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Role of prion protein (PrP_1hnC) in the mammalian dentition [Elektronische Ressource] / vorgelegt von Kurt Schneider

143 pages
CRole of Prion Protein (PrP )in the Mammalian DentitionInaugural-Dissertationzur Erlangung des Doktorgradesder Mathematisch-Naturwissenschaftlichen Fakult¨ atder Heinrich-Heine-Universit¨at Dussel¨ dorfvorgelegt vonKurt Schneideraus Nurn¨ bergD¨ usseldorf, Mai 2010Aus der Poliklinik fur¨ Zahnerhaltung,Parodontologie und Endodontologieder Heinrich-Heine-Universit¨at Dussel¨ dorfTeile dieser Arbeit wurden bereits ver¨ offentlicht:Kurt Schneider, Yuk¨ sel Korkmaz, Klaus Addicks, Hermann Lang, WolfgangH.-M. Raab. Prion Protein (PrP) in Human Teeth: An Unprecedented Pointerto PrP’s Function. Journal of Endodontics, 33(2): 110–113, February 2007.Gedruckt mit Genehmigung derMathematisch-Naturwissenschaftlichen Fakult¨ at derHeinrich-Heine-Universit¨ at Dussel¨ dorfReferent: Univ.-Professor Dr. med. dent. Wolfgang H.-M. RaabKoreferent: Univ.-Professor Dr. rer. nat. Dieter WillboldTag der mundlic¨ hen Prufung:¨ 28.05.2010Die hier vorgelegte Dissertation habe ich eigenst¨ andig und ohne unerlaubteHilfe angefertigt. Die Dissertation wurde in der vorgelegten oder in ahnlic¨ herForm noch bei keiner anderen Institution eingereicht. Ich habe bisher keineerfolglosen Promotionsversuche unternommen.D¨ usseldorf, den 28.05.2010(Kurt Schneider)1AcknowledgementsI would like to thank my venerable teacher, Professor Wolfgang H.–M. RaabD.D.S., for taking care of and the continuous support for my work, as well asthe numerous valuable suggestions and discussions.
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CRole of Prion Protein (PrP )
in the Mammalian Dentition
Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakult¨ at
der Heinrich-Heine-Universit¨at Dussel¨ dorf
vorgelegt von
Kurt Schneider
aus Nurn¨ berg
D¨ usseldorf, Mai 2010Aus der Poliklinik fur¨ Zahnerhaltung,
Parodontologie und Endodontologie
der Heinrich-Heine-Universit¨at Dussel¨ dorf
Teile dieser Arbeit wurden bereits ver¨ offentlicht:
Kurt Schneider, Yuk¨ sel Korkmaz, Klaus Addicks, Hermann Lang, Wolfgang
H.-M. Raab. Prion Protein (PrP) in Human Teeth: An Unprecedented Pointer
to PrP’s Function. Journal of Endodontics, 33(2): 110–113, February 2007.
Gedruckt mit Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakult¨ at der
Heinrich-Heine-Universit¨ at Dussel¨ dorf
Referent: Univ.-Professor Dr. med. dent. Wolfgang H.-M. Raab
Koreferent: Univ.-Professor Dr. rer. nat. Dieter Willbold
Tag der mundlic¨ hen Prufung:¨ 28.05.2010Die hier vorgelegte Dissertation habe ich eigenst¨ andig und ohne unerlaubte
Hilfe angefertigt. Die Dissertation wurde in der vorgelegten oder in ahnlic¨ her
Form noch bei keiner anderen Institution eingereicht. Ich habe bisher keine
erfolglosen Promotionsversuche unternommen.
D¨ usseldorf, den 28.05.2010
(Kurt Schneider)
1Acknowledgements
I would like to thank my venerable teacher, Professor Wolfgang H.–M. Raab
D.D.S., for taking care of and the continuous support for my work, as well as
the numerous valuable suggestions and discussions.
I’m deeply indepted to Professor Istvan Szelenyi, V.M.D., who was the first to
make me unambiguously aware of the benefits of scientific work. Without his
effective encouragement, this thesis would never have come into existence.
Thanks go to Professor Stefan Pohlmann,¨ Ph.D. for carefully reading the
manuscript and giving me invaluable tips how to lay out this thesis.
I thank Yuksel¨ Korkmaz D.D.S., who tought me everything I know about
immunohistochemistry. Furthermore, Dr. Korkmaz was an invaluable help in
interpreting the histological results.
