Misfolded SOD1 Accumulation and Mitochondrial Association

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Misfolded SOD1 accumulation and mitochondrial association contribute to the selective vulnerability of motor neurons in familial ALS: correlation to human disease Salah Abu-Hamad, Joy Kahn, Marcel F. Leyton-Jaimes, Jonathan Rosenblatt, and Adrian Israelson ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00140 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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ACS Chemical Neuroscience

Misfolded SOD1 accumulation and mitochondrial association contribute to the selective vulnerability of motor neurons in familial ALS: correlation to human disease Salah Abu-Hamada, Joy Kahna, Marcel F. Leyton-Jaimesa, Jonathan Rosenblattb and Adrian Israelsona,c1

a

Department of Physiology and Cell Biology, Faculty of Health Sciences, b Department of Industrial Engineering and Management c The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, P.O.B. 653 Beer Sheva, 84105, Israel

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corresponding author:

Dr. Adrian Israelson Dept. of Physiology and Cell Biology Faculty of Health Sciences Ben-Gurion University of the Negev P.O.B. 653 Beer Sheva 84105,Israel Tel: +972-86477343 E-mail: [email protected] Keywords: mutant SOD1, misfolded SOD1, ALS, mitochondrial dysfunction Running title: Misfolded SOD1 accumulation correlates with ALS patient survival

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Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, with a 10% genetic linkage, of which 20% of these cases may be attributed to mutations in superoxide dismutase (SOD1). Specific mutations in SOD1 have been associated with disease duration, which can be highly variable ranging from a life expectancy of three to beyond ten years. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form, binds to intracellular organelles disrupting their function, or forms insoluble toxic aggregates. In order to understand whether these biophysical properties of the mutant protein may influence disease onset and duration, we generated 19 point mutations in the SOD1 gene, based on available clinical data of disease onset and progression from patients. By overexpressing these mutants in motor neuron-like NSC-34 cells, we demonstrate a variability in misfolding capacity between the different mutants with a correlation between the degree of protein misfolding and mutation severity. We also show a clear variation of the different SOD1 mutants to associate with mitochondrial-enriched fractions with a correlation between mutation severity and this association. In summary, these findings reveal a correlation between the accumulation of misfolded SOD1 species and their mitochondrial association with disease duration but not with disease onset, and they have implications for the potential therapeutic role of suppressing the accumulation of misfolded SOD1.

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Introduction Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is the most common form of motor neuron disease, characterized by the progressive loss of the upper and lower motor neurons in the cortex, brain stem and spinal cord. Muscle weakness, spasticity, atrophy and paralysis are some of the common symptoms among ALS patients, culminating in respiratory failure and subsequent death 1, 2. Most cases of ALS are sporadic (SALS) lacking any apparent genetic linkage, but 10% are inherited in a dominant manner (familial ALS; FALS). Twenty percent of these cases may be attributed to mutations in Cu2+/Zn2+ superoxide dismutase (SOD1). More than 178 mutations have been identified within SOD1 that are linked to ALS. These are primarily point mutations which are spread throughout the 153 amino acid protein sequence occurring at highly conserved amino acids 3. While diverse mutations show similar clinical manifestations, specific mutations have been associated with consistent disease durations of either short (up to 3 years)-or long (longer than 10 years)-clinical course. The A4V mutation, for example, the most common mutation in the United States, demonstrates a life expectancy of about 2 years while patients with the H46R mutation live for more than 10 years 4. The enigma of this diversity still needs to be resolved. Mutations in SOD1 result in the destabilizing and loosening of protein structure resulting in some degree of protein unfolding or “misfolding”. An additional universal feature of mutant SOD1 is its irreversible assembly into an insoluble structure known as aggregation. Comprehensive studies in patients 5, murine models 6-12, cell culture 13, 14and other in vitro assays

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have attributed this aggregation to mutation-induced misfolding of SOD1. In

