In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration

Abstract

Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuroinflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and functions. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) may cause structural deviations that impair GTP binding, and plausibly prevent or destabilize tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies predict structural perturbations and disruption of GTP binding, culminating in failure to form a stable tubulin heterocomplex, which may furnish an important pathogenic mechanism to trigger motor neuron degeneration in ALS.

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“…The default equilibration type NVT/NPT with the temperature of 300 K, pressure of 1 bar with 100 ps was performed before the production dynamics in order to equilibrate the system. Finally, the Leapfrog integrator with the time period of 10 ns was carried out for the production dynamics with the 5000 frames 66 .…”

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“…Mutations in the tubulin gene tubulin alpha 4a ( TUBA4A ) have been identified as likely pathogenic in several rare cases of fALS and FTLD-TDP ( Smith et al, 2014 ; Mol et al, 2021 ). TUBA4A is a tubulin alpha chain, one of the basic building blocks of microtubules and the cytoskeleton ( Ganne et al, 2023 ). A mutation in the C. elegans TUBA4A homolog, mec-12(e1605) , leads to progressive neuronal dysfunction in touch receptor neurons but does not alter lifespan ( Pan et al, 2011 ).…”

“…A mutation in the C. elegans TUBA4A homolog, mec-12(e1605) , leads to progressive neuronal dysfunction in touch receptor neurons but does not alter lifespan ( Pan et al, 2011 ). Structural and biological studies suggest TUBA4A mutations have both a loss of function component, disrupting microtubule polymerization, as well as a gain of function component, where mutant TUBA4A aggregates and disrupts microtubule dynamics in fALS ( Smith et al, 2014 ; Ganne et al, 2023 ).…”