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Infektionen des Zentralnervensystems (ZNS) können durch unterschiedliche Erreger verursacht werden, wobei Viren das Hauptpotential bilden. Bei der Abklärung der Ätiologie von Infektionen des ZNS nimmt die Labordiagnostik eine zentrale Rolle ein. Die Kenntnis des ätiologischen Agents ist von hoher prognostischer und therapeutischer Relevanz und für die Optimierung des Patientenmanagements bedeutend. Es wurden molekularbiologische Methoden zur Identifizierung und Charakterisierung ZNS-assoziierter Viren etabliert und zur Gewinnung aktueller Prävalenzdaten eingesetzt. Enteroviren (EV) waren mit 21,8% das häufigste Pathogen, gefolgt von Adenoviren. HSV und VZV spielten nur eine untergeordnete Rolle. Eine Bedeutung von West Nil-Virus bei ZNS-Infektionen in der Region Vorpommern konnte ausgeschlossen werden. Die genotypische Charakterisierung zirkulierender Stämme zeigte für EV Cluster mit hoher Homologie zur Gruppe der Coxsackie B-Viren. Weiterhin wurden Vertreter von Coxsackievirus A und von Echovirus identifiziert. Isolierte EV-Stämme wiesen gegenüber Pleconaril eine hohe Empfindlichkeit auf. Ein unerwartet hoher Anteil wurde für Adenoviren gefunden. Die identifizierten Serotypen waren ADV-2, ADV-5 und ADV-41. Untersuchungen zum Proteinprofil EV-infizierter Zellen zeigten signifikante Veränderungen in der Expression für Proteine des Zytoskeletts, für Bestandteile von metabolischen Prozessen und für Proteine, die in Signal- und Transportprozesse sowie die Stress-Abwehr involviert sind und bieten Ansätze für die Entwicklung neuer therapeutischer Strategien.
Impact of proteostasis and the ubiquitin proteasome system on myeloid cell function in the CNS
(2023)
Cellular protein homeostasis (proteostasis) maintains a functional proteome and thus proper cell function. Proteostasis is facilitated by the ubiquitin-proteasome system (UPS), an intracellular protein turnover machinery ensuring clearance of damaged, misfolded, old and/or unneeded regulatory proteins. This is particularly important in the central nervous system (CNS), where it is linked to neurodegeneration. Disruptions of the proteostasis systems cause the accumulation of misfolded proteins which are commonly seen in progressive neurodegenerative diseases also linked to neuroinflammation. Proper UPS function can protect cells from the accumulation of defective proteins, neurodegeneration and neuroinflammation. Furthermore, it has been found that loss of function mutations in the genes encoding UPS components are linked to systemic inflammation including neuroinflammation and/or neurodevelopmental disorders. Proteasome defects in patients suffering from these disorders cause decreased proteasome activity, accumulation of proteins, activation of proteotoxic stress responses and systemic inflammation. However, the molecular link between proteotoxic stress and the initiation of inflammatory signalling remained unclear. In Article 2, we summarized the importance of the UPS in immune cell proteostasis and function including activation of innate and adaptive immune responses. Although UPS function is notably important in innate immune signalling, the current understanding of the role of UPS in myeloid cell function in the CNS is limited. We also indicated the involvement of impaired UPS function in sterile systemic inflammation including neuroinflammation as well as tumour diseases and pathogen manipulation of immune cells.
To investigate the molecular link behind proteasome impairment and systemic inflammation in the brain, we focused on microglia cells as the only immune residents of the CNS. In Article 1, we used a pharmacological inhibitor called bortezomib which targets β5 and β5i/LMP7 subunit activities in standard proteasome (SP) and immunoproteasome (IP), respectively. We showed for the first time on the molecular level that inhibition of proteasome activity by bortezomib triggers the accumulation of ubiquitylated proteins, proteotoxic stress responses and innate immune signalling activation depending on the induced proteotoxic stress response called unfolded protein response (UPR) in murine microglia. In particular, activation of the inositol-requiring protein 1α arm of UPR upon bortezomib treatment leads to systemic inflammation as indicated by type I interferon (IFN) response.
IP enhance the proteolytic capacity of UPS by rapid clearance of proteins upon immune signalling activation. Microglia, like other immune cells, exhibit constitutive expression of IP as well as SP to maintain their cellular proteostasis. In Manuscript 3, we studied the particular impact of IP impairment on microglial cellular function. We showed accumulation of ubiquitin-modified proteins and activation of proteotoxic stress responses in IP-impaired mouse and human microglia models. Moreover, we identified possible IP substrates in microglia using β5i/LMP7 knockout mice as an IP deficiency model and, examined how IP deficiency affects microglia function. IP deficient microglia affected the ubiquitylation levels of proteins involved in multiple pathways such as immune responses, energy metabolism, cytoskeleton organisation, cell cycle and ribosome function. Based on the molecular analysis, we confirmed sterile activation of innate immune signalling mechanisms in IP impaired microglia. This is driven by the proteotoxic stress sensor protein kinase R (PKR). In addition, we were able to show that IP impairment altered levels of the microglial activation markers, which are also involved in motility, adhesion and phagocytosis of microglia.
In this thesis, we highlight that UPS function is necessary to maintain microglial proteostasis and, that impairment of proteasome activities triggers sterile inflammation in microglia via activation of proteotoxic stress responses. The described activation of innate immune signalling mechanisms in microglia upon proteasome impairment may be considered as new therapeutic targets for patients suffering from rare protesomapathies or other disorders linked to dysregulated immune signalling.
Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.