Abstract
Posttranslational glutamylation/deglutamylation balance in tubulins influences dendritic maturation and neuronal survival of cerebellar Purkinje neurons (PNs). PNs and some additional neuronal types degenerate in several spontaneous, independently occurring Purkinje cell degeneration (pcd) mice featuring mutant neuronal nuclear protein induced by axotomy (Nna1), a deglutamylase gene. This defective deglutamylase allows glutamylases to form hyperglutamylated tubulins. In pcd, all PNs die during postnatal “adolescence.” Neurons in some additional brain regions also die, mostly later than PNs. We show in laser capture microdissected single PNs, in cerebellar granule cell neuronal clusters, and in dissected hippocampus and substantia nigra that deglutamase mRNA and protein were virtually absent before pcd PNs degenerated, whereas glutaminase mRNA and protein remained normal. Hyperglutamylated microtubules and dimeric tubulins accumulated in pcd PNs and were involved in pcd PN death by glutamylase/deglutamylase imbalance. Importantly, treatment with a microtubule depolymerizer corrected the glutamylation/deglutamylation ratio, increasing PN survival. Further, before onset of neuronal death, pcd PNs displayed prominent basal polylisosomal masses rich in ER. We propose a “seesaw” metamorphic model summarizing mutant Nna1-induced tubulin hyperglutamylation, the pcd’s PN phenotype, and report that the neuronal disorder involved ER stress, unfolded protein response, and protein synthesis inhibition preceding PN death by apoptosis/necroptosis.
Highlights
Tubulins are present in all eukaryotic animal and plant organisms from living single-cell organisms to humans, with free dimeric α- and β-tubulins plus microtubules, both types being crucial multifunctional cytoplasmic constituents
With an integrated portfolio of molecular and cellular techniques, including laser capture microdissection (LCM) global gene expression in single quantitative PCR, IHC, and immunoblots, we have systematically examined major members of the glutamylation and glycylation families in LCM-isolated single Purkinje neurons (PNs), in enriched PN cultures, and in intact isolated cerebellar cortices
To verify and confirm the adverse effects of hyperglutamylated free tubulins on ER regulators and polyribosome-associated initiators, we found that treatment with Nna1 shRNA and/or tubulin tyrosine ligase–like 1 (Ttll1) cDNA increased calreticulin 3 (Calr3) and activating transcription factor 6 (Aft6), and decreased Elf2β2 in cultured PNs (Figure 4F)
Summary
Tubulins are present in all eukaryotic animal and plant organisms (those with intracellular nuclei) from living single-cell organisms to humans, with free dimeric α- and β-tubulins plus microtubules, both types being crucial multifunctional cytoplasmic constituents. Glutamylation and glycylation are 2 of the most common genetic and enzymatic types of posttranslational modification of tubulin proteins [1,2,3,4]. Glutamylases bind postnatally to tails of both α- and β-tubulins. Glutamylase and deglutamylase, respectively, add and remove polyglutamyl side chains of variable length on or from the exposed γ-carboxyl receptor groups of microtubule subsets of tubulin [5, 7, 8].
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