ALS-linked protein disulfide isomerase variants cause motor dysfunction

Abstract

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

Keywords: ERp57; PDIA1; amyotrophic lateral sclerosis; protein disulfide isomerase.

Figure 1. Expression of ALS‐linked variants of PDIA1 and ERp57 causes motoneuron dysfunction and impairs neurite outgrowth

1. A

   Location of missense variants of PDIA1 and ERp57 identified in ALS cases. PDI primary structure: catalytic a and a′ domains containing the active‐site motif CXXC sequence (dark purple and dark green), non‐catalytic domains b and b′ containing ligand‐binding sites (light purple and light green), and x‐linker region (light gray). The constructs used in this study contained a V5‐tag (orange) at the C‐terminus that was inserted previous to the ER‐retention signal (dark gray).

2. B

   Expression of PDI variants in zebrafish. Zebrafish embryos at 1–2‐cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1^WT, PDIA1^D292N, and PDIA1^R300H: 200 pg/embryo; ERp57^WT, ERp57^D217N, and ERp57^Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti‐V5 antibody in total embryo extracts at 24 h post‐fertilization (hpf). Actin was used as a loading control. In the left panel, 80 μg protein extract was used, and in the right panel, 120 μg protein extract was used.

3. C, D

   Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS‐linked PDI mutants and wild‐type controls (PDIA1^WT and PDIA1^R300H: 80 pg mRNA/embryo; ERp57^WT, ERp57^D217N, ERp57^D217N, and ERp57^Q481K: 30 pg mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.

4. E, F

   Quantification of motoneuron axon length and axon branching in 36 hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (E) and ERp57 (F) mutants.

5. G

   Touch‐evoked escape responses of 48 hpf zebrafish embryos injected with ALS‐linked PDI mutants (Kabashi et al, 2009, 2011). The number of touches necessary to evoke an escape response (left) and speed (right, in mm/s) of the escape response was determined for each condition.

Data information: The total number of analyzed embryos is indicated in parenthesis. Experiments of (D–F) were performed in selected animals sharing normal overall embryo morphology and viability, to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and/or animal behavior. Statistical analyses were performed using one‐way ANOVA and Bonferroni's post hoc tests. Mean ± SEM with only statistically significant P‐values are shown: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Abbreviations: A (anterior), D (dorsal), P (posterior), V (ventral). Scale bars represent 500 μm (C and D: a, d, g, j), 200 μm (C and D: b, e, h, k), and 100 μm (C and D: c, f, i, l).Source data are available online for this figure.

Figure EV1. NMJ morphology in zebrafish expressing mutant PDIs

Co‐immunostaining of SV2 (red) and α‐bungatoxin (α‐BTX; green) in the neuromuscular junction. Merge: overlay of SV2 and α‐BTX signals. Expression of PDIA1^R300H and ERp57^Q481K induces a change in the shape of myotomes from “V” to “U shape” and a decrease and disorganization of SV2 and α‐BTX staining (arrowhead) compared to controls. Scale bar represents 100 μm.

Figure 2. Motoneuron morphology upon expression of ALS‐linked PDI variants

1. A

   NSC34 cells were transiently transfected with expression vectors for V5‐tagged wild‐type and mutant PDIs. After 48 h, overexpressed PDI variants were assessed under reducing conditions in an 8% SDS–PAGE. An antibody detects total PDIA1 (endogenous mouse PDIA1 and exogenous human V5‐tagged PDIA1) (first panel). A second antibody detects only human PDIA1, therefore only V5‐tagged PDIA1 appears (second panel). A mouse‐ and human‐specific antibodies were used to detect total ERp57 (third panel). Anti‐V5 was used to detect the exogenous PDI variants. Anti‐β‐actin was used as a loading control.

2. B, C

   NSC34 cells were transiently transfected with the indicated PDI constructs together with a GFP expression plasmid. Cells were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. (B) Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment were analyzed. In addition, (C) the percentage of cells with neurites was quantified in the three independent experiments (right panel).

