Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 14:13:346.
doi: 10.3389/fncel.2019.00346. eCollection 2019.

A VDAC1-Derived N-Terminal Peptide Inhibits Mutant SOD1-VDAC1 Interactions and Toxicity in the SOD1 Model of ALS

Anna Shteinfer-Kuzmine  1 , Shirel Argueti  2 , Rajeev Gupta  1 , Neta Shvil  2 , Salah Abu-Hamad  2 , Yael Gropper  2 , Jan Hoeber  3 , Andrea Magrì  4 , Angela Messina  4 , Elena N Kozlova  3 , Varda Shoshan-Barmatz  1 , Adrian Israelson  2
Affiliations

A VDAC1-Derived N-Terminal Peptide Inhibits Mutant SOD1-VDAC1 Interactions and Toxicity in the SOD1 Model of ALS

Anna Shteinfer-Kuzmine et al. Front Cell Neurosci. .

Abstract

Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.

Keywords: ALS; N-terminal peptide; VDAC1; misfolded SOD1; mutant SOD1.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Interaction of wild type and mutant SOD1 with VDAC1 and VDAC1-N-terminal derived peptides. (A) Coomassie blue staining of mitochondrial VDAC1, and recombinant SOD1WT, SOD1G93A, and SOD1G85R expressed in sf-9 cells, purified as described in the Methods section. Molecular weight standards are presented. (B) Purified VDAC1 was fluorescently labeled using a NanoTemper blue protein-labeling kit and incubated with SOD1WT (∘), SOD1G93A (∙), or SOD1G85R (Δ; 0.1–100 μM). After 20 min of incubation, 3–5 μl aliquots were loaded into MST-grade glass capillaries (NanoTemper Technologies) and thermophoresis was measured with a NanoTemper Monolith-NT115 system. The percentage change in normalized fluorescence (ΔF Norm %) is plotted as a function of protein concentration. (C) Fluorescently labeled SOD1WT (∘, 165 nM), SOD1G85R (Δ, 230 nM) or SOD1G93A (∙, 56 nM), was incubated with different concentrations of the VDAC1 N-terminal peptide (0.001–50 μM) in 10 mM HEPES buffer, pH 7.4 and analyzed for binding as in (B). (D) Summary of the binding affinity (dissociation constants) of the peptide to SOD1WT, SOD1G93A and SOD1G85R, as derived from a fitted curve of the percentage change in normalized fluorescence (ΔF Norm %) as a function of peptide and purified VDAC1 concentration. (E) Fluorescently labeled SOD1G93A (675 nM) was incubated with the indicated concentrations of N-terminal (∙, 0.01–40 μM) or LP3 (∘, 6–40 μM) peptides in HEPES buffer, and binding was assayed as in (B). KD values were calculated using the mass action equation in the NanoTemper software from duplicate reads of triplicate experiments.
FIGURE 2
FIGURE 2
Mutant SOD1 but not wild type SOD1 interacts with VDAC1 and inhibits channel conductance. (A,B) Recombinant full length VDAC1 purified from E. coli was reconstituted into a PLB and channel currents through VDAC1, in response to a voltage step from 0 to 10 mV (A) or to –10 mV (B), before and 15–20 min after addition of 40 μg/ml (final concentration) of SOD1 were recorded. (C) VDAC1 relative conductance as a function of voltage in a 60 to –60 mV step before (∙) and after addition of SOD1WT (∘). Relative conductance (conductance/maximal conductance) was determined as the average steady-state conductance at a given voltage normalized to the conductance at 10 mV, considered the maximal conductance. (D) Histogram of VDAC1 current amplitudes at 10 mV before and after addition of SOD1WT. (E–H) Similar experiments as in (A–D), except that mutant SOD1G93A was used. (I–L) Similar experiments as in (A–D), except that mutant SOD1G85R was used. (M) Coomassie blue staining of recombinant full length and residue 1-26-truncated VDAC1 (ΔN-VDAC1).
FIGURE 3
FIGURE 3
The N-terminal domain of VDAC1 is required for mutant SOD1 interaction with VDAC1 and inhibition of channel conductance. (A,B) Currents passing through bilayer-reconstituted recombinant N-terminally truncated VDAC1 (ΔN-VDAC1) were recorded in response to voltage step from 0 to 10 mV (A) or –10 mV (B) before and 15–20 min after the addition of 40 μg/ml (final concentration) of SOD1WT. (C) Relative conductance of ΔN-VDAC1 as a function of voltage in a step from 60 to –60 mV before (▲) and after addition of SOD1WT (Δ). Relative conductance (conductance/maximal conductance) was determined as the average steady-state conductance at a given voltage normalized to the conductance at 10 mV, taken as the maximal conductance. (D) Histogram of VDAC1 current amplitudes at 10 mV before and after addition of SOD1WT. (E–H) Similar experiments as in (A–D), except that mutant SOD1G93A was used. (I–L) Similar experiments as in (A–D), except that mutant SOD1G85R was used.
FIGURE 4
FIGURE 4
