Medizin - Open Access LMU - Teil 20/22 Archiv

Overexpression or mutation of alpha-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human A53T] alpha-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from A53T] alpha-Synuclein expression. In addition, decreased TRAP1 expression in A53T] alpha-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued A53T] alpha-Synuclein-induced sensitivity to rotenone treatment. In human (non) neuronal cell lines, small interfering RNA directed against TRAP1 enhanced A53T] alpha-Synuclein-induced sensitivity to oxidative stress treatment. A53T] alpha-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by A53T] alpha-Synuclein overexpression in human SH-SY5Y cells. These results indicate that A53T] alpha-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to alpha-Synuclein.

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins.

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

Wnt/β-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/β-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/β-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/β-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/β-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7.