Research ArticleAmyotrophic Lateral Sclerosis

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis

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Science Translational Medicine  03 May 2017:
Vol. 9, Issue 388, eaad9157
DOI: 10.1126/scitranslmed.aad9157
  • Fig. 1. Annexin A11 mutations identified in ALS patients after stringent filtering of exome sequencing data.

    (A and B) Pedigrees of UK. Family 1 and 2, respectively, carrying the p.D40G mutation. Family members for whom DNA was available for segregation analysis are labeled with M/W for a heterozygous mutation (A > G allele) or W/W for a homozygous reference allele. The gender has been anonymized for each individual. Affected individuals with ALS are denoted by a solid black diamond, unaffected individuals by white diamonds, and unaffected carriers with a black dot. Affected family members who underwent exome sequencing as part of this study are marked with a blue star. (C) The p.G175R mutation segregates in both an index case and affected sibling. (A to C) The age at death, where data are available, is listed above each p.D40G carrier (affected and unaffected). (D) Schematic representation of the annexin A11 protein highlighting the clustering of mutations in the N terminus (first 196 residues). Mutation positions in the N terminus are fully conserved in mammals and conserved in birds, amphibians, and reptiles if the mutations are in annexin domains. Mutations in FALS are indicated by red boxes and those in SALS by blue boxes. The p.D40G mutation found in two index FALS cases and an affected sibling from each UK. family, an Italian index case, and a UK. SALS case (n = 6) clusters with the p.G38R mutation in the N terminus of the annexin A11 molecule. The binding site of calcyclin (CACY) is located in the N terminus (residues 50 to 62).

  • Fig. 2. Annexin A11 immunohistochemical analysis in postmortem spinal cord tissue from a SALS case with the p.D40G mutation.

    (A) Phospho–TDP-43–positive cytoplasmic inclusion from the anterior horn of the spinal cord. (B to E) Annexin A11–positive inclusions in motor neurons of the spinal cord include skein-like structures (B) and filamentous and tubular-shaped structures (C and D). Occasional basket-like inclusions were seen in the spinal cord (E). (F) Abundant annexin A11–positive torpedo-like neuritic structures were present in the neuropil of the motor cortex (red arrows). Representative spinal cord staining for annexin A11 inclusions in a SALS case (n = 15) who is negative for ANXA11 mutations (G) and two C9ORF72 expansion–positive ALS cases (spinal cord and frontal cortex) (H and I). (J) Staining for annexin A11 inclusions was also negative in an ALS case harboring a p.D101G mutation in SOD1 (spinal cord). Similarly, frontotemporal lobar degeneration (FTLD)–TDP-43 cases (spinal cord; n = 3) (K) and Alzheimer’s disease (AD) cases (n = 3) (L) and control individuals (n = 13) (M) were also negative for staining for annexin A11 inclusions. (N and O) Double labeling of spinal cord tissue in the p.D40G SALS case for phospho–TDP-43 (green) and annexin A11 aggregates (red). (P) Costaining for ubiquitinated aggregates (red, annexin A11; green, ubiquitin) showed occasional colocalization (white arrow). Scale bars, 30 μm (A, E, G, and H), 20 μm (B and C), 15 μm (D), 50 μm (F, I, J, K, and L), 25 μm (M and N), and 50 μm (O and P).

  • Fig. 3. Transfection of WT and mutant annexin A11 into mouse primary motor neurons and HEK cells.

    (A) HA tagged annexin A11WT and mutant annexin A11G38R/D40G/R235Q constructs were transfected into mouse primary motor neurons. Transfected cells displayed (A) cytoplasmic vesicle–like structures for annexin A11WT/G38R/D40G and (B) increased localization of smaller foci in the cytoplasm of annexin A11R235Q transfected neurons [***P = 0.0004, one-way analysis of variance (ANOVA) and Dunnett’s post hoc test; bars represent mean and SEM]. Vesicles were defined as structures with a diameter between 0.5 and 1.9 μm (mean, 1 μm) and foci as structures with a diameter between 0.16 and 0.5 μm (mean, 0.3 μm). (C) A significant proportion of foci were associated with annexin A11R235Q (****P < 0.0001, one-way ANOVA and Dunnett’s post hoc test); a smaller proportion of vesicles were associated with annexin A11G38R and were absent with annexin A11R235Q (**P = 0.0051 and ****P < 0.0001, respectively, one-way ANOVA and Dunnett’s post hoc test). (D) Transfection of HEK cells with GFP tagged annexin A11WT/G38R/D40G/G189E/R235Q constructs followed by a solubility assay [showing lysate (L), soluble (S), and insoluble (I) fractions] revealed that annexin A11R235Q formed detergent-resistant insoluble aggregates. (E) The amount of the annexin A11R235Q insoluble fraction is statistically significant when compared with that for the WT protein (***P = 0.007, one-way ANOVA with Dunnett’s post hoc test).

