Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in α-synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in α-synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo , providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients. Here, to further interrogate α-synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of α-synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro , demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding. Overall, our studies use a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

High-resolution analysis of differential gene expression during skeletal muscle atrophy and programmed cell death.

Skeletal muscles can undergo atrophy and/or programmed cell death (PCD) during development or in response to a wide range of insults, including immobility, cachexia, and spinal cord injury. However, the protracted nature of atrophy and the presence of multiple cell types within the tissue complicate molecular analyses. One model that does not suffer from these limitations is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. Three days before the adult eclosion (emergence) at the end of metamorphosis, the ISMs initiate a nonpathological program of atrophy that results in a 40% loss of mass. The ISMs then generate the eclosion behavior and initiate a nonapoptotic PCD during the next 30 h. We have performed a comprehensive transcriptomics analysis of all mRNAs and microRNAs throughout ISM development to better understand the molecular mechanisms that mediate atrophy and death. Atrophy involves enhanced protein catabolism and reduced expression of the genes involved in respiration, adhesion, and the contractile apparatus. In contrast, PCD involves the induction of numerous proteases, DNA methylases, membrane transporters, ribosomes, and anaerobic metabolism. These changes in gene expression are largely repressed when insects are injected with the insect steroid hormone 20-hydroxyecdysone, which delays death. The expression of the death-associated proteins may be greatly enhanced by reductions in specific microRNAs that function to repress translation. This study not only provides fundamental new insights into basic developmental processes, it may also represent a powerful resource for identifying potential diagnostic markers and molecular targets for therapeutic intervention.