RNA suppresses protein aggregation and reduces neuronal cytotoxicity

protein

Key Points – Direct observation of cytoplasmic translocation of the nuclear protein TDP43 immediately after cleavage; Discovery that the formation of protein aggregates is suppressed by RNA; Discovery that protein aggregates in the cytoplasm, rather than in the nucleus, induce neuronal cell death

Outline
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the motor neurons that give commands to muscles uniquely degenerate and are lost. In ALS, the protein named TDP431 is known to be a causative gene product2 that forms intracellular protein inclusion bodies3. It is also known that carboxyl-terminal fragments (CTFs) of TDP43 are found in the protein inclusion bodies within the motor neurons of ALS patients. In this research, by using a fluorescence imaging technique and fluorescence correlation spectroscopy4 with singlemolecule sensitivity, in addition to discovering that TDP43 quickly translocated from the nucleus into the cytoplasm after cleaving, we also discovered that the formation of toxic aggregates of TDP25, which is one of the CTFs of TDP43, is suppressed by RNA. Furthermore, there are indications that these TDP25 aggregates exert cytotoxicity in the cytoplasm. These results have uncovered a new pathway for the formation of aggregates and inclusion bodies of proteins in ALS pathology. We believe that RNA is an important target in the elucidation of ALS pathology as well as in treatment to suppress ALS progression.

All of this research was performed in the research laboratory of Hokkaido University Faculty of Advanced Life Science, Functional Cell Science (Professor Masataka KINJO), under Assistant Professor Akira KITAMURA, and has been published in the journal scientific-reports/” title=”View all articles about Scientific Reports here”>Scientific Reports.

This research was performed with the assistance of a Japan Society for the Promotion of Science (JSPS) Grant-In-Aid for Scientific Research, category of Scientific Research C and Grant-in-Aid for Young Scientists B, and the Japan Agency for Medical Research and Development (AMED) Development of Advanced Measurement and Analysis Systems.

Terms
TDP43 (TAR RNA/DNA-binding protein 43 kDa): The causative gene product in ALS, and a protein mainly localized to the nucleus. TDP43, in addition to having motifs for binding to RNA and DNA, also has a nuclear localization signal sequence and a nuclear export signal sequence, therefore it is believed to have the function of binding RNA while shuttling between the nucleus and the cytoplasm. It has been identified as a protein that forms protein aggregates within the motor neurons of ALS patients, although what forms the aggregates is not necessarily the intact length of TDP43 but also fragments of various lengths. Since TDP43 carries, at its carboxyl-terminal region, a domain that promotes assembly and aggregation of proteins, called a prion-like domain, it has been hypothesized that the cause of ALS onset is a loss of TDP43 function that is involved in its aggregation.
Causative Gene Product: The protein that is produced by the transcription and translation of a gene (the causative gene) that causes a genetic disease (in this research, ALS).
Protein Inclusion Bodies: Almost all proteins function by folding from a single polypeptide chain, and the misfolded protein causes non-specific assembly with multiple molecules, which changes them into a form that is non-functional and does not dissolve well in water. This condition in which multiple proteins are non-specifically assembled and lose their function is called a protein aggregate. If protein aggregates that
form within a cell are degraded to amino acids then they can be reused to synthesize new polypeptides, but if aggregates remain without degradation then they may be partitioned in a certain compartment and/or deposit. Such intracellular structures where aggregates are partitioned are called protein inclusion bodies.

Fluorescence Correlation Spectroscopy: A method that can measure the dynamics (diffusion coefficient) of fluorescent molecules in solution with single-molecule sensitivity, and can also measure the average fluorescent intensity per particle. By comparing the measured value of the diffusion coefficient with a reference substance, it is possible to measure changes in molecular weight in a short time and with high sensitivity. In particular, the ability to measure the change in fluorescent intensity per particle is an extremely useful technique to determine aggregate formation of protein-of-interest.

Inquiries
Masataka KINJO, Professor

Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University

E-mail: kinjo[at]sci.hokudai.ac.jp

Akira  KITAMURA, Assistant Professor

Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University

E-mail: akita[at]sci.hokudai.ac.jp