  • Fig. 4. Annexin A11 with the p.R235Q mutation sequesters WT annexin A11.

    (A) Coexpression of ANXA11WT tagged with HA and ANXA11R235Q tagged with GFP in SH-SY5Y cells demonstrated colocalization by immunocytohistochemistry, suggesting that the mutant protein sequestered the WT protein. Scale bars, 10 μm. (B) To confirm this observation, we conducted solubility assays, generating lysate, soluble, and insoluble fractions, followed by Western blot. Expression of GFP-tagged ANXA11WT and GFP-tagged ANXA11R235Q in HEK cells replicated that seen in Fig. 3D, with aggregates seen only in the insoluble fraction (labeled as I) of ANXA11R235Q. Coexpression of GFP-tagged ANXA11WT or ANXA11R235Q with an equal amount of HA-tagged ANXA11WT in HEK cells resulted in accumulation of HA-tagged ANXA11WT in the insoluble fraction only when coexpressed with GFP-tagged ANXA11R235Q (indicated by a red cross) (n = 3). (C) A further IP assay also confirmed sequestering of ANXA11WT by ANXA11R235Q. Cotransfection of HEK cells with GFP-tagged ANXA11R235Q and HA-tagged ANXA11WT followed by IP with rabbit anti-GFP antibody, and probing with mouse anti-HA antibody demonstrated direct sequestering of ANXA11WT in the IP fraction. This included higher insoluble molecular weight species compared to control empty plasmid vector and empty plasmid vector plus GFP (top). Verification of the IP was shown by staining with mouse-GFP (input total protein lysate and IP; middle) and positive staining of HA-tagged ANXA11WT [input and nonbound bead flow-through (FT); top].

  • Fig. 5. Annexin A11 mutations disrupt binding to calcyclin.

    (A) Western blot demonstrating lack of binding of FLAG-tagged calcyclin to GFP-tagged mutant annexin A11 (ANXA11D40G, ANXA11G189E, and ANXA11R235Q) compared to ANXA11WT after IP (n = 3). Top: GFP intensities of input and immunoprecipitated fractions, with immunoglobulin G (IgG) heavy [50 kDA (*)] and light [25 kDa (**)] chains indicated. Bottom: Input and IP of FLAG-tagged calcyclin alone. Ctrl, control. (B) Cross section of postmortem spinal cord tissue showing calcyclin expression in (i) controls and (ii) a SALS case with the p.D40G mutation. High calcyclin expression can be seen in the lateral corticospinal tracts (black arrows). (iii) SALS case with no known ALS causing mutation but displaying strong expression of calcyclin in the lateral corticospinal tracts in postmortem spinal cord (black arrows). Scale bars, 40 μm (i), 30 μm (ii), and 25 μm (iii). (C) To determine the effect of calcyclin overexpression on aggregation, we conducted an NP-40 solubility assay by coexpressing 500 ng of GFP-tagged ANXA11WT or ANXA11R235Q and 500 ng of FLAG-tagged calcyclin in HEK cells. Clearance of insoluble p.R235Q mutant annexin A11 aggregates by calcyclin was observed. Addition of the proteasomal inhibitor MG132 restored the aggregation event by blocking degradation of the aggregated proteins by the ubiquitin proteosomal pathway. An equivalent amount of empty GFP vector (500 ng) was added to HEK cells expressing WT or p.R235Q mutant annexin A11 as a loading control. (D) Quantification of the proportion of insoluble fractions from (C) relative to untreated ANXA11GFP (WT) including calcyclin overexpression (+C) and calcyclin overexpression plus MG132 treatment (+C + M). Bands were quantified with ImageJ, and intensity was calculated with respect to untreated GFP-tagged ANXA11WT. A one-way ANOVA test demonstrated significance for increased aggregation due to the p.R235Q mutation and rescue of aggregation in p.R235Q mutant cells by calcyclin overexpression and MG132 treatment (R235Q + C + M). *P < 0.05 and ***P < 0.001, respectively, one-way ANOVA and Dunnett’s post hoc test (n = 3).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/388/eaad9157/DC1

    Fig. S1. The ANXA11 p.D40G mutation shares a common haplotype composed of four exonic SNPs and two microsatellites.

    Fig. S2. Haplotype gene map of the ANXA11 p.D40G locus.

    Fig. S3. Annexin A11 Western blot of lysates made from postmortem tissue of the SALS patient harboring the p.D40G mutation and control individuals.

