restricted expression of MuSK and AChR
   Dok-7 is essential for mid-muscle expression of MuSK and AChR
                          WT   Dok-7Tg Dok-7 -/-           WT   Dok-7Tg Dok-7 -/-




                  E14.5




                                                   E14.5
                  E18.5




                                                   E18.5
in situ hybrid.
  (Diaphragm)                   MuSK                            AChRa




    Immunostaining
        (Diaphragm)                                                          scale bar: 100 mm
   Dok-7 is essential for mid-muscle expression of MuSK and AChR
                           WT   Dok-7Tg Dok-7 -/-            WT        Dok-7Tg Dok-7 -/-




                  E14.5




                                                     E14.5
                  E18.5




                                                     E18.5
in situ hybrid.
  (Diaphragm)                    MuSK                                  AChRa



 Dok-7 regulates the mid-muscle NMJ formation by controlling
    MuSK activity, expression pattern, and responsiveness to Agrin.

             L                     L                                     L                    L
             R                     R                                     R                    R
             P                     P      Dok-7                          P                    P
             4                     4                                     4                    4
                                             PH                   PH
                                                                  PH                              PH

                                             PTB              PTB                                 PTB
                                                    Dok-7
                  Rapsyn
                                             Y
                                                                               AChR cluster       Y
  AChR                           MuSK                         Y
                                             Y                Y                                   Y
                          Myotube                                                 Myotube
A simplified model of Dok-7-MuSK-Agrin signaling in the NMJ formation

  Ⅰ                                                Ⅲ                                  Motor nerve

                                                                    ACh
                                                                                            Agrin
                                                                                      ACh
                            MuSK                                                                    MuSK
                   LRP4

                                    Dok-7                                                            Dok-7


                                (myotube)                                                            (myotube)

      Dok-7 is expressed in the central region           ACh disperses non-innervated AChR clusters
       of skeletal muscle, where it activates              but the local mediator Agrin fully activates
      MuSK and induces MuSK’s mid-muscle               Dok-7-primed/activated MuSK and counteracts ACh.
                 pattern formation.


  Ⅱ                                                Ⅳ                      Motor nerve


                             MuSK                                             Agrin
                AChR                                                   ACh
                                                                                      MuSK
                                     Dok-7
          Rapsyn
                                                                                        Dok-7
                                (myotube)

        Activated MuSK induces postsynaptic                                             (myotube)
       specialization in the central region of  
      muscle independently of nerve and Agrin.                 Dok-7-MuSK-Agrin triad signaling
                                                            promotes and reinforces NMJ formation.
                                                                      Nerve   AChR    Merge
   Nerve       AChR        Merge
                                                           WT
                                        WT
                                                     Dok-7 -/-

                                        Dok-7        Dok-7 -/-
                                         Tg        + Dok-7 Tg
( Inoue et al. Science Signaling, 2009 )
                                                                E14.5 Diaphragm (unpublished)

     motor
     nerve                                  Dok-7 Tg rescues NMJs in Dok KO mice
.. .. ..
                                    Agrin
             L Agrin            L                                 L                    L
             R                  R                                 R                    R
             P                  P       Dok-7                     P                    P
             4                  4                                 4                    4
                                            PH             PH
                                                           PH                                 PH

                                            PTB            PTB                             PTB
                                                   Dok-7
                  Rapsyn
                                            Y
                                                                       AChR cluster        Y
AChR                       MuSK                            Y
                                            Y              Y                               Y
                    Myotube                                              Myotube
                                                           Paris, December 1-2, 2009




                International Conference on Myasthenia




ok-7, MuSK, and the development of the neuromuscular junction


  •   Dok-7 is an essential, muscle-intrinsic activator of MuSK, in NMJ
      development

  •   Enhanced Dok-7 signaling may ameliorate DOK7 and MuSK-related
      myasthenia
Collaborators
                        David Beeson
                        Jackie Palace
Masakatsu Motomura Angela Vincent
Irokazu Shiraishi       (Oxford Univ.)
Katsumi Eguchi
                        Peter Fawcett
(Nagasaki Univ.)
                        Clarke Slater
Takashi Okada           (Univ. Newcastle)
Shin’ichi Takeda
                        Juliane S. Muller
(Natl. Inst. Neurosci.)
                        Hanns Lochmuller
Shigeru Kakuta          (Ludwig-Maxim. Univ.)   ・Osamu Higuchi
Sachiko Kubo                                    ・Akane Inoue
                        Frederic Chevessier
Yoichiro Iwakura                                  Kiyoko Setoguchi
                        Veit Witzemann
Takafumi Jigami         (Max-Planck Inst.       ・Johko Hamuro
Tetsu Akiyama           Med.)                   ・Kumiko Okada
(Univ. Tokyo)
                        Daniel Hantai

				
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