PMP22-overexpressing mice as a model for Charcot-Marie-Tooth 1A neuropathy implicate a role of immune-related cells [Elektronische Ressource] / vorgelegt von Bianca Dorothea Kohl

Bianca Kohl

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

PMP22-overexpressing mice
as a model for
Charcot-Marie-Tooth 1A neuropathy
implicate a role of immune-related cells







Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg

vorgelegt von
Bianca Dorothea Kohl
aus Rüdenau



Würzburg, 2009























Eingereicht am: 03.08.2009



Mitglieder der Promotionskommission:

Vorsitzender: Prof. Dr. Martin Müller

Gutachter: Prof. Dr. Rudolf Martini

Gutachter: Prof. Dr. Erich Buchner



Tag des Promotionskolloquiums: 04.12.2009



Doktorurkunde ausgehändigt:

- 2 -





















Eidesstattliche Erklärung

Hiermit erkläre ich, die vorliegende Arbeit selbständig angefertigt und keine anderen als die
angegebenen Hilfsmittel verwendet zu haben.

Diese Arbeit hat weder in gleicher noch in ähnlicher Form in einem anderen
Prüfungsverfahren vorgelegen.

Ich habe in keinem früheren Verfahren einen akademischen Grad erworben oder zu
erwerben versucht.



Würzburg, 03.08.2009
Bianca Kohl

- 3 -












Für
Martin und meine Familie




- 4 -Table of contents

1. Summary ------------------------------------------------------------------------------------------- 8 -

2. Zusammenfassung --------------------------------------------------------------------------- 10 -

3. Introduction ------------------------------------------------------------------------------------- 12 -
3.1 The myelin sheath-------------------------------------------------------------------------- 12 -
3.1.1 Formation and structure of the myelin sheath --------------------------------------- - 12 -
3.1.2 Proteins of the peripheral nervous system ------------------------------------------- - 14 -
3.2 Disorders of the peripheral nervous system ----------------------------------------- 15 -
3.2.1 Polyneuropathies --------------------------------------------------------------------------- - 15 -
3.2.2 Charcot-Marie-Tooth disease------------------------------------------------------------ - 16 -
3.2.3 PMP22 mutations as models for CMT1A --------------------------------------------- - 18 -
3.2.4 The immune system as a mediator of pathology in inherited
demyelination -------------------------------------------------------------------------------- - 20 -
3.3 Aim of the study--------------------------------------------------------------------------- 22 -

4. Materials and methods ---------------------------------------------------------------------- 23 -
4.1 Equipment, reagents, solutions, buffers, media and antibodies ---------------- 23 -
4.2 Mutant mice and genotyping ------------------------------------------------------------ 23 -
4.3 Cell culture ----------------------------------------------------------------------------------- 25 -
4.4 Bone marrow transplantation- 25 -
4.5 Flow cytometry------------------------------------------------------------------------------ 26 -
4.6 Isolation of RNA and protein from peripheral nerves------------------------------ 26 -
4.7 Reverse transcription and quantitative real-time PCR ---------------------------- 27 -
4.8 Western blot analyses--------------------------------------------------------------------- 28 -
4.9 Immunohistochemical analyses -------------------------------------------------------- 29 -
4.10 Electron microscopic analyses- 31 -
4.11 Neurographic recordings---------------------------------------------------------------- 32 -
4.12 Functional tests --------------------------------------------------------------------------- 32 -
4.13 In vivo inhibition of the MEK1/2/ERK1/2 signaling cascade by CI-1040 ---- 33 -
4.14 Statistical analyses----------------------------------------------------------------------- 33 -





