ALS mutations shift the isoelectric point of the KIF5A C-terminal inducing protein aggregation and TDP-43 mislocalization.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by death of lower and upper motor neurons. Although the mechanism behind the selective neuron loss is still unclear, several heterogenous genes have been causally linked to ALS. encodes for a neuronally enriched kinesin involved in protein transport and mutations within this gene have been causally linked to different motor neuron diseases. The mutations identified in ALS patients are mostly predicted to alter its mRNA splicing, leading to a frameshift mutation and an aberrant 39 amino acid-long sequence in the C-terminal domain of KIF5A.Here we found that ALS-related KIF5A mutations induce the accumulation of the mutant form of the protein in human motoneurons, which are also characterized by the cytosolic mislocalization of TDP-43. This ALS hallmark was even exacerbated upon overexpression of the ALS-KIF5A protein in cells differentiated from healthy controls and primary neurons, suggesting a pathological connection between the cellular load of the mutant protein and TDP-43 pathology. While the terminal domain of the WT isoform is characterized by an acid isoelectric point (pI), the ALS variant presents a basic pI due to the altered aminoacidic composition of this sequence. We thus generated a KIF5A ALS isoform that retained part of the aberrant sequence but with lower pI. The overexpression of this mutated variant led to significantly lower protein aggregation and TDP-43 mislocalization than the ALS mutant. Our data show that re-establishing the correct pI rescues KIFA aggregation and significantly reduces the cytoplasmic mislocalization of TDP-43. Amyotrophic Lateral Sclerosis is a lethal neurodegenerative disease to which no cure is still known. Heterogenous genes have been causally linked to ALS, yet, the exact pathomechanism responsible for neuronal death remains unclear. One such gene is KIF5A which encodes for a neuronally enriched kinesin. Identified mutations cause incorrect mRNA splicing resulting in an aberrant C-terminal aminoacidic sequence. Here, we identified TDP-43 cytosolic enrichment, a hallmark common to many ALS models, in two distinct hiPSC-derived motoneuron lines harboring the ALS mutation KIF5A Moreover, we generated a KIF5A isoform that retained most of the aberrant sequence but did not promote protein aggregation nor TDP-43 mislocalization upon overexpression. These results shed further light on the pathobiochemistry of the ALS-KIF5A cases.