Mutations in TrkA Causing Congenital Insensitivity to Pain with Anhidrosis (CIPA) Induce Misfolding, Aggregation, and Mutation-dependent Neurodegeneration by Dysfunction of the Autophagic Flux

Abstract

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder characterized by insensitivity to noxious stimuli and variable intellectual disability (ID) due to mutations in the NTRK1 gene encoding the NGF receptor TrkA. To get an insight in the effect of NTRK1 mutations in the cognitive phenotype we biochemically characterized three TrkA mutations identified in children diagnosed of CIPA with variable ID. These mutations are located in different domains of the protein; L213P in the extracellular domain, Δ736 in the kinase domain, and C300stop in the extracellular domain, a new mutation causing CIPA diagnosed in a Spanish teenager. We found that TrkA mutations induce misfolding, retention in the endoplasmic reticulum (ER), and aggregation in a mutation-dependent manner. The distinct mutations are degraded with a different kinetics by different ER quality control mechanisms; although C300stop is rapidly disposed by autophagy, Δ736 degradation is sensitive to the proteasome and to autophagy inhibitors, and L213P is a long-lived protein refractory to degradation. In addition L213P enhances the formation of autophagic vesicles triggering an increase in the autophagic flux with deleterious consequences. Mouse cortical neurons expressing L213P showed the accumulation of LC3-GFP positive puncta and dystrophic neurites. Our data suggest that TrkA misfolding and aggregation induced by some CIPA mutations disrupt the autophagy homeostasis causing neurodegeneration. We propose that distinct disease-causing mutations of TrkA generate different levels of cell toxicity, which may provide an explanation of the variable intellectual disability observed in CIPA patients.

Keywords: ER quality control; TRK1-transforming tyrosine kinase protein (Trk-A); autophagic flux; autophagy; congenital insensitivity to pain with anhidrosis; neurodegeneration; protein aggregation.

FIGURE 1.

Mutations in NTRK1 used in this study. A, pedigree of the family and results of the NTRK1 genetic analysis in CIPA patients. As indicated by the symbols in the pedigree, individuals II-1 and II-2 are affected by CIPA, whereas the parents and the sister are all carriers. The NTRK1 mutation detected in each individual is indicated under the corresponding symbol. Electropherograms demonstrating the presence of the NTRK1 point mutations. The position of each mutation is indicated by an asterisk. B, the mutations described in this report are named following the recommendations from the Human Genome Variation Society. C, location of the different mutations in the TrkA protein is shown in a scheme of the NTRK1 gene. Exons are numbered and shown as boxes.

FIGURE 2.

Inactivation of the signaling and neurite differentiation mediated by TrkA mutations. A, Western blotting of PC12nnr5 cells transfected with the indicated constructs after stimulation with NGF (50 ng/ml) during different time points. B, differentiation of PC12nnr5 cells co-transfected with GFP and the indicated TrkA constructs upon stimulation with NGF (50 ng/ml) during 72 h. Presence of neurites is observed in TrkA-wt (white arrows). C, percentage of PC12nnr5 transfected with the indicated construct cells having neurites longer than the cell body.

FIGURE 3.

TrkA CIPA mutants have impaired traffic to the plasma membrane. A, biotinylation protocol. Avidin-HRP immunodetection of TrkA immunoprecipitates from HeLa cells transfected with TrkA-wt and TrkA mutants. Mock indicates HeLa cells transfected with empty control plasmid. B, Western blotting of lysates from cells transfected with TrkA-wt and TrkA mutants treated with buffer (c), End-H (H), and N-glycosidase (N). C, immunolocalization of HeLa cells co-transfected with TrkA mutants (red) and GFP-Ras (green) in the absence and presence of 0.1% Triton X-100. DAPI nuclear staining and merge of three channels are shown. WB, Western blot.

FIGURE 4.

TrkA CIPA mutants are retained in the ER. A, TrkA mutants (red) co-localization with calnexin (green) in transfected HeLa cells. DAPI nuclear staining and merge of three channels are shown. B, Pearson's coefficient to assess the co-localization of TrkA-wt and mutants with calnexin. C, TrkA mutants (red) co-localization with giantin (green) in transfected HeLa cells. DAPI nuclear staining and merge of three channels are shown. D, Pearson's coefficient to assess the co-localization of TrkA-wt and mutants with giantin. Error bars are S.E.; *, p < 0.05; **, p < 0.01; ***, p < 0.001. t test compared with wt.