VDAC1 N-terminal peptides inhibit the cell death of NSC-34 cells mediated by mutant SOD1G93A. NSC-34 (A), U-87MG (B), or A549 (C) cells were incubated with the indicated concentrations of (1-26)N-Ter-Antp (∙), D-(15-26) (19-26)N-Ter-Antp (▲), (1-20)N-Ter-Antp (∘), (5-20)N-Ter-Antp (□) or (10-20)N-Ter-Antp (◆) peptide for 5 h. Cell death was analyzed using PI staining and flow cytometry. (D–F) NSC-34 cells were transfected to express human SOD1WT, the human mutant SOD1G93A, or neither (empty), in each case either without or with addition of increasing concentrations of the indicated VDAC1 N-terminal-derived peptide for 5 h. Cell viability analysis was performed with the CellTiter 96 AQueous one-solution cell proliferation assay with ELISA at 490 nm. (G) The rescuing effects of the VDAC1 N-terminal-derived peptides are shown as a percentage of cell death. The significance of quantitative analysis of triplicates of different biological repeats (n = 3) was performed by Student’s t-test; ∗∗P < 0.01. (H) SH-SY5Y cells (4.5 × 104 cells/well in 24-well plates) were transfected with an empty plasmid or a plasmid encoding for mutant SOD1G93A or SOD1G37R. Twenty-four hours post-transfection, the cells were incubated for 5 h with (10-20)N-Ter-Antp peptide (20 μM) and then analyzed for apoptosis using acridine orange and ethidium bromide staining, as described previously (McGahon et al., 1995). Fluorescence microscopy images were analyzed and about 100 to 300 cells were counted for each treatment in representative microscopic fields. The significance of quantitative analysis of triplicates of different biological repeats (n = 3) was performed by one-way Anova; ∗∗P < 0.01.
FIGURE 5
FIGURE 5
The VDAC1 N-terminal peptide induces detachment of misfolded SOD1 from its mitochondrial binding site. Co-localization of VDAC1 and misfolded SOD1 in SH-SY5Y cells was assayed by indirect immunofluorescence with antibodies for VDAC1 along with simultaneous identification of misfolded SOD1 (with the B8H10 antibody). Twenty-four hours post-transfection, the cells were incubated for 5 h with (10-20)N-Ter-Antp peptide (20 μM) and then analyzed for co-localization using a confocal microscope. Representative images of SH-SY5Y cells transfected to express human mutant SOD1G93A (A) or SOD1G37R (C) in each case either without (A,C) or with (B,D) addition of the (10-20)N-Ter-Antp peptide are shown. Scale bar, 10 μm.
FIGURE 6
FIGURE 6
The VDAC1 N-terminal peptide ameliorates SOD1G93A-mediated motor neuron toxicity. SOD1G93A-expressing mESC-derived MNs were incubated with the indicated concentrations of (1-20)N-Ter-Antp peptide for 1–4 days following initiation of final differentiation. (A,B) (1-20)N-Ter-Antp at a concentration of 10 μM significantly increased MN neurite outgrowth over the first 24 h. Scale bar, 100 μm. (C) Twenty-four hours after the initiation of differentiation, 5 and 10 μM of (1-20)N-Ter-Antp peptide significantly increased the number of MNs present in the culture. (D) (1-20)N-Ter-Antp at a concentration of 10 μM significantly increased the number of surviving MNs over the course of final differentiation. After 96 h, MNs showed the typical cellular morphology of maturing neurons with extension of a singular long process and branching resembling a dendritic tree. GFP fluorescence indicates the expression of the motor neuron-specific transcription factor HB9. Gross cellular morphology of surviving MNs remained unaffected by the (1-20)N-Ter-Antp peptide. Scale bar, 25 μm. (E) Quantification of MNs survival. For all assays, results are expressed as the mean ± SEM (n = 3). *P < 0.05.
FIGURE 7
FIGURE 7
Mutant SOD1 binds to VDAC1 and inhibits VDAC1 activities, with addition of a VDAC1 N-terminal peptide preventing such inhibition. Schematic model showing mutant SOD1 binding to VDAC1, with the N-terminal peptide serving as a decoy. (A) Mutant SOD1 is proposed to bind to the VDAC1 N-terminal domain and inhibit VDAC1 conductance, thereby suppressing both influx and efflux of different mitochondrial metabolites and ions, including Ca2+, and ROS. This reduction in metabolite flux results in reduced energy production and increased oxidative stress, leading to mitochondrial dysfunction and cell death. (B) VDAC1 N-terminal-derived peptides bind mutant SOD1 and prevent its association with VDAC1, thereby preventing mitochondria dysfunction. The N-terminal peptide thus provides a new therapeutic approach for inhibiting mutant SOD1 toxicity in ALS. OMM and IMM indicate outer and inner mitochondrial membrane, respectively while IMS indicates, the intermembrane space.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abu-Hamad S., Arbel N., Calo D., Arzoine L., Israelson A., Keinan N., et al. (2009). The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins. J. Cell Sci. 122 1906–1916. 10.1242/jcs.040188 - DOI - PubMed
    1. Abu-Hamad S., Kahn J., Leyton-Jaimes M. F., Rosenblatt J., Israelson A. (2017). Misfolded SOD1 accumulation and mitochondrial association contribute to the selective vulnerability of motor neurons in familial als: correlation to human disease. ACS Chem. Neurosci. 8 2225–2234. 10.1021/acschemneuro.7b00140 - DOI - PubMed
    1. Aggarwal T., Hoeber J., Ivert P., Vasylovska S., Kozlova E. N. (2017). Boundary cap neural crest stem cells promote survival of mutant sod1 motor neurons. Neurotherapeutics. 14 773–783. 10.1007/s13311-016-0505-8 - DOI - PMC - PubMed
    1. Arbel N., Ben-Hail D., Shoshan-Barmatz V. (2012). Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein. J. Biol. Chem. 287 23152–23161. 10.1074/jbc.M112.345918 - DOI - PMC - PubMed
    1. Arbel N., Shoshan-Barmatz V. (2010). Voltage-dependent anion channel 1-based peptides interact with Bcl-2 to prevent antiapoptotic activity. J. Biol. Chem. 285 6053–6062. 10.1074/jbc.M109.082990 - DOI - PMC - PubMed