3. D

   Primary rat ventral spinal cord neurons were prepared and after 4 days in vitro (DIV) transfected with GFP alone or together with the indicated PDI constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP‐ and SMI32‐positive cells was quantified. Results are compiled from three independent experiments.

4. E

   Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDI‐expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP‐ and HB9‐positive neurons was obtained. Neurite number and assessment of the length of each neurite were performed in a similar manner as for primary rat motoneurons (see Materials and Methods). Four independent experiments were performed.

5. F

   NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment were analyzed.

Data information: Statistical analyses were performed using one‐way ANOVA and Bonferroni's post hoc tests. Mean ± SEM with only statistically significant P‐values are shown: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale bars represent 25 μm (B, D and E).Source data are available online for this figure.

Figure EV2. Sensitivity of NSC34 cells expressing wild‐type and mutant PDIs to ER stress

1. A

   V5‐tagged wild‐type and mutant PDIs were transiently expressed in NSC34 cells. After 44 h, cells were treated with 1 μg/ml tunicamycin for 16 h and the ER stress markers XBP1s, ATF4, and BiP were detected using the respective antibodies. V5‐tagged PDIs were detected in the cell extract using anti‐V5. HSP90 was used as a loading control.

2. B, C

   Cell viability after treatment with thapsigargin (B) or tunicamycin (1 μg/ml) (C) was determined by the MTT assay. The dose–response curve of the mock is the same, since all treatments were performed in the same experiment. Mean and SEM of triplicate measurements of one representative experiment are shown.

3. D

   BDNF‐GFP was co‐expressed with V5‐tagged PDIs (wild types and mutants) in N2a cells. BDNF secretion to the cell medium was assessed by Western blot after 18 and 42 h. As a control, V5‐tagged PDIs were detected in the cell extract using anti‐V5.

4. E

   V5‐tagged wild‐type and mutant PDIs were overexpressed in Neuro2a cells. Cell medium was collected after 48 h and tested for the presence of progranulin by ELISA.

Data information: Mean and SEM are shown. For statistical analysis, Student's t‐test was performed. ***P ≤ 0.001.Source data are available online for this figure.

Figure 3. Effects of ALS‐linked mutations in PDIA1 on the structure and enzymatic activity

1. PDI mutants form abnormal disulfide‐dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5‐tagged wild‐type and mutant PDIA1. After 48 h, differential disulfide‐dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non‐reducing (−DTT) conditions in an 8% SDS–PAGE. Anti‐V5 was used for detection in Western blot.

2. Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg³⁰⁰ located to the b′ domain of PDIA1 with Trp³⁹⁶ located to the a′ domain adjacent to the active‐site motif CGHC (designated as AS in yellow) is shown in comparison with the mutated version of PDIA1^R300H highlighting the same residues. A potential stabilization of the interaction between the b′ and a′ domain is shown that may be caused by the interaction of the imidazole rings of mutated His³⁰⁰ with Trp³⁹⁶.

3. ALS‐linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of PDIA1^WT and mutants are shown.

4. Averages for CD spectroscopic thermal denaturation of recombinant PDIA1^WT and mutants.

5. Representative electrophoresis analysis of proteinase K‐treated recombinant PDIA1 variants. Mass spectrometric analysis of proteinase K‐digested samples from total sample and from in‐gel trypsin‐digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x‐linker region. Bottom panel: ratios of band 1 to band 2 were quantified in four independent experiments.

6. Representative fluorescence spectra of recombinant PDIA1 b′x^WT fragments and the equivalent ALS‐linked mutants (n = 6). Ratios from the two peak areas and change compared to PDIA1 b′x^WT. Both mutants analyzed show a significant shift in peak position compared to wild type, but in opposite directions, suggesting that PDIA1^D292N shifts equilibrium toward the capped version (x‐region over the binding pocket) and the PDIA1^R300H mutant toward the uncapped version (Nguyen et al, 2008).

