Introduction: Astrocytopathy is known to be an early feature of different neuroinflammatory diseases. However, the impact of astrocyte loss and repopulation on the development and progression of demyelinating lesions in complex etiologies, such as multiple sclerosis, is difficult to determine. To more easily analyse astrocyte- oligodendrocyte-interactions during lesion formation and progression in the human brain, diseases like Central pontine myelinolysis (CPM) can be used as a less complex model of demyelinating disorders. CPM is a rare neurological condition characterized by damage to the myelin sheath of pontine nerves after osmotic shifts in serum. Astrocytopathy is regarded to be the first event in the pathogenesis of CPM lesions. Methods: Histological investigation of autopsy tissue from human CPM patients was performed. Lesions were staged considering the myelination and the appearance of different astrocyte subtypes, which was used to judge behaviour of the astrocytic compartment. Further, dynamics of oligodendrocyte loss and repopulation were analysed and compared to the astrocytic repopulation. Results: Early-staged lesions were largely demyelinated and showed an overall reduction of astrocyte densities. The few astrocytes present showed a bipolar morphology and were APQ4-negative, indicating an immature state. Intermediate- stage lesions were still largely demyelinated, but had increased overall densities of astrocytes, which did not yet reflect densities observed in the perilesion. Astrocytes appeared mostly ramified and AQP4-positive, indicating maturity. Nevertheless, bipolar astrocytes were still observable, indicating that repopulation was not yet finalized. Late-stage CPM-lesions were at least partially remyelinated. Astrocytes were detectable in overall densities comparable to the perilesion and showed a ramified (or even reactive morphology), as well as regular expression of AQP4. Investigating the oligodendrocytes, intralesional densities were reduced in early- and intermediate-stage lesions when compared to the perilesion. Re-increase in oligodendrocyte densities was first observable in late-stage lesions, but did not reach perilesional levels. Conclusion: The study at hand indicates that the recovery of demyelinated osmolyte- induced pontine lesions follows a distinct time-course. Repopulation of the lesion with oligodendrocytes is not carried out until lesions are completely repopulated with functional resident astrocytes, as indicated by the ramified morphology and the expression of AQP4. Further studies will be needed to determine, whether the appearance of immature astrocytes, indicating an ongoing repopulation of lesions with astrocytes, correlates with an inefficient repair of demyelinated lesions.:List of Abbreviations.................................................................................................................6 1 Introduction................................................................................................................7 1.1 Osmotic Demyelinating Syndrome......................................................................... 7 1.2 Clinical manifestation............................................................................................. 9 1.3 Diagnosis and Management of CPM.....................................................................11 1.4 Aetiology of Central Pontine Myelinolysis.................................. ......................... 14 1.5 The brain, its adaptation to hyponatraemia and response to correction – pathophysiology of CPM............................................................................................16 1.6 Pathology of myelin............................................................................................. 19 1.6.1 Astrocytopathy and oligodendrocytopathy.................................................................................................20 1.7 Aims of the study................................................................................................. 23 2 Material und Methods............................................................................................. 24 2.1 Patient tissue........................................................................................................ 24 2.2 Histology and immunohistochemistry................................................................................................24 2.2.1 Basic concepts........................................................................................... ......24 2.2.2 Hematoxylin and Eosin (HE)............................................................................. 26 2.2.3 Luxol Fast Blue/ Periodic Acid Schiff stain........................................................27 2.2.4 Immunohistochemistry. Application and Protocol.............................................28 2.3 Implementation.................................................................................................... 31 2.4 Estimation of demyelination................................................................................. 32 2.5 Analysis of cell density and proliferation.............................................................. 32 2.6 Data plotting and statistical analysis.................................................................... 32 3 Results..................................................................................................................... 33 3.1 Patient cohort....................................................................................................... 33 3.2 Characteristics of demyelination.......................................................................... 35 3.3 CPM lesion and disease staging.......................................................................... 