Biochemical and single-cell gene expression analyses of ALS-causing KIF5A mutations
Final Report Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder leading to paralysis and death within 3–5 years after diagnosis. To date, over 30 genes have been identified as monogenic causes of ALS. In 2018, we discovered splice site mutations concentrated in a hot spot of KIF5A in ALS patients, presenting KIF5A as a novel ALS gene. These mutations all caused a frameshift in the cDNA, which would result in a consistently identical 40-amino acid “neopeptide” sequence at the C-terminus if translated. We hypothesized that the neopeptide led to a toxic gain of function. As the experimental findings were lacking, we aimed to investigate the effects of the KIF5A mutants in ALS causation on cellular and biochemical levels. Therefore, we utilized various methods in this research project and worked with transient cell expression models, IPSC-differentiated motor neurons from ALS patients carrying heterozygous KIF5A mutations, and primary motor neurons derived from mutant knock-in mice. We demonstrated the expression of mutant KIF5A protein in cells, observing the formation of aggregates. Characterization of these aggregates, including size, number, intensity, and shape, was conducted using a machine learning algorithm. Additionally, we found that the aggregates tested positive for p62 and contained wild-type KIF5A, suggesting a dominant negative role of the mutants. Interestingly, our study revealed that not all ALS mutations lead to the skipping of exon 27, emphasizing the importance of the presence of neopeptide at the C-terminus of the protein. Next, we identified the differential interaction partners of KIF5A by mass spectrometry and BRET assays. Our findings suggest the abrogated ribosomal pathway could play a pivotal role in KIF5A-caused ALS pathology. Finally, employing snRNA-Seq on wild-type and knock-in mouse tissues, we observed significant alterations in differentially expressed genes in neurons, but not in other cell types, implying a neuron-autonomous mechanism. In conclusion, this project complements our identification of KIF5A as a novel ALS gene with molecular studies, providing insights into how ALS-causing mutations may lead to cellular defects.
Publications
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ALS ‐linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain‐of‐function. EMBO reports, 23(8).
Pant, Devesh C.; Parameswaran, Janani; Rao, Lu; Loss, Isabel; Chilukuri, Ganesh; Parlato, Rosanna; Shi, Liang; Glass, Jonathan D.; Bassell, Gary J.; Koch, Philipp; Yilmaz, Rüstem; Weishaupt, Jochen H.; Gennerich, Arne & Jiang, Jie
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ENCALS (European Network to Cure ALS) 2022 Meeting in Edinburgh, United Kingdom. Title: ALS-causing mutant KIF5A proteins form aggregates
Rüstem Yilmaz
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Brain Aging, Neurodegeneration, and the Role of Natural Molecules in Maintaining Brain Health Meeting, 2023 in Paris, France.Title: The role of KIF5A in neurodegeneration
Rüstem Yilmaz
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Fall Meeting 2023 of International Graduate School in Molecular Medicine Ulm, in Ulm, Germany. Title: The role of KIF5A in neurodegeneration: from gene discovery to pathomechanisms
Rüstem Yilmaz
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Frequency of C9orf72 and SOD1 mutations in 302 sporadic ALS patients from three German ALS centers. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 24(5-6), 414-419.
Yilmaz, Rüstem; Grehl, Torsten; Eckrich, Lukas; Marschalkowski, Ines; Weishaupt, Kanchi; Valkadinov, Ivan; Simic, Melita; Brenner, David; Andersen, Peter M.; Wolf, Joachim & Weishaupt, Jochen H.
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A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice. Journal of Experimental Medicine, 221(5).
Brenner, David; Sieverding, Kirsten; Srinidhi, Jahnavi; Zellner, Susanne; Secker, Christopher; Yilmaz, Rüstem; Dyckow, Julia; Amr, Shady; Ponomarenko, Anna; Tunaboylu, Esra; Douahem, Yasmin; Schlag, Joana S.; Rodríguez, Martínez Lucía; Kislinger, Georg; Niemann, Cornelia; Nalbach, Karsten; Ruf, Wolfgang P.; Uhl, Jonathan; Hollenbeck, Johanna ... & Weishaupt, Jochen H.