7. The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.

8. Measurement of H₂O₂ levels at the ER lumen of living cells. Left panel: reduced PDIs can be oxidized by ERO1Lα, which then transfers electrons to molecular oxygen (O₂) to generate hydrogen peroxide as product from PDI activity. Right panel: NSC34 cells were transiently co‐transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h, the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n = 55–74) monitored in four independent experiments are shown.

9. HEK293T cells were transfected with expression vectors for V5‐tagged wild‐type and mutant PDIA1, as well as empty vector. After 48 h, V5‐tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by Western blot. The inputs and elutions are shown as control. Right panel: quantification of the degree of interaction is presented.

Data information: Statistical analyses were performed using Student's t‐test in (F, G and I) or one‐way ANOVA and Bonferroni's post hoc tests in (H). Mean ± SEM with P‐values: n.s., P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.Source data are available online for this figure.

Figure 4. ALS‐linked mutations in ERp57 lead to altered interactions with calnexin and calreticulin

1. PDI mutants form abnormal disulfide‐dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5‐tagged wild‐type and mutant ERp57. After 48 h, differential disulfide‐dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non‐reducing (−DTT) conditions in an 8% SDS–PAGE. Anti‐V5 was used for detection in Western blot.

2. ALS‐linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57^WT and mutants are shown.

3. A thermal denaturation curve of ERp57^WT and mutants was performed.

4. Representative electrophoresis of proteinase K‐treated ERp57 recombinant proteins.

5. Surface plasmon resonance analysis was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P domain. K_D values are expressed as percentage of ERp57^WT. Values are derived from four independent experiments. For absolute K_D values, please see Appendix Table S2.

6. HEK293T cells were transfected with expression vectors for V5‐tagged wild‐type and mutant ERp57, as well as empty vector. After 48 h, V5‐tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: quantification of the degree of interaction is presented.

7. The gain of an N‐glycosylation site of ERp57^D217N was predicted after the analysis of the protein sequence since the change of Asp²¹⁷ to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5‐tagged wild‐type and mutant ERp57, as well as empty vector and treated with 1 μg/ml tunicamycin (Tm) for 20 h to inhibit N‐glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N‐glycosylation was analyzed by Western blot using anti‐V5 antibody.

Data information: Statistical analyses were performed using Student's t‐test in (E) and (F). Mean ± SEM with P‐values: *P ≤ 0.05; ***P ≤ 0.001.Source data are available online for this figure.

Figure 5. ERp57 deficiency in the nervous system leads to motor dysfunction and premature death

1. ERp57^flox/flox mice were crossed with Nestin‐Cre transgenic mice to generate nervous system‐specific ERp57‐deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57^WT: n = 4, ERp57^Nes+/−: n = 5, and ERp57^Nes−/−: n = 4 mice. HSP90 levels were used as a loading control. Right panel: quantification of ERp57 levels was performed relative to Hsp90 levels.

2. Body weight measurements were performed at the indicated time points in ERp57^WT (n = 50), ERp57^Nes+/− (n = 32) and ERp57^Nes−/− (n = 19) mice.

3. Rotarod performance was performed ERp57^WT (n = 20), ERp57^Nes+/− (n = 15) and ERp57^Nes−/− (n = 8) mice.

4. Hanging test performance was assessed in ERp57^WT (n = 41), ERp57^Nes+/− (n = 32), and ERp57^Nes−/− (n = 12) mice.

5. Kaplan–Meier survival curve for ERp57^Nes−/− mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57^WT (n = 50) and ERp57^Nes+/− (n = 32) mice are shown as a reference.

6. Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57^WT and ERp57^Nes−/− mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.

7. Stereological analysis of the spinal cord from ERp57^WT (n = 4), ERp57^Nes+/− (n = 4) and ERp57^Nes−/− (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker choline acetyltransferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.