    Fig. S4. p.R235Q ANXA11-GFP colocalizes with ubiquitin and p62 in HEK cells.

    Fig. S5. Modeling of annexin A11 identifies two amphipathic helices in the N terminus of annexin A11 that overlap with the G38 and D40 residues.

    Fig. S6. Twenty-two ExAC polymorphisms spanning the D40 locus do not disrupt the formation of amphipathic helices.

    Fig. S7. Rare and common ANXA11 polymorphisms do not disrupt calcyclin binding compared to ALS-specific variants.

    Fig. S8. N-terminal annexin A11 mutations do not alter binding of sorcin and ALG-2.

    Table S1. New variants shared between members of each familial ALS family.

    Table S2. New variants shared between ANXA11 p.D40G families.

    Table S3. Homogeneity of the FALS cohort and ExAC NFEs.

    Table S4. Primer sequences used in the ANXA11 p.D40G haplotype study and for Sanger sequencing of ANXA11 exons.

    Table S5. Exon coverage data of genes within the minimal p.D40G haplotype region.

    Table S6. All variants found within ANXA11 in FALS and SALS.

    Table S7. Clinical details of patients with ANXA11 mutations.

  • Supplementary Material for:

    Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis

    Bradley N. Smith, Simon D. Topp, Claudia Fallini, Hideki Shibata, Han-Jou Chen, Claire Troakes, Andrew King, Nicola Ticozzi, Kevin P. Kenna, Athina Soragia-Gkazi, Jack W. Miller, Akane Sato, Diana Marques Dias, Maryangel Jeon, Caroline Vance, Chun Hao Wong, Martina de Majo, Wejdan Kattuah, Jacqueline C. Mitchell, Emma L. Scotter, Nicholas W. Parkin, Peter C. Sapp, Matthew Nolan, Peter J. Nestor, Michael Simpson, Michael Weale, Monkel Lek, Frank Baas, J. M. Vianney de Jong, Anneloor L. M. A. ten Asbroek, Alberto Garcia Redondo, Jesús Esteban-Pérez, Cinzia Tiloca, Federico Verde, Stefano Duga, Nigel Leigh, Hardev Pall, Karen E. Morrison, Ammar Al-Chalabi, Pamela J. Shaw, Janine Kirby, Martin R. Turner, Kevin Talbot, Orla Hardiman, Jonathan D. Glass, Jacqueline de Belleroche, Masatoshi Maki, Stephen E. Moss, Christopher Miller, Cinzia Gellera, Antonia Ratti, Safa Al-Sarraj, Robert H. Brown Jr., Vincenzo Silani, John E. Landers, Christopher E. Shaw*

    *Corresponding author. Email: chris.shaw{at}kcl.ac.uk

    Published 3 May 2017, Sci. Transl. Med. 9, eaad9157 (2017)
    DOI: 10.1126/scitranslmed.aad9157

    This PDF file includes:

    • Fig. S1. The ANXA11 p.D40G mutation shares a common haplotype composed of four exonic SNPs and two microsatellites.
    • Fig. S2. Haplotype gene map of the ANXA11 p.D40G locus.
    • Fig. S3. Annexin A11 Western blot of lysates made from postmortem tissue of the SALS patient harboring the p.D40G mutation and control individuals.
    • Fig. S4. p.R235Q ANXA11-GFP colocalizes with ubiquitin and p62 in HEK cells.
    • Fig. S5. Modeling of annexin A11 identifies two amphipathic helices in the N terminus of annexin A11 that overlap with the G38 and D40 residues.
    • Fig. S6. Twenty-two ExAC polymorphisms spanning the D40 locus do not disrupt the formation of amphipathic helices.
    • Fig. S7. Rare and common ANXA11 polymorphisms do not disrupt calcyclin binding compared to ALS-specific variants.
    • Fig. S8. N-terminal annexin A11 mutations do not alter binding of sorcin and ALG-2.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). New variants shared between members of each familial ALS family.
    • Table S2 (Microsoft Excel format). New variants shared between ANXA11 p.D40G families.
    • Table S3 (Microsoft Excel format). Homogeneity of the FALS cohort and ExAC NFEs.
    • Table S4 (Microsoft Excel format). Primer sequences used in the ANXA11 p.D40G haplotype study and for Sanger sequencing of ANXA11 exons.
    • Table S5 (Microsoft Excel format). Exon coverage data of genes within the minimal p.D40G haplotype region.
    • Table S6 (Microsoft Excel format). All variants found within ANXA11 in FALS and SALS.
    • Table S7 (Microsoft Excel format). Clinical details of patients with ANXA11 mutations.

    [Download Tables S1 to S7]

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