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addition, there seems to be a clear correlation between the presence of mutant SOD1 aggregate formation, which varies considerably among the SOD1 mutants, and human disease progression 16, 17. Mutant, misfolded SOD1 in its non-aggregated, soluble form also plays a role in cell cytotoxicity, through its ability to engage in aberrant interactions, thereby enabling a gain of toxic function. More specifically, mutant SOD1 has been shown to associate with the mitochondria. It has been found deposited on the cytoplasmic face of spinal cord mitochondria

18-20

, accompanied by altered mitochondria shape and distribution

21

. This

may, in part, be attributed to its binding to the mitochondrial protein, VDAC1, inhibiting its conductance across the outer mitochondrial membrane and possibly compromising the energy supply 22. Additionally, mutant SOD1 alters the mitochondrial protein composition and affects the protein import mechanism

23

. Mitochondria have been implicated as a

possible target for toxicity in ALS in a number of studies

18, 20, 24-30

. Mitochondrial

dysfunction has been reported in motor neurons and in skeletal muscles (the cell types most affected in ALS) of patients with SALS or FALS, as well as in some ALS mouse models in the pre-symptomatic phase of the disease

18, 27, 31-34

. Furthermore, we recently

demonstrated a significant accumulation of mutant SOD1 within the mitochondrial and ER membranes of the spinal cord at disease onset in a murine model of ALS 35. A few selected studies have focused their attention on understanding which biochemical/biophysical properties of the mutant protein may influence its effect on disease progression and subsequent patient life expectancy. These studies failed to show a correlation between disease duration and the position of the mutation

36

. However, they

have shown a correlation between duration of disease and aggregation propensity where 4


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patients with short life expectancy for example, have mutations in SOD1 resulting in high propensity for aggregation 16, 17, 37. These studies have thus paved the way to understanding the mechanistic relationship between diverse SOD1 mutations and highly variable disease progression and severity. However, variations in aggregation rates of the specific mutations could not be explained by differences in known protein properties such as enzyme activity, protein thermostability, mutation position, or changes in protein charge 16. Moreover, the mean rates of fibrillary and amorphous nucleation are not uniformly increased by mutations that cause ALS compared to wild type SOD1 38. Since these studies, little progress has been made in further determining the properties of specific SOD1 mutations that may affect variations in patient life expectancy. In the present study, we have generated 19 different SOD1 mutants for which clinical data (onset and duration of disease in patients) is available from the literature in an adequate number of patients 16, with the aim of further understanding which mutant properties may influence clinical outcome. By overexpression of these mutant SOD1 proteins in motor neuron-like cells (NSC-34), we show a correlation between amount of misfolded SOD1 accumulation and association with mitochondrial-enriched fractions and disease duration. Specifically, we report that mutations associated with short life expectancy demonstrate significantly increased misfolded SOD1 accumulation compared to those with longer clinical outcome.

Results and Discussion Generation of specific SOD1 mutations related to variability in patient life expectancy

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Published clinical data from Prudencio et al 2009, relating to variability in ALS disease duration and patient life expectancy with specific mutations in SOD1, prompted us to divide these mutations into three groups, determined by disease duration: 1. Severe aggressive mutations (red block), correlating with a short life expectancy of up to 3 years; 2. Moderate aggressive mutations (blue block), correlating with a life expectancy of 3 to 7 years; and 3. Low aggressive mutations (green block), correlating with a longer life expectancy of more than 8 years. We have summarized these subdivisions in Figure 1a and Table 1. Following this, we established a model of the SOD1 protein sequence (Figure 1b) and 3D structure (Figure 1c), with the aim of identifying whether these mutation subdivisions exist within specific domains of the protein. In accordance with previous reports

16, 36, 39

, we observed that these subdivisions could not be correlated to common

specific domains and were randomly dispersed along the protein sequence (Figure 1b) or 3D structure (Figure 1c), emphasizing that there is no correlation between the mutation location and the severity of the disease. In our current research we have used these subdivisions of SOD1 mutations as the basis of our studies, with the aim of shedding more light on how different mutations lead to such diversity in disease duration. From previous studies, it appears that it is the biophysical properties of mutant SOD1 that may govern its role in clinical outcome and disease progression. A correlation has been found between specific mutants, their aggregation propensity and disease duration 16, 17. Thus, we have generated 19 different point mutations in the pciNeo mammalian expression vector as described in Methods. These mutations were chosen according to the existing

