Aggregate formation and phosphorylation of neurofilament-L Pro22 Charcot–Marie–Tooth disease mutants

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13- cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.

Neurofilament light chain polypeptide gene mutations in Charcot–Marie–Tooth disease: nonsense mutation probably causes a recessive phenotype

“…13 Functional studies have shown that missense mutant NEFL proteins disrupt the assembly with wild-type NEFL and with the NEFM and NEFH, and cause aggregation, resulting in the disruption of axonal neurofilament translocation and anterograde or retrograde axonal transport including mitochondria. [14][15][16] In this study, we found a homozygous nonsense mutation, Glu140-Stop, in one patient (case 2) in addition to four heterozygous missense mutations in five other patients. The parents of case 2 were first cousins, but they never complained of muscle weakness.…”

NEFL Pro22Arg mutation in Charcot-Marie-Tooth disease type 1

“…In previous studies, Pro22Ser and Pro22Thr mutants showed defects in filamentous network formation irregular aggregation in the SW13-cell, and the mutations abolish the Thr-Pro PDPK phosphorylation site in the NEFL head domain, which regulates filament assembly via phosphorylation (Pérez-Ollé et al 2005;Sasaki et al 2006). In our study, patients with the Pro22Arg mutation had the same demyelinating neuropathies as did patients with the Pro22Thr mutation; therefore, it seems that the Pro22Arg mutation also may abolish the Thr-Pro phosphorylation site of the head domain by PDPKs.…”

“…It has been suggested that Pro22 mutations abolish the Thr-Pro phosphorylation sequence of the head domain of NEFL by proline-directed protein kinases (PDPKs) (Sasaki et al 2006). In this study, we identified another NEFL Pro22Arg mutation, which was not reported in the inherited peripheral neuropathies mutation database (http://www.molgen.ua.ac.be/CMTMutations), and sought to determine its clinical and pathological characteristics.…”

“…Neurofilament light chain polypeptide (NEFL), which consists of an N-terminal head, a central road, and a C-terminal tail domain, is one of the most abundant cytoskeletal components in the neuron (Brownlees et al 2002). Several missense mutations in the NEFL gene have been reported, and a number of these have been predicted to produce alterations in the formation of the intermediate filament network in neurons (Pérez-Ollé et al 2005;Sasaki et al 2006). Mutations in the NEFL gene were originally reported to be associated with CMT2E (De Jonghe et al 2001;Fabrizi et al 2004).…”

Intermediate Charcot-Marie-Tooth disease

“…It is supposed that NEFL mutants not only infl uence the normal assembly of neurofi laments, but also cause aggregate formation that prevents transport or other physiological functions of the axon [53] . Moreover, NEFL mutants impede neurofilament assembly and transport in vivo and affect mitochondrial morphology by interfering with mitochondrial dynamics in a cellular model of the CMT2E/1F phenotype [54,55] .…”