Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy

Abstract

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Figure 1

NEFH Frameshift Variants in CMT2-Affected Families Asterisks indicate probands. (A) Pedigree and Sanger sequence traces of the CMT-affected family carrying the NEFH variant c.3010_3011delGA (p.Asp1004Glnfs^∗58). Abbreviations are as follows: M, mutant c.3010_3011delGA allele; and +, wild-type allele. (B) Pedigree and Sanger sequence traces of the CMT-affected family carrying the NEFH variant c.3017_3020dup (p.Pro1008Alafs^∗56). Abbreviations are as follows: M, mutant c.3017_3020dup allele; and +, wild-type allele. (C) Diagram shows NEFH domains and variants associated with diseases. Coding KSP deletions and insertions from reported ALS individuals are represented by triangles. CMT frameshift variants from families UK1 and F2 are indicated by arrows.

Figure 2

Identification of CAEs Encoded by the NEFH and NEFL 3′ UTRs (A) Clustal Omega multiple-sequence alignment of wild-type NEFH and frameshift variants harbored by the CMT-affected families. Translation of the 3′ UTR open reading frames (ORFs) is illustrated. (B) TANGO score of NEHF 3′ UTR ORFs. (C) Consensus sequence of positive residues (asterisks) for all aggregation predictors tested for NEFH 3′ UTR ORF3. (D) NEFL 3′ UTR ORF sequences. (E) TANGO score of NEFL 3′ UTR ORFs. (F) Consensus sequence of positive residues (asterisks) for all aggregation predictors tested for NEFL 3′UTR ORF1.

Figure 3

Protein Aggregation in Cultured Neuro-2a Cells Expressing NF Genes with CAE-Encoding 3′ UTRs (A) Perinuclear aggregates in GFP-FS-NEFH-transfected cells after 24 hr. Graphs show the number of transfected cells containing NEFH aggregates and the percentage of cells with neuronal projections from six independent experiments. Error bars represent the SD, and the scale bar represents 10 μm. (B) A small proportion of cells transfected with the truncated construct, GFP-NEFH-Stop4, showed protein aggregation, and cells transfected with GFP-WT-NEFH-CAE showed high levels of aggregation. Scale bars represent 10 μm. (C) Prominent protein aggregation in cells transfected with GFP-NEFL-ORF. The graph shows the number of cells expressing NF-like structures. Error bars represent the SD, and scale bars represent 10 μm.

Figure 4

Evaluation of Cultured Neuro-2a Cells Expressing the NEFH 3′ UTR-Encoded CAE (A) Western blot shows comparable levels of GFP in cells transfected with GFP-WT-NEFH and GFP-FS-NEFH. (B–D) Sample time-lapse phase-contrast and GFP live-cell images obtained from IncuCyte imager system. (B) Calculation of the average area (μM2) of green (GFP) objects per cell at different time points. (C) Quantification of GFP-positive cell confluence. (D) Quantification of the average eccentricity of green objects measures object roundness from 0 to 1 (a perfect circle has a value of 0). Error bars represent the SD. (E) Fibers of approximately 10 nm in diameter in a Neuro-2a cell transfected with GFP-FS-NEFH. Filaments are visible in the cytoplasm in longitudinal sections (blue box) and cross-sections (red box). Approximately 10% of cells transfected with GFP-FS-NEFH showed filaments. Scale bars represent 200 nm. (F) Aggregates from GFP-FS-NEFH cells co-localized with thioflavin T staining. Scale bars represent 10 μm.

Figure 5

Confocal Microscopy Shows NEFH Subcellular Co-localization (A) Cells co-transfected with GFP-WT-NEFH and NEFL-Myc showed co-localization of NEFH and NEFL in NF network structures. Cells co-transfected with GFP-FS-NEFH and NEFL-Myc showed co-localization in the aggregates. Scale bars represent 10 μm. (B) Co-immunuoprecipitation assay: cell lysates were immunoprecipitated with an antibody against either GFP or Myc and IgG as a negative control. Both GFP-WT-NEFH and GFP-FS-NEFH were co-immunoprecipitated with NEFL-Myc. Kinesin was also present in the co-immunoprecipitate. (C) Cells transfected with either GFP-WT-NEFH or GFP-FS-NEFH showed normal distribution of the microtubule network in cells stained with anti-tubulin. Scale bars represent 10 μm. (D) Subcellular localization of mitochondria: cells transfected with GFP-WT-NEFH showed an even cytoplasmic distribution of mitochondria, whereas GFP-FS-NEFH-transfected cells showed mitochondria accumulation next to aggregates. Scale bars represent 10 μm.

Figure 6

Phenotypic Analysis in Zebrafish Embryos Injected with Either Wild-Type or Mutant RNAs (A) Zebrafish embryos injected with RNA encoding either GFP-WT-NEFH or GFP-FS-NEFH did not show major morphological defects at 48 hpf. (B) Motor neurons labeled in the transgenic line Tg(Olig2:Dsred). Embryos injected with GFP-WT-NEFH showed normal motor neuron development, whereas zebrafish injected with GFP-FS-NEFH showed examples of stunted axons. The scale bar represents 100 μm. (C) Quantification of the average axon length shows shorter lengths in GFP-FS-NEFH-injected fishes than in both GFP-WT-NEFH-injected and uninjected larvae. Axon length was not significantly different between GFP-WT-NEFH-injected fishes and uninjected control fish. The average axon length per fish was calculated from the first four myotomes. Data were compiled from three independent experiments, and significance was determined by a one-way ANOVA with a Bonferroni post-test. p values are ^∗0.024 and ^∗∗0.014. (D) Confocal images depict relative GFP fluorescence of the tagged NEFH proteins. Scale bars represent 100 μm. (E) Semiquantitative assessment of relative fluorescence intensity as mean gray values 0–255. (F) Western blot shows the presence of both wild-type and mutant NEFH proteins at 24 hpf. GFP-FS-NEFH-injected larvae were also incubated in 50 μM chloroquine (Chq) in an attempt to increase the amount of the mutant protein. (G) The presence of both GFP-WT-NEFH and GFP-FS-NEFH mRNAs was confirmed by RT-PCR.

Figure 7

Protein Aggregation in Cultured Neuro-2a Cells Expressing FUS Fused in Frame with Its Predicted 3′ UTR-ORF1-Encoded CAE (A) TANGO score of ORF1 in the FUS 3′ UTR. (B) Consensus sequence of positive residues (asterisks) for all aggregation predictors tested for ORF1 in the FUS 3′ UTR. (C) GFP-WT-FUS localized to the nucleus, whereas GFP-FUS-CAE aggregated in the cytoplasm and neuronal-like projections. A magnified view of the data (M) is shown in the white box. Scale bars represent 10 μm.