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clinical data (onset and duration of disease in patients) which is available from the literature 16

in an adequate number of patients (n > 5) to perform a comprehensive study. We

expressed these mutations in motor neuron-like NSC-34 cells to study the influence of these mutations on SOD1 biophysical properties. Then, we verified protein expression of the different mutants by immunoblot using the anti-SOD1 antibody (Figure 2). SOD1-associated ALS mutants present a large variability in the accumulation of misfolded SOD1 species recognized by B8H10 and A5C3 antibodies We first analyzed the 19 different SOD1 mutants for their ability to form and accumulate misfolded SOD1 protein within NSC-34 cells. This was achieved via immunoprecipitation with the B8H10 monoclonal antibody that was previously shown to specifically recognize a misfolded form of the protein

40, 41

. We observed a variability in misfolding capacity

between the different mutants, which was significantly different from wild type SOD1, where misfolded protein was hardly seen (Figure 3a). Quantification of the misfolded protein accumulation, calculated from the ratio of bound to unbound protein fractions further illustrates this diversity of misfolded SOD1 accumulation between the different mutants (Figure 3b). Surprisingly, even in different mutations which occur at a particular codon (for example G93A, G93S, G93C), we observed a high variability in the accumulation of misfolded SOD1 for each one of the individual mutations (Figure 3b), suggesting that the position of the mutation does not dictate the misfolding potential of the protein. Severe aggressive SOD1 mutations induce higher protein misfolding and mitochondrial-enriched fractions association

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Since we have shown that there is a high variability in misfolding capacity of the 19 different mutants as analyzed by immunoprecipitation with B8H10 antibody (Figure 3b), we next wanted to determine whether this variability might correlate with disease duration revealed from the existing clinical data. We showed that cells expressing SOD1 mutations from the severe aggressive mutation group (short life expectancy), accumulate the most misfolded SOD1, recognized by B8H10 antibody (Figure 3b, red bars), when compared to cells expressing mutations from the low aggressive mutation group (longest life expectancy), which have a significantly lower amount of accumulation of the misfolded protein (Figure 3b, green bars). The mean values of the misfolded SOD1 accumulation among the different mutations show a statistically significant difference between the groups (Figure 3b, inset). Since it is well established that different species of misfolded SOD1 exist

42-45

, and that the B8H10 antibody may

recognize some of these species but not others, we used an additional antibody, A5C3, which was also shown to specifically recognize misfolded SOD1 40. Immunoprecipitation of misfolded SOD1 with the A5C3 antibody in representative mutants from the three different groups showed a similar pattern as observed for the B8H10 antibody (Figure 3c). We obtained similar results when we used the B8H10 antibody to detect misfolded SOD1 accumulation by immunocytochemistry (Figure 4). In cells expressing SOD1G37R, a low aggressive mutation, a significant reduction in misfolded SOD1 accumulation was clearly observed, in contrast to cells expressing the high aggressive mutation SOD1G93A (Figure 4a and 4b).

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The misfolded SOD1 protein also elicits a toxic function in its soluble non-aggregated form by its association with intracellular organelles such as the mitochondria

20-22, 41

. We

therefore aimed to determine whether the different mutations we generated showed variability in their mitochondrial associating capacity. To this end, we expressed each mutant SOD1 separately in NSC-34 cells, and purified fractions enriched for mitochondria (Figure 5a). Immunoblot data verifies the presence of mutant SOD1 in the mitochondrialenriched fractions for of all the mutant expressing cells. These blots show a clear variation in the association of the different mutants with mitochondrial-enriched fractions (Figure 5b). Quantification of this association of mutant SOD1, calculated from the ratio of mitochondrial bound to total protein fractions, further emphasizes the diversity of mutant SOD1 mitochondrial association between the different mutants (Figure 5c). Importantly, we observed a similar pattern of mutant SOD1 association with the mitochondrial–enriched fraction and misfolded SOD1 accumulation among the different mutants. We further analyzed whether a correlation exists between the association of the different mutants with mitochondrial-enriched fractions and their degree of severity (Figure 5c). Our data reveal a higher ratio of mutant SOD1 associated with the mitochondrial-enriched fractions in cells that expressed mutants from severe aggressive mutation group (Figure 5c, red bars), when compared with those that expressed mutants from the other groups (Figure 5c, blue and green bars). The mean values of the SOD1 association with the mitochondrial-enriched fractions in the different mutations show a statistically significant difference between the groups (Figure 5b, inset), similarly to the data presented for SOD1 misfolding species recognized by B8H10 or A5C3 antibodies.

