Defective presynaptic choline transport underlies hereditary motor neuropathy

Abstract

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies.

Figure 1

Family Pedigree and c.1497delG Cosegregation The c.1497delG variant results in the creation of a novel SspI restriction site that facilitates cosegregation analysis by restriction digestion of exon 9 PCR products resolved by polyacrylamide gel electrophoresis (PCR primers used for amplification of genomic DNA were 5′-CCCTGGCTATTACCCTGATG-3′ and 5′-CACAAGTGCAAGTTCATCTAATTT-3′). The c.1497delG variant results in the digestion of the WT 218 bp product into fragments of 198 bp and 20 bp (not shown). All subjects with the 198 bp fragment are heterozygous and thus also have an undigested 218 bp band. These genotypings were also confirmed by dideoxy sequence analysis. For cross reference with the previously published pedigree, refer to Table S1.

Figure 2

Immunoblotting and Cell-Surface Biotinylation of WT and FS CHT (A) Levels of total WT and FS CHT after SDS-PAGE and immmunoblotting with HA antibody confirm FS-CHT truncation and reveal reduced expression of total FS CHT at both 2× (lanes 1 and 2) and 1× (lanes 3 and 4) protein. Stripping and blotting for β-actin reveal equal protein loaded across lanes. All experiments were conducted with the same total amount of transfected plasmid and were balanced with a pcDNA3 vector. (B) Saturation analysis of HC-3-sensitive choline-transport activity in transfected HEK 293 cells demonstrates significantly reduced transport activity for FS CHT than for WT CHT. Transport deficits of FS-CHT-transfected wells are accounted for by a reduction in choline transport V_MAX (33.4% reduction, p = 0.024, Student’s unpaired t test) as opposed to a change in choline K_M (WT = 5.15 ± 1.12 μM, FS = 6.12 ± 1.72 μM, p = 0.66, Student’s unpaired t test). Specific choline uptake was determined by subtraction of the uptake obtained from transfected cells from that obtained from mock (vector) transfected cells acquired from assays conducted in parallel. (C) Levels of total and surface WT and FS CHT are lower in cells transfected with both WT and FS CHT at 1× levels (as presented in A) than in cells separately transfected at 1× levels. All transfections were performed with identical amounts of total plasmid and were balanced by a pcDNA3 vector. Equivalent findings were obtained with cells transfected with the tagged WT and FS CHT constructs. (D) Choline-transport activity is lower in FS CHT than in WT CHT (n = 5, ^∗p = 0.035, Student’s unpaired t test). Observed transport activity of cells coexpressing WT and FS CHT (WT + FS_(o)) falls significantly below that expected (WT + FS_(e)) from a sum of the independent expression of these isoforms (n = 5,^∗p = 0.005, Student’s unpaired t test). (E) HC-3-sensitive choline-transport activity in whole-blood monocytes of dHMN-VII subjects (n = 4) and controls (n = 11) reveals a dramatic loss in dHMN-VII subjects (^∗p = 0.72 × 10⁻⁶, Student’s one-tailed t test).