I’m indepted to Wiebke Wetzel Ph.D., who gave me the chance to learn many
molecular biological methods.
I thank Mrs. Vasiliki Liakakou for her permanent and unfatiguable support in
the procurement of instruments, chemicals and reagents.
Thanks go to Ms. Britta Michaelsen for translating historical papers about
scrapie from French to German.
Furthermore, thanks go to Professor Donald Knuth, Dr. Leslie Lamport and
Dr. Oren Patashnik, for supplying us with the extraordinary tools named T X,E
ALT X, and BibT X, respectively, which make writing a thesis a real pleasure.E E
I would like to express my apologies to the many people who accompanied me
on my way to earning this degree and whom I’ve failed to mention for lack of
space. I trust the omission does not offend.
2Contents
Summary 15
Zusammenfassung 17
I Introduction 19
1 PrP is found in different isoforms 19
1.1 FeaturesofthePrionProteinanditsGene............20
1.2 PrionProteinsinVertebrates ...................24
C1.3 PrP andtheMammalianDentition ....25
1.3.1 TethGrinding.......................25
1.3.2 Threat of Disease Transmission during Dental Treatment 26
1.3.3 HistologicStudiesofPrPExpresion...........30
II Aims of this Study 32
III Materials and Methods 33
2 Mouse Teeth 33
2.1 Genotypingofmice.........................3
2.1.1 Tisuepreparation..........3
2.1.2 DNAIsolationandPCR..................3
2.2 DeterminationofCopperandManganeseContents .......34
2.2.1 Tisuepreparation.....................34
2.2.2 CuandMnDetermination......34
32.3 ScanningElectronMicroscopy...................35
2.3.1 Tisuepreparation..........35
2.3.2 ScanningEM.............35
2.3.3 Quantificationofdata ...................36
2.3.4 Statisticalanalysis..........36
2.4 HistologyandImmunohistochemistry....36
2.4.1 H&EStaining........................37
2.4.2 Immunohistochemistry........38
2.4.3 ImmunohistochemicalControls...38
3 Human Teeth 38
3.1 CelCulture.............................38
3.1.1 ColectionofSamples.........38
3.1.2 IsolationofCels...........39
3.1.3 CultivationofCels.....................39
3.1.4 RNAIsolationandPCR.......39
3.1.5 Western-Blot.............40
3.1.6 Induction and Detection of Calcifying Potential . . . . . 41
3.2 HistologyandImmunohistochemistry...............41
3.2.1 ColectionofSamples.........41
3.2.2 TissuePreparation..........41
3.2.3 H&EStaining........................42
3.2.4 Immunohistochemistry........42
3.2.5 ImmunohistochemicalControls...43
3.2.5.1 Immunohistochemical Controls by Omission of
Antibodies ....................43
3.2.5.2 Immunohistochemical Controls by Preabsorp-
tionTechnique.......43
43.2.5.3 Immunohistochemical Controls using Non-Relevant
Antibodies ....................43
3.2.5.4 Immunohistochemical Controls by Immunoblot 43
3.2.6 Densitometry.............44
3.2.7 StatisticalAnalysis.....................4
IV Results 45
4 Mouse Teeth 45
0/04.1 Characterization of PrP Mice by Genotyping and Western-Blot 45
0/04.1.1 Genotyping of PrP Mice.................45
0/04.1.2 Western-Blot Characterization of PrP Mice......46
4.2 DeterminationofCuandMncontents...46
4.2.1 CuandMnDetermination.................46
4.2.2 Quantificationofdata ........46
4.3 ScanningElectronMicroscopy........47
4.3.1 ScanningEM........................47
4.4 HistologyandImmunohistochemistry....50
4.4.1 Immunohistochemical Localization of PrP in Mouse Teeth 50
4.4.2 Characterization of Dentin and Bone Structures in Wild-
0/0Type and in PrP MicebyH&EStaining........51
5 Human Teeth 53
5.1 Expression and Detection of PrP in Different Types of Human
Cels.................................53
5.1.1 Detection of PrP at the mRNA Level in Different Types
ofHumanCelLines.........53
5.1.2 Western-Blot Detection of PrP in Different Types of Hu-
manCellLines.......................54
55.2 Immunohistochemical Detection of PrP in Different Cell Types
ofHumanTeth...........................56