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SOD1 misfolding and association with mitochondrial-enriched fractions inversely correlate with human disease duration but not with disease onset Since patient data is available for the mutations that we generated, the relevant question was whether our biophysical data from the cell culture model of mutant SOD1 expressing NSC-34 cells, correlates with human disease onset and duration. Analysis of ratio of misfolded SOD1 species, recognized by B8H10 antibody, from total cell extracts expressing the various mutants to patient life expectancy, showed a clear inversed correlation between the misfolded protein generated from each particular mutant and disease duration (Figure 6a). Specifically, in patients with mutations resulting in a short life expectancy, we observed a higher misfolding capacity of the various SOD1 mutants as recognized by B8H10 antibody (Figure 6a, red circles), whereas in patients with mutations leading to a longer disease course, we observed a lower misfolding capacity of the various mutants (Figure 6a,blue triangles and green squares). Furthermore, we also observed a similar inversed correlation between association of the various mutants with mitochondrial-enriched fractions and patient life expectancy. In particular, in patients with mutations presenting a correlation between short life expectancy and higher misfolded SOD1, we observed a higher association of mutant SOD1 with these mitochondrial-enriched fractions (Figure 6b, red circles). Moreover, in patients with mutations leading to a longer life expectancy and lower levels of misfolded SOD1, there was a lower association of the mutant proteins within these mitochondrial-enriched fractions (Figure 6b, blue triangles and green squares).

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On the other hand, no correlation was observed between misfolded SOD1 accumulation as recognized by B8H10 antibody (Figure 7a) or SOD1 association with mitochondrialenriched fractions (Figure 7b) and disease onset, suggesting that these biophysical properties of the different mutants have no effect on the onset of the human disease. Similar results were shown for the lack of correlation between mutant SOD1 aggregation and disease onset16. Further evidence supporting our findings is the variation in disease onset among patients who carry the same SOD1 mutation, suggesting that the variation in the age of onset has a very significant familial component

46

, similar to other

neurodegenerative diseases 47, 48, and may therefore be determined by factors other than the mutation site in the SOD1 gene or the biophysical properties of the different mutants. Our observations revealing a correlation between variable accumulation of misfolded SOD1 species in the different mutants with patient life expectancy, which is accompanied by variable mutant SOD1 association with fractions enriched for mitochondria, suggests that the aggressive progression of the disease may be attributed to mutant SOD1misfolding and its association to intracellular organelles, such as the mitochondria. This association may lead to disruption of mitochondrial function as we and others have previously proposed

19, 22, 23

, culminating in motor neuron degeneration. Indeed, mitochondrial

dysfunction has been consistently reported in motor neurons and skeletal muscles of ALS patients as well as in ALS mice models in which it was observed in the pre-symptomatic phase of the disease 18, 27, 31-34. Our findings correlating mutations in SOD1 patients with accumulation of a misfolded form, and subsequent disease progression are reinforced by studies showing that ALSlinked mutations in SOD1 lead to an increased propensity of the protein to misfold and/or 11



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aggregate and this misfolded protein has been detected in SOD1 FALS

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49-53

and mutant

SOD1 mouse models 40, 53-57, accumulating preferentially in spinal cord motor neurons. Further supporting our findings, are a number of studies showing an inverse correlation between the aggregation propensity of the SOD1 mutants and disease duration16, 17, 58, however no correlation to disease onset has been identified in these studies. Since it was previously shown that eliminating the aggregated misfolded SOD1 has no effect in slowing disease progression in different mutant SOD1 mouse models

