Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

CK1BP Reduces α-Synuclein Oligomerization and Aggregation Independent of Serine 129 Phosphorylation.

The pathological accumulation of α-Synuclein (α-Syn) is the hallmark of neurodegenerative α-synucleinopathies, including Parkinsons's disease (PD). In contrast to the mostly non-phosphorylated soluble α-Syn, aggregated α-Syn is usually phosphorylated at serine 129 (S129). Therefore, S129-phosphorylation is suspected to interfere with α-Syn aggregation. Among other kinases, protein kinase CK1 (CK1) is known to phosphorylate α-Syn at S129. We overexpressed CK1 binding protein (CK1BP) to inhibit CK1 kinase activity. Using Bimolecular Fluorescence Complementation (BiFC) in combination with biochemical methods, we monitored the S129 phosphorylation and oligomerization of α-Syn in HEK293T cells. We found that CK1BP reduced the overall protein levels of α-Syn. Moreover, CK1BP concomitantly reduced S129 phosphorylation, oligomerization and the amount of insoluble α-Syn. Analyzing different α-Syn variants including S129 mutations, we show that the effects of CK1BP on α-Syn accumulation were independent of S129 phosphorylation. Further analysis of an aggregating polyglutamine (polyQ) protein confirmed a phosphorylation-independent decrease in aggregation. Our results imply that the inhibition of CK1 activity by CK1BP might exert beneficial effects on NDDs in general. Accordingly, CK1BP represents a promising target for the rational design of therapeutic approaches to cease or at least delay the progression of α-synucleinopathies.

Monitoring α-synuclein multimerization in vivo.

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.