Early and gender-specific differences in spinal cord mitochondrial function and oxidative stress markers in a mouse model of ALS
© Cacabelos et al. 2016
Received: 25 November 2015
Accepted: 27 December 2015
Published: 13 January 2016
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A.
Results and Conclusions
Our results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS.
KeywordsMotor neuron Complex I Respirometry Fatty acid composition Oxidative damage Estrogens
Amyotrophic lateral sclerosis (ALS, OMIM #105400), the motor neuron disorder with the highest level of occurrence in adult humans, is characterized by a progressive loss of upper and lower motor neurons that leads to muscular atrophy, paralysis, and death after an average disease duration of 3 years . The most common form of ALS is sporadic, having no apparent inheritability, whereas the dominantly inherited familial ALS accounts for only about 5–10 % of all ALS patients [2, 3]. Among the genetic causes of ALS, 15–20 % of familial ALS cases and about 5 % of sporadic ALS cases are associated with multiple mutations in the gene for Cu,Zn-superoxide dismutase (SOD1), which imparts an increase in a novel toxic function to this otherwise superoxide radical scavenging enzyme in normal conditions . Mutated SOD1 forms misfold, aggregate, and accumulate primarily in spinal cord motor neurons and glial cells of ALS patients . Furthermore, transgenic mice overexpressing mutant forms of the human sod1 gene develop a progressive motor neuron syndrome similar to the human ALS phenotype [6, 7] and they have been extensively used as an experimental model to gain insight into the pathogenesis of ALS. In particular, one of the most studied mutations is the substitution of glycine for alanine in the 93th residue (hSOD1-G93A).
Nonetheless, individual factors could help to explain the great clinical heterogeneity of this disease, with similar mutations leading to markedly clinically different disease forms. Among these factors, gender differences in many neurodegenerative diseases are observed across epidemiologic studies, pathophysiology, and treatments [8, 9]. ALS is no exception; its incidence (male: female ratio 3:1) and prevalence are higher in men than in women, with a predominance of men with younger disease onset. Clinical phenotypes are also different in male and female patients, especially regarding the site of onset of weakness, as well as cognitive impairment [10, 11]. The aforementioned gender differences have been reported both in studies that included all ALS patients (sporadic and familial) and in familial ALS cases studied separately . Lending further support to the greater occurrence of familial ALS in men than in women, data extracted from the ALSoD website (http://alsod.iop.kcl.ac.uk) revealed a 1.5 male/female ratio for most mendelian ALS-related mutant genes, including SOD1. Moreover, it has been reported that gender can account for variations in the course of disease in familial ALS .
Most neurodegenerative disorders involve either causally or consequently mitochondrial abnormalities [14–17]. Although the underlying causes of motor neuron degeneration in ALS remain largely unknown, ALS-causing SOD1 mutations lead to mitochondrial dysfunction, among multiple pathogenic pathways also present in sporadic ALS, such as oxidative stress and endoplasmic reticulum stress [18–21]. Mitochondrial dysfunction may also directly provoke cell death by activating the apoptotic cascade, due to misfolded or aggregated SOD1 that results either in aberrant localization and release of proapoptotic factors or binding to apoptotic inhibitors .
Studies in different species have reported that mitochondria in females are more differentiated, and as a result, the mitochondria show greater capacity for and efficiency in substrate oxidation than in males. These features of female mitochondria have been described in nervous tissue, among other organs and tissues [23–27]. On the other hand, results of several authors suggest a greater sensitivity of males to mitochondrial dysfunction compared to females, including higher permeability of the outer and inner mitochondrial membranes with increased translocation of apoptosis-inducing factor to the nucleus in neurons from immature rat brains submitted to hypoxia-ischemia . In brain, among other mitochondrial properties more pronounced in males than females are lower mitochondrial electron transport chain complex activities, diminished mitochondrial mass, and lower mitochondrial membrane potential [29, 30].
Taking this background into account, the aim of this work was to study whether gender-related differences in spinal cord mitochondrial function of a mouse model of ALS could contribute to less severe female phenotypes. We hypothesized that differences in the degree of spinal cord mitochondria dysfunction between male and female hSOD1-G93A mice could explain, at least in part, their different clinical features. These data will be helpful to further understanding of whether different degrees of spinal cord mitochondrial dysfunction are related to the earlier onset and faster disease progression in male ALS patients than in their female counterparts. Moreover, understanding the causes of the sex differences in ALS could yield clues concerning the pathophysiology of this devastating disease.
