Analysis of JCPy virus spread in human CNS tissue and establishment of PML models
von Adriane Kutllovci
Datum der mündl. Prüfung:2023-10-19
Erschienen:2023-12-07
Betreuer:Prof. Dr. Imke Metz
Gutachter:Prof. Dr. Imke Metz
Gutachter:Prof. Dr. Klaus-Armin Nave
Dateien
Name:Adriane Kutllovci Dissertation Complete_without CV.pdf
Size:4.39Mb
Format:PDF
Diese Datei ist bis 18.10.2024 gesperrt.
Zusammenfassung
Englisch
Progressive multifocal leukoencephalopathy (PML) is a demyelinating CNS disease caused by the reactivation of the JC polyomavirus (JCPyV) in immunosuppressed individuals. The disease course is typically severe and in up to 50% of cases, even fatal. Through the use of novel immunomodulatory drugs, PML has increasingly become a complication, and its treatment remains a challenge since its pathogenesis is still poorly understood. The JCPy virus infects only human cells with a specific glial tropism, which has hampered the development of suitable in vitro and in vivo models to study disease mechanisms. The first part of this study focused on the analysis of JCPyV infection and spread in human CNS tissue. A new classification system has been established for PML lesions (stages 1 - 6) to characterize PML lesion evolution and to identify the target cells of JCPyV. This has been achieved by evaluating the temporal changes in JCPyV-infected/replicating cells, micro-glia/macrophages, and the degradation of myelin proteins. I observed that lesion initiation and earliest lesion development occurs exclusively in P25+ oligodendrocytes, P25+ Olig2+ oligodendrocytes and P25- Olig2+ oligodendrocyte progenitor cells. In contrast, GFAP+ astrocytes are neither infected nor allow the replication of JCPyV during initial viral propagation. In these early lesion stages, oligodendrocyte reduction and axonal damage are already present and lead to a subtotal loss of oligodendrocytes along with extensive axonal damage in advanced demyelinated stages of PML. Notably, astrocytes were shown to become susceptible to JCPyV infection and replication during this advanced lesion stage. Taken together, the data demonstrate the importance of preventing viral infection and replication specifically of oligodendrocytes. Further experimental studies should focus on oligodendroglial lineage cells in the search for new treatments of PML that could take effect early in the disease course to prevent irreversible axonal damage. Furthermore, the present insights provide a thorough understanding of PML pathology as well as guidance on how in vitro and in vivo models should be designed to properly mimic PML. We aim to establish human glial chimeric mice with a chimerism restricted to the forebrain. These human glial chimeric mice will be used in further studies with the goal to create a new mouse model of PML, including both immunodeficient and immunocompromised animals. Thus, the second part of my thesis focused on the initial steps to generate human glial chimeric mice. In prior studies it was shown that human-induced pluripotent stem cells (hiPSCs) can be dif-ferentiated into glial progenitor cells and intracerebrally transplanted into conditional myelin basic protein (MBP) knockout mice after birth, resulting in myelination in target brain areas. I established the differentiation of hiPSC into hiPSC-derived glial progenitor cells. Cells were injected into Foxg1-IRES-Cre MBP flox/flox mice that were characterized by a myelin deficiency restricted to the forebrain but that have a normal life span. In immunocompetent mice, despite the adjustment of cell numbers to ensure graft acceptance, the transplanted cells were consistently rejected by an eosinophilic and CD4+ T cell-dominated inflammation, termed eosinophilic graft rejection. In contrast, cells injected into immunodeficient mice did not elicit an immune reaction against the xenograft, as evidenced by vital and migrating donor cells 20 days after neonatal injection. Overall, these results indicate that inducing graft acceptance in immunocompetent mice is challenging and needs optimization, e.g. by enhancing perinatal tolerization. Immunodeficient mice demonstrate potential for the development of glial chimerism, which is expected to develop 3-4 months after neonatal injec-tion. The third part of this study investigated the permissivity of JCPyV-MAD1 in COS-7, hiPSC derived glial progenitor cells and bioengineered neuronal organoids (BENOs). COS-7 cells, which are immortalized monkey kidney-derived cells, have been engineered to express SV40 T-Ag, an early viral gene that is essential for facilitating JCPyV infection. BENOs exhibit a variety of CNS cell populations including neurons, astrocytes, and oligodendrocytes. It was observed that COS-7 are suited to efficiently propagate JCPyV-MAD1 and yield virus for further infection studies. The hiPSC-derived glial progenitor cells have been shown to be susceptible to JCPyV-MAD1 infection and replication, a prerequisite for the set-up of our planned PML mouse model. Finally, astrocytes within the BENOs were highly susceptible to JCPyV-MAD1 infection and replication. The cell composition in the BENOs needs to be optimized with higher numbers of myelinating oligodendrocytes to mimic the human situation. In conclusion, all three cell sources were found to be susceptible to virus propagation, and the investigation of BENOs may serve as a promising means of studying JCPyV propagation.
Keywords: PML; Progressive multifocal leukoencephalopathy; JC virus
Schlagwörter: PML; Progressive multifocal leukoencephalopathy; JC virus