37 3.4 Astrocytes within human CPM lesions................................................................. 42 3.4.1 Astrocyte densities are decreased in early CPM lesions....................................42 3.4.2 Astrocytes in CPM– morphological distinctions.................................................45 3.5 Oligodendrocyte densities within human CPM lesions.........................................48 3.6 Macrophages and activated microglia.................................................................. 54 3.6.1 KiM1P – a marker for infiltrating macrophages and activated microglia............54 3.6.2 Proliferating Iba1+ cells are observed in all lesion stages..................................58 4 Discussion................................................................................................................ 61 4.1 Lesion Staging...................................................................................................... 61 4.2 Astrocytes in the pathogenesis of CPM............................................................... 65 4.3 Oligodendrocyte pathology in CPM..................................................................... 69 4.4 Mechanisms of regeneration in human CPM lesions............................................72 4.5 Summary, interpretation and limitations of our study............................................78 5 Conclusion and Outlook.......................................................................................... 80 6 Bibliography............................................................................................................. 82 7 List of Tables.............................................................................................................91 8 List of Figures.......................................................................................................... 92 9 Appendix.................................................................................................................. 94 9.1 Declaration of Authenticity.....................................................................................94 9.2 Acknowledgements...............................................................................................95

La maladie d'Alexander est une maladie neurodégénérative due à des mutations hétérozygotes du gène GFAP codant le principal filament intermédiaire des astrocytes matures. Nous avons étudié l'effet des mutations GFAP dans l'hippocampe d'un patient avec AxD infantile et de deux souris knockin, l'une portant une mutation dans le rod domain (p.R85C) et l'autre dans le tail domain (p.T409I). Chez le patient, nous décrivons pour la première fois: (i) des changements morphologiques sévères des cellules GFAP+ dans la zone subgranulaire du gyrus denté, qui ont perdu la plupart de leurs processus radiaux; (ii) une réactivité microgliale; (iii) et un déficit de la neurogénèse hippocampique postnatale. Nous avons trouvé des anomalies similaires dans les deux souris knockin, plus sévères chez les homozygotes. La comparaison de ces modèles a montré que ces anomalies prédominent chez les souris GFAPT409I, tandis que l’accumulation de GFAP est supérieure chez les souris GFAPR85C. La comparaison des deux modèles de souris a montré que les conséquences pathologiques dépendent la localisation de la mutation dans la GFAP. Ces résultats suggèrent qu'en plus du gain évident de fonction, d'autres dysfonctions astrocytaires dans peuvent être dues à une perte de fonction. De plus, nous avons traité les souris mutantes avec de la ceftriaxone, connu pour son effet neuroprotecteur, mais nous n'avons observé aucun effet significatif. Enfin, la mégalencéphalie étant fréquente chez les patients AxD, nous avons mesuré la quantité d'eau cérébrale chez les souris mutantes GFAP. Nous avons trouvé une augmentation significative de la teneur en eau chez les souris GFAPR85C/R85C âgées d'un an. Nous avons observé une localisation anormale de l'AQP4 dans les astrocytes des asouris mutées, pouvant participer au déséquilibre hydrique cérébral. / Alexander disease is a neurodegenerative disorder caused by heterozygous mutations of GFAP gene coding the major intermediate filament of mature astrocytes. We studied the effect of GFAP mutation in the hippocampus of infantile onset AxD patient and two novel knockin mouse models, one bearing a mutation located in the rod domain (p.R85C), and the other bearing a mutation located in the tail domain (p.T409I) of mouse Gfap. In the AxD patient, we describe for the first time: (i) obvious morphological changes of GFAP+ cells in the subgranular zone of the dentate gyrus, which have lost most of their radial processes; (ii) microglial reactivity; (iii) and deficit in postnatal hippocampal neurogenesis. We found similar abnormalities in the two knockin mouse lines, more obvious in homozygous mice. A comparison of these mouse models showed that pathological findings predominated in the GFAPT409I mice, whereas GFAP accumulated in larger amounts in the GFAPR85C mice. The comparison of the two mouse models showed that their pathological consequences depend on the location of the mutated residues in GFAP. These findings suggest that in addition to the evident gain of GFAP function, other astrocyte dysfunctions in this disease may be due to a loss of function of GFAP. In addition, we treated the mice mutants with ceftriaxone, which has been reported to have a neuroprotective effect, but we observe no significant effect. Finally, AxD patients have often megalencephaly, therefore we measured the brain water content in AxD mouse models. We found a significant increase in brain water content in the one year old GFAPR85C/R85C mice vs controls. We observed mislocalization of AQP4 in mutant mice astrocytes that can participated to water imbalance in brain.