Tab. 7 (zu Abb. 17a): mRNA-Expression von HSPs in Neuronen in vitro nach HS und OS. Mittelwerte (MW) in Prozent zu „HS 3h“ in Relation zu GAPDH und Standardabweichungen (SD).
HSP90 HSP70 HSP32 HSP25 αB-Crystallin MW [%]
Co 86,7 12,6 549,8 32,5 53,1
HS 3h 100,0 100,0 100,0 100,0 100,0
HS 7h 81,0 64,7 727,8 77,4 48,7
OS 3h 70,5 32,9 144,8 36,9 44,9
OS 7h 64,7 23,6 1779,8 36,9 38,9
SD [%]
Co 21,2 13,3 10,2 21,9 16,4
HS 3h 0,0 0,0 0,0 0,0 0,0
HS 7h 13,4 5,1 103,8 6,2 22,1
OS 3h 23,4 18,9 91,0 14,0 24,1
OS 7h 25,5 25,5 242,7 28,1 23,5
Tab. 8 (zu Abb. 17b): mRNA-Expression von HSPs in Astrozyten in vitro nach HS und OS. Mittelwerte (MW) in Prozent zu „HS 3h“ und Standardabweichungen (SD) in Relation zu GAPDH.
HSP90 HSP70 HSP32 HSP25 αB-Crystallin MW [%]
Co 120,5 19,3 26,8 60,1 43,6
HS 3h 100,0 100,0 100,0 100,0 100,0
HS 7h 83,9 64,6 24,4 81,1 62,7
OS 3h 72,7 20,2 53,7 51,0 55,2
OS 7h 85,2 15,4 66,9 61,3 68,8
SD [%]
Co 22,2 6,0 14,1 15,5 28,5
HS 3h 0,0 0,0 0,0 0,0 0,0
HS 7h 0,9 1,1 10,9 10,0 14,4
OS 3h 16,8 4,5 24,1 24,0 31,7
OS 7h 7,3 0,6 14,7 13,9 20,3
Tab. 9 (zu Abb. 24b): Einfluss von MG132 auf die HSP-Induktion in Neuronen und Astrozyten. Prozentuale Werte der HSP-Proteine in Bezug auf die Astrozyten Kontrolle (ein Versuch).
HSP25 αB-Crystallin
Astrozyten Co 100,0 100,0
Astrozyten + MG132 125,5 184,0
Neurone + MG132 38,9 12,6
Anhang 123 Tab. 10 (zu Abb. 26a,b): mRNA-Expression von HSPs in Neuronen und Astrozyten in vitro nach Behandlung mit MG132. Mittelwerte (MW) aus drei Versuchen in Prozent zu „MG132 3h“ in Relation zu GAPDH, mit Standardabweichungen (SD).
Neurone Astrozyten
HSP32 HSP25 αB-Crystallin HSP32 HSP25 αB-Crystallin MW
Co 38,9 52,7 50,7 18,6 75,4 73,1
MG132 3h 100,0 100,0 100 100,0 100,0 100,0
MG132 7h 1303,1 187,7 96,3 88,2 100,4 141,4
SD
Co 1,2 11,7 37,0 5,9 12,6 30,0
MG132 3h 0,0 0,0 0,0 0,0 0,0 0,0
MG132 7h 406,8 56,9 13,7 21,6 10,0 49,6
Curriculum vitae
Grit Vollmer Heinrich-Neu-Str. 2 69123 Heidelberg
Geburtsdatum: 24.11.1976 Geburtsort: Recklinghausen Staatsangehörigkeit: deutsch
Familienstand: ledig
Ausbildung:
1996 Abitur, SZ Rübekamp, Bremen
1996 – 2001 Studium der Biologie, Universität Bremen 1998 Vordiplom
2001 Diplomarbeit, AG Molekulare Neurobiologie/
Neurochemie, Frau Prof. Dr. C. Richter-Landsberg, Universität Oldenburg, „Proteinveränderungen bei demyelinisierenden Erkrankungen“
2002 – 2005 Promotionsstudium, Universität Oldenburg
AG Molekulare Neurobiologie/Neurochemie, Prof. Dr.
Christiane Richter-Landsberg
Kooperation: Institut für Neuropharmakologie, Prof. Dr.