8. Representative images of the spinal cord myelination of ERp57^WT and ERp57^Nes−/− mice.

Data information: For statistical analysis, two‐way ANOVA with Bonferroni's post hoc test was performed in (B) and one‐way ANOVA with Bonferroni's post hoc test was performed in (A, C and D). Mean ± SEM with P‐values: n.s., non‐significant, P > 0.05; *P ≤ 0.05; ***P ≤ 0.001.Source data are available online for this figure.

Figure 6. ERp57 deficiency results in loss of neuromuscular connectivity

1. Representative EMG recordings of ERp57^WT (n = 16), ERp57^Nes−/+ (n = 12) and ERp57^Nes−/− (n = 5) mice is presented. The presence of positive sharp waves (PSW) in ERp57^Nes+/− and ERp57^Nes−/− mice indicated muscle denervation.

2. Analysis of NMJ and muscle morphologies were performed in ERp57^WT (n = 5), ERp57^Nes+/− (n = 5) and ERp57^Nes−/− (n = 4) mice. Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 μm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.

3. Whole‐mounted diaphragms from ERp57^WT and ERp57^Nes−/− mice were co‐stained with anti‐neurofilament (red) and α‐BTX to reveal the postsynaptic densities (green). Three‐dimensional reconstructions (lower panel) of higher magnification are shown. ERp57^WT NMJs are fully innervated pretzel‐like, whereas ERp57^Nes−/− NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Videos EV4, EV5, EV6, EV7).

4. Representative 3D reconstructions of z‐stacks showing the five categories in which NMJ morphologies were grouped: pretzel, fragmented, O‐shaped, C‐shaped, and dismantled NMJs. Histogram of the distribution frequency of > 200 different NMJ per animal is shown.

5. Quantitative morphometry of > 60 NMJs in the different genotypes analyzed.

Data information: For statistical analysis, one‐way ANOVA with Bonferroni's post hoc test was performed in (B, D, and E). Mean ± SEM with P‐values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 7. ERp57‐deficient mice display aberrant skeletal muscle fibers

1. The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57^WT (n = 4), ERp57^Nes+/− (n = 5), and ERp57^Nes−/− (n = 4) mice. HSP90 was used as a loading control.

2. Tibialis anterior muscles from ERp57^WT (n = 5), ERp57^Nes+/− (n = 5), and ERp57^Nes−/− (n = 4) mice were dissected and cryosectioned (20 μm) for the analysis of several parameters of muscle physiology. Upper panel: hematoxylin/chromotrope (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasma membrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.

3. Tibialis anterior cryosections (20 μm) from ERp57^WT, ERp57^Nes+/−, and ERp57^Nes−/− mice were stained for NADH‐TR activity to identify oxidative myofibers (left panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: NADH‐TR‐positive (slow twitch) and NADH‐TR‐negative (fast twitch) myofibers were quantified as percentage of total for every genotype (upper right panel). The mean diameter of slow‐ and fast‐twitch muscle fibers was evaluated at the light microscope using the ImageJ software (lower right panel). Data represent the average ± SEM of > 300 fibers per animal.

Data information: Scale bars represent 50 μm (B, C). For statistical analysis one‐way ANOVA with Bonferroni's post hoc test was performed in (C). Mean ± SEM with P‐values: *P ≤ 0.05.Source data are available online for this figure.

Figure 8. Loss of ERp57 in the cortex results in lower levels of the synaptic protein SV2

1. The levels of synaptic proteins SV2, NMDAR2A, PSD95, and synaptophysin in the cortex were monitored by Western blot. ERp57^WT (n = 4), ERp57^Nes+/− (n = 5) and ERp57^Nes−/− (n = 4) mice. HSP90 and β‐actin were used as loading controls.

2. Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β‐actin was used as the reference.

Data information: For statistical analysis one‐way ANOVA with Bonferroni's post hoc test was performed in (B). Mean ± SEM with P‐values: *P ≤ 0.05,**P ≤ 0.01.Source data are available online for this figure.