Molecular characterization of disease-causing TUBB4A mutations reflecting the phenotypic spectrum

Abstract

While the broad phenotypic spectra or even pleiotropy caused by pathogenic variants in the same gene remains a poorly understood enigma, they are of great translational importance for a better understanding of potential differential mechanisms on the one hand, and for more individualized patient counseling on the other. In this thesis, TUBB4A mutations with a clinical spectrum ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) were functionally characterized. Whether this broad spectrum represents a phenotypic continuum or a pleiotropic pattern is controversial. While the former would imply that a common pathway is impaired to varying degrees, the latter could imply that different cellular functions are impaired to varying degrees. Another unresolved question is how different TUBB4A mutations affect the dynamic properties of microtubules in affected cells such as oligodendrocytes. To this end, mutations representative of the phenotype were examined for their specific effects at the molecular and cellular levels. For this thesis, an advanced cell lysate method was established that allows detailed characterization of microtubule dynamics, combining the advantages of live-cell imaging and TIRF microscopy - without the need to isolate tubulin beforehand. In addition, in silico simulations were performed to determine the influence of TUBB4A mutations on heterodimer conformations, which has not been done before for this tubulin isoform. Furthermore, a CRISPR/Cas9-generated heterozygous TUBB4A knockout iPSC line was established. The derived neurons showed severe impairment of mitochondrial motility, providing an ideal positive control to compare TUBB4A mutations regarding intracellular trafficking. The major finding of this thesis is the demonstration of a differential effect of TUBB4A mutations (H-ABC vs. DYT-TUBB4A) on microtubule growth, possibly indicating a pleiotropic manifestation. The comprehensive combination of in vitro and in silico approaches enabled to obtain a holistic picture of the potential effects of TUBB4A mutations and provided mechanistic insights into their differential phenotypic expression. In particular, the thesis reveals that: 1) The analyzed TUBB4A variants exert distinct effects on microtubule dynamics. Importantly, the H-ABC-associated TUBB4A variants (p.R2W and p.D249N) severely disrupt microtubule polymerization compared to variants causing the milder dystonic phenotype. 2) Mutants are less likely to adopt a straight conformation in silico. Instead, the bent conformation appears to be sterically favored in H-ABC mutants, consistent with the impaired microtubule polymerization of these mutants. 3) Anti-morphic variants per se do not prevent the morphological maturation of oligodendrocytes. Instead, these mutations may disrupt mitosis during the proliferation of oligodendrocyte progenitors, most likely leading to hypomyelination in H-ABC patients in infancy. Therefore, altered microtubule growth appears to be differentially affected by TUBB4A mutations, which may interfere with various cellular functions, suggesting a pleiotropic spectrum rather than a phenotypic continuum in TUBB4A-related diseases. The work was mainly performed at the University of Lübeck at the Institute of Neurogenetics (laboratory of Prof. Christine Klein, supervised by Dr. Aleksander Rakovic). For specific methods, collaborations were established at the University of Lübeck with the Institute of Cardiogenetics (laboratory of Prof. Jeanette Erdmann, supervised by Dr. Stephanie Tennstedt), the Institute of Experimental and Clinical Pharmacology and Toxicology (laboratory of Prof. Markus Schwaninger, supervised by Dr. Helge Müller-Fielitz) and the Institut Curie, Université PSL, CNRS UMR3348, Orsay, France (laboratory of Dr. Carsten Janke "Control of microtubule dynamics and function with the tubulin code").