Biochemical and single-cell gene expression analyses of ALS-causing KIF5A mutations

• Applicant: Dr. Rüstem Yilmaz
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 492655220

Amyotrophic lateral sclerosis (ALS) is an adult-onset lethal neurodegenerative disease. In a considerable proportion of ALS cases a monogenic cause is detected. We identified KIF5A as a novel ALS gene, in which mutations leading to splice site defects accumulate in a hot spot around exon 27. These mutations result in a frameshift, and give rise to a new predicted protein tail with an always-identical 40 amino acid sequence, referred to as neopeptide. The uniform consequence of all ALS-causing KIF5A mutations at the protein level strongly suggests a toxic gain of function of the neopeptide. However, experimental proof is lacking until now. Therefore, I will investigate the effects of the KIF5A mutants in ALS causation on cellular and biochemical levels. To that end, I will utilize IPSCs from ALS patients harboring heterozygous KIF5A mutations, mouse-derived primary motor neurons, and additional in vitro models. I will search for altered characteristics of the mutants, for example their cellular distribution, protein turnover rate, and function. As our preliminary data show that the mutations at least partially alter the cargo-binding domain of the protein, I will validate differential interaction partners of KIF5A by mass spectrometry and BRET. The novel, abrogated or altered-in-affinity interactions could be a key in understanding the KIF5A ALS pathology. Finally, I want to use the advantage of exclusively neuronal expression of KIF5A to study the neuron-autonomous pathogenesis connected to it, and address how malfunction of a neuronal protein affects the other cell types in the mouse brain by performing single-cell RNA sequencing analysis in the neopeptide knock-in mouse brain. The cause and effect relationship in ALS has been a long-standing question and this project will shed light on neuron-glia interactions. Overall, my objective is to complement our identification of a novel ALS gene with the functional studies to understand the pathology that KIF5A mutants exert. This will also have substantial relevance for the understanding of ALS pathogenesis in general.

• DFG Programme: WBP Position