54

, we

suggest that the toxic species is derived from the soluble form of misfolded SOD1 (which was investigated in this study) rather than the highly aggregated insoluble form. In this context, we have shown that these soluble species of misfolded SOD1 can be reduced by the 12 kDa macrophage migration inhibitory factor (MIF) 1, 35, 41. Finally, different studies have reported misfolded SOD1 accumulation in motor neurons 50, 59, 60 and glia 49of spinal cords as well as in peripheral blood mononuclear cells 61 and lymphocytes 62from SALS patients. However, these findings were disputed by others showing undetectable levels of misfolded SOD1 in spinal cord and cortex tissues from patients with sporadic or nonSOD1 inherited ALS

51, 52, 63-65

, suggesting that mutations in SOD1 may be the primary

factor leading to its misfolding. Taken together, our observations provide evidence that misfolded SOD1 accumulation and its mitochondrial association may be important biophysical properties that are influenced by specific mutations and have an impact on FALS human disease duration. Thus, strategies to modulate SOD1 misfolding could be useful therapeutics for slowing the progression of the disease.

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Methods Cell culture and transfection The spinal cord neuroblastoma hybrid (NSC-34) cells were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% tetracycline-free FBS, L-Glutamine (2 mM), and penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (All reagents purchased from Biological Industries, Kibbutz Beit-Haemek, Israel).Transfection was performed using TurboFect transfection reagent (Thermo Fisher Scientific, Inc, Lithuania) according to the manufacturer’s protocol. Mutagenesis A one-step PCR-assisted site-directed mutagenesis technique 66 was performed to produce the various SOD1 point mutations with pCI-neo WT hSOD-1 vector as template, using a pair of complementary primers bearing the desired mutations in the middle of the sequences (Table 1) and the Q5 high fidelity DNA polymerase according to manufacturer's instructions (NEB, Ipswich, MA, USA). PCR reaction was treated with 1 μlDpnI (NEB, Ipswich, MA, USA), transformed to E. coli, plasmid DNA was isolated using a TIANprepRapid Mini Plasmid Kit (Tiangen Biotech, Beijing, China) and sequenced. Immunoprecipitation Cell extracts were treated with immunoprecipitation (IP) buffer [50 mMTris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.5% Nonidet P-40 plus protease inhibitors] and incubated overnight with the B8H10 or A5C3 (MediMabs, Quebec, Canada) antibody which was crosslinked to DynabeadsTM Protein G (Invitrogen, Thermo Fisher Scientific, Norway) 13



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with dimethyl pimelimidate (Pierce, Thermo Fisher Scientific Inc, IN, USA) prior to incubation according to the manufacturer’s instructions. The beads were magnetically isolated and washed three times with IP buffer. Samples were eluted by boiling in 2X sample buffer. Mitochondria purification Transfected cells were grown in 60 mm dishes. Forty eight hours after transfection, cells were scraped and homogenized on ice in 300 l ice-cold homogenization buffer (HB), composed of 210 mM mannitol, 70 mM sucrose, 1 mM EDTA-(Tris), (1:50) protease inhibitor cocktail tablets (Roche, IN, USA) and 10 mMTris-HCl (pH 7.2). Homogenates (cell extracts) were centrifuged at 1000 g for 10 min, supernatant was stored on ice, and pellet resuspended with half the previous volume of HB, re-centrifuged and once again supernatant was collected. The supernatants were combined, and centrifuged at 12,000 g for 15 min to yield a mitochondrial pellet. Supernatant was collected and re-centrifuged as previously described. The pellets from each centrifugation were combined by resuspending in a 100ul HB. The proteins were quantified and run on a 13% SDS-PAGE gel. Immunoblotting Proteins were separated on 13% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with various antibodies, including goat anti-SOD1 (C-17) (Santa Cruz Biotechnology, Texas, USA), rabbit anti-human SOD1 (Abcam), monoclonal antiVDAC/porin 31HL (Calbiochem, MA, USA). Horseradish peroxidase-conjugated IgG secondary antibodies (Jackson Immunochemicals, PA, USA) were used and detection was done with the EZ-ECL Chemiluminescence Detection kit for HRP (Biological Industries, 14