- 5 -Table of contents

5. Results-------------------------------------------------------------------------------------------- 34 -
5.1 Morphological analyses of peripheral nerves of juvenile PMP22 mutant
mice- 34 -
5.1.1 Investigations of peripheral nerves of PMP22tg mice at the age P7 reveal
no alterations in axonal sorting---------------------------------------------------------- - 34 -
5.1.2 Overexpression of PMP22 leads to thicker myelin sheaths in peripheral
nerves of PMP22tg mice at the age P7 ----------------------------------------------- - 36 -
5.2 The functional contribution of T-lymphocytes to the pathogenesis of
PMP22tg mice------------------------------------------------------------------------------ 38 -
5.2.1 Pathological alterations are non-significantly increased in peripheral
nerves of PMP22tg/RAG-1-/- mice at the age of 12 months--------------------- - 38 -
5.2.1.1 The absence of mature lymphocytes does not influence
macrophage numbers within nervous tissue of PMP22tg mice ------ - 38 -
5.2.1.2 Peripheral nerves of PMP22tg/RAG-1-/- mice disclose a non-
significant increase of pathological alterations at 12 months of age- - 40 -
5.2.2 Deficiency of PD1 (PD-1-/-) in PMP22tg mice do not cause an aggravated
neuropathological phenotype ------------------------------------------------------------ - 43 -
5.2.2.1 The numbers of immune cells are not altered in peripheral
nerves of PMP22tg BMC PD-1-/- compared to PMP22tg BMC wt --- - 43 -
5.2.2.2 The degree of pathological alterations is equal in nervous tissue
of PMP22tg BMC PD-1-/- and of PMP22tg BMC wt --------------------- - 45 -
5.2.2.3 Sciatic nerve conduction properties are not altered in
PMP22tg BMC PD-1-/- compared to PMP22tg BMC wt ---------------- - 47 -
5.2.2.4 exhibit stride properties similar to
PMP22tg BMC wild type -------------------------------------------------------- - 48 -
5.3 The functional contribution of macrophages to the pathogenesis of
PMP22tg mice------------------------------------------------------------------------------- 50 -
5.3.1 MCP-1 mRNA expression is increased in peripheral nerves of
PMP22tg mice ------------------------------------------------------------------------------- - 50 -
5.3.2 Nervous tissue of PMP22/MCP-1 double mutants show differences in
the demyelinating phenotype ------------------------------------------------------------ - 51 -
5.3.2.1 MCP-1 deficiency causes a reduced number of macrophages in
peripheral nerves of PMP22tg mutants compared to wild type mice - 52 -


- 6 -Table of contents

5.3.2.2 PMP22/MCP-1 double mutant mice show different cytokine
expression -------------------------------------------------------------------------- - 54 -
5.3.2.3 Reduction and deficiency of MCP-1 leads to amelioration of
disease in PMP22tg mice------------------------------------------------------- - 56 -
5.3.2.4 Properties of axonal integrity are only improved in the complete
absence of MCP-1 in PMP22tg mice ---------------------------------------- - 59 -
5.3.3 The MEK1/2/ERK1/2 signaling cascade is involved in the expression of
MCP-1 in peripheral nerves of PMP22tg mice -------------------------------------- - 63 -
5.3.3.1 The MEK1/2/ERK1/2 signaling cascade is activated in peripheral
nerves of PMP22tg mice-------------------------------------------------------- - 63 -
5.3.3.2 In vivo inhibition of the MEK1/2/ERK1/2 signaling cascade leads to
a reduction of macrophage numbers in nerves of PMP22tg mice---- - 66 -

Discussion ------------------------------------------------------------------------------------------ 68 -
6.1 Pathological alterations in juvenile PMP22tg mice--------------------------------- 68 -
6.2 Lymphocytes do not have a crucial role in the neuropathology of
PMP22tg mice------------------------------------------------------------------------------- 71 -
6.3 Macrophages have a modulating capacity in the neuropathology of
PMP22t- 73 -
6.4 PMP22tg and P0+/- mice: comparison of two models for inherited
neuropathies--------------------------------------------------------------------------------- 79 -
6.5 Closing remarks ----------------------------------------------------------------------