5.2.1 Dentin-PulpComplex.........56
5.2.1.1 Odontoblasts...................56
5.2.1.2 NerveFibers........56
5.2.2 PeriodontalLigament....................56
5.2.2.1 Cementoblasts .......57
5.2.2.2 EpithelialRestsofMalasez...........57
5.2.2.3 NerveFibers...................58
5.3 Increased Expression of PrP beneath a Dentin-Cavity Filling . . 58
5.3.1 Characterization of Dentin-Pulp Complex Cells by H&E 58
5.3.2 Expression of PrP in Pulp Stroma Cells and Odonto-
blastsBeneathaDentin-Cavity..............59
5.4 ImmunohistochemicalControls........60
5.4.1 Immunohistochemical Controls by Omission of Antibodies 60
5.4.2 Immunohistochemical Controls by Preabsorption Tech-
nique.............................62
5.4.3 Positive Immunohistochemical Controls Using Non-Relevant
Antibodies....63
5.5 DensitometryandStatisticalAnalysis...............65
5.6 Characterization of Cultivated Human Dental Cells by Alizarin-
RedStaining .................6
V Discussion 67
6 Methodical Considerations 67
6.1 ScanningElectronMicroscopy...................67
66.2 Determination of Copper and Manganese Contents in Mouse
Teth.................................69
6.3 DetectionofPrPExpresionbyPCR....69
6.4 Comparison of Knockout-, Wild-Type Mouse Brain and Cell
CultureCels ............................69
6.5 Involvement of PrP in the Regulation of Different Cells in the
Dentin-PulpComplexandPeriodontalLigament.........70
6.5.1 Immunohistochemistry...................71
6.5.1.1 Immunohistochemical Controls with Non-Relevant
Antibodies .........71
6.5.1.2 Immunohistochemical Controls by Preabsorp-
tionofPrPAntibodies..............72
6.5.1.3 Immunohistochemical Controls by Staining with
Antibodies Directed Against Different Epitopes
ofPrP.......................72
6.5.1.4 Positive and Negative Immunohistochemical Con-
trols.............72
6.5.1.5 Immunohistochemical Incubations and H&E Stain-
ing Comparing Wild-Type with Knockout Mouse
Teth.......................73
6.5.1.6 Demonstration of the Specificity of the 6H4
AntibodybyWestern-Blot............73
7 Involvement of PrP in Matrix Formation, Matrix Biomin-
eralization and in Neurotransmission in Cells of the Dentin-
Pulp Complex and in Cells of the Desmodontal (Cement, Pe-
riodontal Ligament and Alveolar Bone) Unit 74
7.1 CopperandManganeseContentsinMouseTeth........74
7.1.1 CopperContentsinSoftTisue..............75
7.1.2 CopperContentsinTeeth......76
7C7.1.3 Suggested Role(s) of PrP by Copper Binding in the
Desmodontal Connective Tissue Formation and Tissue
Maintenance as well as in the Dentin- and Bone-Matrix
FormationandMatrixBiomineralization.........7
7.2 Cellular Regulation of Odontoblasts and Nerve Fibers by PrP . 79
7.2.1 PrP may be Involved in the Formation and Biomineral-
isationofDentinMatrix..................79
7.2.2 PrP may be Involved in the Regulation of Nerve Fiber
Formation and/or Neurotransmission in the Dentin-Pulp
ComplexDuringHumanToothDevelopment.......80
7.3 PrPinCelsofthePeriodontalLigament.............81
7.3.1 Formation of Cementum may be Regulated by PrP dur-
ingHumanToothDevelopment..............81
7.3.2 PrP Localization in the ERM is Compatible with an
Involvement of PrP in the Epithelial Differentiation to
Development of Enamel or Epithelial Transformation to
CystsandAmeloblastoma.................82
7.3.3 PrP may be Involved in the Formation Mechanisms of
Nerve Fibers and/or Neurotransmission in the Periodon-
tal Ligament during Human Tooth Development . . . . . 83
7.3.4 Alveolar Bone Matrix Formation and Matrix Biominer-
alisationmaybeRegulatedbyPrP............83
8 Considerations Concerning the Transmission of TSEs 84
8.1 RisksbyandforAnimals......................84
8.1.1 SheddingofTeth...84
8.1.2 WearofTeth........................85
8.2 RiskbyandforHumans...........85
8.2.1 PreparationofCavities...................86
8

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