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Kibbutz Beit-Haemek, Israel) on an ImageQuant LAS 4000 digital imaging system (General Electric Healthcare Bio-Sciences, PA, USA). Immunocytochemistry Cover slices were coated with PDL (Poly-D-Lysine) for 30 minutes. Approximately, 40,000 cells were plated in 24 well plates, containing coated cover slices. 24 hours later, NSC-34 cells were transfected with pCINeo plasmids carrying a SOD1WT, SOD1G93A, SOD1G85R or SOD1G37R mutant using TurboFect reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. An empty plasmid was used as control. 30 hours after transfection, NSC-34 transfected cells, were washed 3 times with PBS (Biological Industries), fixated with 4% PFA (Paraformaldehyde) for 15 minutes at room temperature. The cells were permeabilized with PBS containing 0.25% triton-X100 for 30 minutes. Blocking with 10% chicken serum, 1% BSA free fatty acid and 0.1% triton-X100 diluted in PBS was performed by 1 hour at room temperature. First antibodies B8H10 (1:100 MédiMabs) and anti-SOD1 (C17, SCB) were left overnight in a cold room. After 24 hour, first antibodies were washed and secondary antibodies were added; anti-mouse Alexa 488 (1:200, Thermo Fisher Scientific) and anti-goat Alexa 637 (1:300, Abcam) for 2 hours in a dark room. Cover slices were removed from the 24 cell plates and placed on top of a slice in upright position with immu-mount solution (Thermo Fisher Scientific). Images were acquired with a Nikon C2Plus laser confocal microscope docked to a Nikon Ti eclipse unit, using 60× oil objectives. Scanning settings were reused across the samples. Statistical analysis

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Values are reported throughout as mean ± SEM. Comparisons of two datasets were performed using the Student’s t test, after a normal distribution was confirmed by the Shapiro-Wilk normality test. For the correlation analyses, the spearman’s correlation coefficient was calculated. Significance was set at a confidence level of 0.05. In all figures, *P< 0.05 and ***P< 0.001. All statistical analysis were performed with SigmaPlot (Systat Software).

Funding Sources This work was supported by grants from the Israel Science Foundation (ISF #124/14), the United States – Israel Binational Science Foundation (BSF #2013325), the FP7 Marie Curie Career Integration Grant (CIG # 333794), the German-Israeli Foundation for Scientific Research and Development (GIF # I-2320-1089.13), and the National Institute for Psychobiology in Israel (NIPI #b133-14/15).

Author contributions: SAH, JK, MFL and AI designed the research. SAH and MFL conducted the experiments. SAH, JK, MFL, JR and AI analyzed the data. SAH, JK and AI wrote the manuscript. Supporting Information Description of the mutations used in this study, including the primers used for the cloning and the age of onset and duration of the disease in patients (Supplementary Table 1).

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Figure legends Figure 1. SOD1 mutations used in this study. a. Mutations were divided into three groups based on their degree of severity: severe aggressive mutations (red block), correlating with a life expectancy of up to 3 years; moderate aggressive mutations (blue block), correlating with a life expectancy of 3 to 7 years; and low aggressive mutations (green block), correlating with long life expectancy of more than 8 years. b. Model showing the localization of each mutation in the SOD1 protein sequence. c. 3D model showing the localization of each mutation in a WT holo-SOD1 homodimer (PDB 1HL5). Positions with severe aggressive mutations are highlighted in red, positions with moderate aggressive mutations in blue, positions with low aggressive mutations in green, positions sharing both moderate and low aggressive mutations in light blue and positions sharing both severe and low aggressive mutations are in yellow.

Figure 2. Overexpression of SOD1 mutants in NSC-34 cells. NSC-34 cells were transfected with each human SOD1 mutant individually, and the overexpression of SOD1 in total cells extracts was analyzed by immunoblot using a specific anti-SOD1 antibody, which recognizes both mouse and human SOD1.