FIGURE 5.

TrkA CIPA mutations induce protein misfolding and aggregation. A, trypsin digestion of Triton X-100-soluble fractions of TrkA mutants. Samples were incubated with the indicated concentrations of trypsin during 10 min on ice, quenched by adding SDS-PAGE sample buffer (2 times), boiled, and analyzed by SDS-PAGE/immunoblot with a specific antibody against the extracellular region of TrkA. B, analysis of disease protein solubility in the non-ionic detergent Triton X-100. Cell extracts were prepared in lysis buffer supplemented with 1% Triton X-100. Total, pellet, and supernatant were analyzed by SDS-PAGE and immunoblotting. C, the ratio of pellet to total (pellet/total) was quantified for two independent experiments. Error bars are S.E. WB, Western blot.

FIGURE 6.

TrkA constructs have different degradation kinetics and differential sensitivity to proteasome and lysosome inhibitors. A, Western immunoblots of a representative experiment showing the levels of TrkA after protein translation inhibition with cycloheximide during the indicated time points. B, protein turnover half-lives of TrkA mutants were determined by quantifying the remaining levels of TrkA protein normalized to the actin blot. Three independent experiments consisting of independent transfections were performed. Data points were adjusted to a one exponential decay curve (black line) using GraphPad Prism software to calculate the half-lives times of each TrkA construct. C, Western blot analysis of cycloheximide treatment for 9 h and the sensitivity of the different TrkA constructs to the indicated inhibitors. D, fraction of the total protein remaining after 9 h. The values were normalized to time 0 h (1.0). Quantification of at least three independent experiments was quantified. Errors bars are S.E. p values are shown on the graph. t test compared with the control (−). ns, not significant.

FIGURE 7.

TrkA CIPA mutants induce the autophagic flux. A, HeLa cells were co-transfected with mock vector and the indicated TrkA constructs with LC3-GFP. A composite of confocal microscopy images for the channel green (LC3-GFP) and red (TrkA) is shown. B, the presence of green puncta indicative of AVs was quantified and plotted as the number of AVs per cell. C, HeLa cells co-transfected with TrkA-L213P and LC3-GFP were mock, wortmannin, and bafilomycin stimulated for 6 h, fixed, and analyzed by confocal microscopy. The number of green puncta of at least 20 cells was plotted in D. Error bars are S.E. *, p < 0.05; ** p < 0.01; ***, p < 0.001. One-way analysis of variance test with Tukey's multiple comparisons test (B) and t test (D) was compared with control.

FIGURE 8.

Cell toxicity of TrkA CIPA mutations in PC12nnr5 cells. Tunicamycin induces inhibition of protein glycosylation of all TrkA mutants. A, Western immunoblotting showing a decrease in the molecular weight due to inhibition of N-glycosylation of all the TrkA constructs upon tunicamycin treatment. B, percentage of PC12nnr5-transfected cells experimenting cell death (apoptotic cells) quantified by annexin V/propidium iodide. 2 μg/ml of tunicamycin were added to increase the basal ER stress. Three independent experiments were quantified. Error bars are S.E. p values are shown on the graph. t test compared with the control (C) is shown. ns, not significant.

FIGURE 9.

Presence of AVs and dystrophic neurites in L213P-transfected embryonic cortical neurons. A, embryonic cortical neurons were co-transfected with LC3-GFP and TrkA-wt or TrkA-L213P. 24 h post-transfection neurons were fixed and stained with TrkA antibody immunofluorescence (red). Several green positive AVs are observed in the soma and in the neurites of the neurons transfected with TrkA-L213P. B, E17 embryonic mouse cortical neurons were co-transfected with TrkA-wt and the CIPA mutants and GFP. Representative transfected neurons are shown. The presence of swollen regions in the neurites is indicated by white arrows. Inserts showed the TrkA staining. C, quantification of the percentage of transfected neurons bearing swollen regions. A positive neuron is counted if has more than 10 swollen regions in the axon or in the neurites. Error bars are S.E. p values are shown on the graph. t test compared with the control (C) is shown. ns, not significant.