Objective: To study the role of tubulin beta 4A (TUBB4A) in mitochondrial motility in isogenic induced pluripotent stem cells (iPSC)-derived neurons.

Background: Heterozygous mutations in the TUBB4A gene have been linked to torsion dystonia-4 (DYT4) and hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC), however, the molecular underpinnings remain elusive. As alpha- and beta-tubulins are the principal components of cellular microtubules, loss-of-function in TUBB4A have an effect on the cytoskeleton and motility of organelles in neuronal cells. We here used a neuronal cell model derived from iPSC to analyze neuronal morphology and axonal transport of mitochondria.

Methods: Heterozygous TUBB4A knock-out iPSC lines were generated using CRISPR/Cas9 technology. To study mitochondrial motility in neuronal cells, we differentiated control and TUBB4A heterozygous knock-out (htTUBB4A KO) iPSC into cortical neurons using a previously established protocol. Mature neurons were transduced with lentiviral particles expressing MitoDsRed. To track mitochondrial motility, live cell imaging was performed on at least 12 spatially distinct positions in each cell line. Pictures were taken every second over a period of 3 minutes. Videos were converted into kymographic images using Fiji and analyzed using the kymograph plugin for ImageJ.

Results: We imaged >200 mitochondria per cell line and quantified the number of stationary and mobile mitochondria. We observed a 3.5 fold reduction in the number of mobile mitochondria in htTUBB4A KO in comparison to control neurons. In addition, the average velocity of mitochondrial transport was 35% lower in htTUBB4A KO compared to control cells. Of note, we did not observe any differences in cell morphology or axonal outgrowth between these two cell lines.

Conclusions: A reduction in copy number of TUBB4A impairs mitochondrial motility and velocity of mitochondrial transport in our human neuronal model of DYT4.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/heterozygous-tubb4a-knock-out-impairs-mitochondrial-motility-in-human-neuronal-model-of-dyt4/