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Figure 3. Analysis of misfolded SOD1 accumulation in NSC-34 cells. a. Each SOD1 mutant was expressed individually in NSC-34 cells. Total cell extracts were collected and analyzed for the accumulation of misfolded SOD1 via immunoprecipitation using the B8H10 monoclonal antibody that recognizes misfolded SOD1 and detected using an antihuman SOD1 antibody. b. Quantification analysis of different biological repeats (n=3) of the ratio of bound to unbound protein fraction in each group was performed. Severe aggressive mutation group (red bars), moderate aggressive mutation group (blue bars) and low aggressive mutation group (green bars) are shown. Bar values represent the means ± SEM of three independent experiments. In the inset are the mean values of the misfolded SOD1 accumulation for the different mutations separated by groups according to their severity. * P< 0.05 and *** P < 0.001. c. Representative SOD1 mutants from each group were expressed individually in NSC-34 cells. Total cell extracts were collected and analyzed for the accumulation of misfolded SOD1 via immunoprecipitation using the A5C3 monoclonal antibody that recognizes misfolded SOD1.

Figure 4. Severe aggressive mutations accumulate higher levels of misfolded SOD1 as recognized by B8H10 antibody. a. Representative micrographs of NSC-34 cells expressing

SOD1WT,

SOD1G93A,

SOD1G85R

or

SOD1G37R

processed

for

immunofluorescence using the B8H10 antibody for misfolded SOD1 (red) and the total SOD1 expression using anti-SOD1 antibody (green). The merge column clearly shows the different levels of misfolded SOD1 accumulation. An empty plasmid was used as control. Scale bar, 20 µm. b. Quantification of the relative fluorescence intensity of the B8H10

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staining of NSC-34 cells expressing SOD1G93A, SOD1G85R or SOD1G37R was performed by un-paired Students’t tests. * P < 0.05 and ** P < 0.01. Figure 5. Analysis of mutant SOD1 association with mitochondrial-enriched fractions in NSC-34 cells. a. Schematic diagram of mitochondrial purification protocol from NSC34 cells. The enrichment of mitochondria is shown by immunoblot analysis using antiVDAC1 antibodies. b. Each SOD1 mutant was expressed individually in NSC-34 cells. The fractions enriched for mitochondria from each transfection were purified and analyzed for mutant SOD1 association by immunoblot analysis. The lanes were not loaded in the same order as shown in this figure and lanes from different gels were placed together, as separated by dashed lines. c. Quantitative analysis of different biological repeats (n=3) of the ratio of mitochondrial associated mutant SOD1 from the total SOD1 in transfected NSC-34 cells was performed for each mutant group. Severe aggressive mutation group (red bars), moderate aggressive mutation group (blue bars) and low aggressive mutation group (green bars) are shown. Bar values represent the means ± SEM of three independent experiments. In the inset are the mean values of the SOD1 mitochondrial association of the different mutations separated by groups according to their severity. *** P < 0.001.

Figure 6. Correlation of SOD1 misfolding and mitochondrial association with patient life expectancy. Analysis of the ratio between misfolded SOD1 accumulation, as recognized by B8H10 antibody, from total cell extracts (a) or ratio between association of the various mutants to mitochondrial-enriched fraction (b) and patient disease duration among the various mutants was performed using the spearman’s correlation test and show

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a clear significant inverse correlation (P < 0.001). Short life expectancy (red circles), moderate life expectancy (blue triangles) and longer life expectancy (green squares) (n=3).

Figure 7. Lack of correlation of SOD1 misfolding and mitochondrial association with human disease onset. Analysis of the ratio between misfolded SOD1 accumulation, as recognized by B8H10 antibody (a) or ratio between association of the various mutants to mitochondrial-enriched fractions (b) and disease onset was performed using the spearman’s correlation test and showed no significance (n=3).

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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Figure 7


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Misfolded SOD1 Accumulation and Mitochondrial Association

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