Michael Koch, Universität Bremen
Publikationen
Abstracts zu Tagungsbeiträgen:
Vollmer G., Goldbaum O., Richter-Landsberg C. (2003). Heat shock proteins in cultured rat brain neurons: Developmental expression and differential regulation after stress. Proceedings of the 5th Meeting of the German Neuroscience Society/ 29th Göttingen Neurobiology Conference, eds:
Elsner N. und Zimmermann H., Thieme Verlag (Poster 880)
Vollmer G. and Richter-Landsberg C. (2004). Differential stress response of cultured rat brain neural cells and heat shock protein association with the cytoskeleton. Annual International Conference GBM Study Group Neurochemistry, Leipzig, Germany. Int J Dev Neurosci
Vollmer G., Schwabe K., Koch M., Richter-Landsberg C. (2005). Effect of
antisense oligonucleotides on reelin translation and synaptic/dendritic protein expression in the rat brain. Proceedings of the 6th Meeting of the German Neuroscience Society/ 30th Göttingen Neurobiology Conference, eds: Zimmermann H. und Krieglstein K., Neuroforum 2005, 1 Suppl.:
(336B)
Erklärung
Hiermit versichere ich, dass ich diese Arbeit selbstständig verfasst und keine anderen als die angegebenen Hilfsmittel und Quellen benutzt habe.
Heidelberg, 20.10.2005
Grit Vollmer
Summary
The investigations in this study were carried out to contribute to the understanding of neurodevelopmental disorders. Neurodevelopmental disorders can develop from a combination of harmful environmental factors and genetic predisposition. Many environmental factors like chemical agents can induce cellular stress. Cells normally react to stress by the induction of heat shock proteins (HSPs). HSPs also have important functions under physiological conditions and in brain development. HSPs may be stress specific or cell type specific expressed, i.e. they can be found constitutively or inducible especially either in neurons or glia or in both cell types. Interactions between neurons and glia are important for normal development and function of the nervous system.
Under pathological conditions and neurodevelopmental disorders this interaction might be disturbed and lead to functional losses. For example changes in the extracellular matrix (ECM) can lead to the disturbance of neuron-glia- or neuron-neuron-interactions.
In the present study, the reaction of neurons to different stressors, like heat shock (HS), oxidative stress (OS) and proteasomal stress, was investigated and compared to the stress respond of astrocytes. Furthermore, the expression of Reelin, a glycoprotein of the ECM, in the neuronal cultures was analyzed, and the consequences of suppressing the synthesis of Reelin with antisense oligonucleotides (AS-ODNs) for the cells were investigated. Reelin normally plays a role in brain development, where it is an extracellular signalling molecule for migrating neurons. In the adult brain, the protein seems to be important for dendritic spine plasticity and synaptic plasticity and therefore for LTP and memory. Reelin is found to be decreased in the neurodevelopmental disorder schizophrenia.
The data showed that, in vitro HSPs were already constitutively differentially expressed in neurons and glia. In contrast to neurons, the sHSPs HSP25 and αB-Crystallin were found constitutively in astrocytes. Additionally, stress specific and cell type specific stress responses were observed. In neurons HS specifically induced HSP70, HSP25 and αB-Crystallin, and OS induced particularly HSP32 and to a lower extend HSP25. In astrocytes after HS HSP70
and αB-Crystallin, and after OS HSP32 and αB-Crystallin were induced. In both cell types proteasomal stress led to the induction of all HSPs induced by HS and OS. Astrocytes in culture respond to stress by a stronger induction of HSPs.
An important finding of these investigations was that under physiological as well as under stress astrocytes contained higher endogenous concentrations of HSPs than neurons. Also, astrocytes were less susceptible to stressful situations than neurons, as shown by morphological examination and investigation of apoptotic cell death. This is probably linked to the high expression of HSPs in astrocytes. Under physiological conditions as well as after stress, particularly high amounts of HSP25 were found in astrocytes.
HSP25 can therefore be used as a marker for astrocytes in histological investigations.
In neurological disorders where stress is involved, cytoskeletal changes can often be observed. HSPs play a role in protecting the cytoskeleton. An important finding of the present study was that astrocytes, but not neurons already constitutively expressed the cytoskeleton-associated sHSPs. HSP25 was associated with the cytoskeleton only in astrocytes. Mainly an association with the MT, but also with glia filaments was observed. In astrocytes the organization of the microtubule network was preserved also under stress, which points to a protective function of HSP25 particularly in astrocytes. In addition other HSPs like HSP70 and αB-Crystallin which can also interact with the cytoskeleton could contribute to the protection of the cytoskeleton. Neurons with their elaborated cytoskeleton are more vulnerable and respond stronger to toxic influences due to the comparatively low endogen concentration of HSPs.
Therefore, aversive influences which interfere with the brain development, mainly affect nerve cells, and their damage can lead to functional losses.
The data presented here indicate that Reelin can be detected in neuronal cell cultures in soluble form in the culture media as well as bound in cellular form.
But it seems not to be necessary for the normal development and differentiation of neurons in vitro, however, suppression of extracellular Reelin by AS-ODNs led to morphological changes of neurons. Possibly the suppression of Reelin with AS-ODNs was too ineffective, what is supported by the finding that intracellular Reelin was not affected. Also, experiments with protease inhibitors