Materials and methods
All reagents, unless stated otherwise, were purchased from Sigmα–Aldrich (St. Louis, MO). Cell culture media, sera, antibiotics, media supplements, and Lipofectamine 2000® were obtained from Invitrogen. 17β-estradiol (E2) was dissolved in ethanol at a concentration of 1 mM, further diluted to 40 μM with culture medium, aliquoted, and stored frozen for up to one month at -20 °C. E2 stock solutions were used for a final concentration of 10 nM.
Animals and phenotype analyses
A colony of the strain B6SJL-Tg (SOD1-G93A)1Gur/J (JAX catalogue stock number 002726; from now on G93A mice) was purchased from The Jackson Laboratories (Bar Harbor, ME), maintained in the B6SJL background, and genotyped as indicated in Additional file 1. Non-transgenic littermates (from now on Control mice) were used as controls. Animals were maintained under a constant 12 h light–dark cycle in individual cages after weaning (day 21) with temperatures around 22 ± 4 °C, and fed standard rodent chow and water ad libitum.
For the phenotype analyses, animals were weighed weekly, and daily for those under stride length analysis. A third-order polynomial curve was fitted to the age-body weight sets of data with Prism 6.0 software (GraphPad, La Jolla, CA) in order to calculate maximum weight and age of attaining maximum weight, and to interpolate age attaining maximum weight minus 10 %. Neurological scoring, paw print analysis, and end-point determination were performed on alternate days starting from day 60; these are further described in the Additional file 1. All experimental procedures were approved by the Ethical Committee for Animal Testing of the Institut de Recerca Biomèdica de Lleida (IRBLleida) and conformed to Directive 2010/63/EU of the European Parliament.
Spinal cord sample preparation
Animals were sacrificed by cervical dislocation at indicated times after being fasted overnight. Spinal cord lumbar sections were rapidly excised and kept in ice-cold saline to perform respirometry, or frozen in liquid nitrogen and stored at -80 °C for further analysis, respectively. For fatty acid, oxidative markers and western-blot frozen samples were thawed on ice and homogenized at 0 °C in a buffer containing 180 mM KCl, 5 mM 3-[N-morpholino]propanesulfonic (MOPS) acid, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM diethylenetriaminepentaacetic acid, 1 μM freshly prepared butylated hydroxyl toluene (BHT), 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), at pH 7.3 using a Potter-Elvehjem motor-driven glass-Teflon homogenizer. After a brief centrifugation (500 x g, 5 min) to pellet cellular debris, protein concentrations were measured in the supernatants using the Bradford assay.
A small set of animals (n = 5 per gender, age 90 days) was employed for immunohistochemistry. Briefly, after rapid extraction of spinal cords, these were fixed using paraformaldehyde and cryoprotected using a sucrose 30 % solution in phosphate buffer. After that, spinal cords sections (16 μm) were obtained using a Leica CM3000 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and immunohistochemistry was performed using anti-p21 (1:500, ref. ab7960, Abcam Co, Cambridge, UK) and SMI-32 (1:500, Stenberger monoclonals; Biolegend, San Diego, CA).
Cell culture and transfection
Neuro-2A cell line was obtained from ATCC (#CCL-131) and grown in Advanced-Minimum Essential Medium (MEM) without phenol red supplemented with 10 % heat inactivated fetal bovine serum, 2 mM L-glutamine, 20 U/ml penicillin, and 20 μg/ml streptomycin. Cells were kept at 37 °C in a humidified atmosphere with 5 % CO2.
After 10 days of pre-treatment with 10nM E2 or vehicle, cells were harvested, counted, and subcultured in 6-well plates (200,000 cells per well). The day after plating, ransfection was performed using Lipofectamine 2000 (Invitrogen, El Prat de Llobregat, Barcelona, Spain) according to the manufacturer’s instructions. Briefly, lipofectamine and selected DNA plasmids were mixed (1 μg of DNA/1 μL of lipofectamine) with Optimen medium (Invitrogen) for 20 min and the resulting mixture was dispensed to the cell cultures (10 μg DNA per well). The plasmids pEGFP-G93-hSOD1 and pEGFP-wt-hSOD1, expressing G93A mutant or non-mutated human SOD1 tagged with enhanced green fluorescent protein (EGFP), respectively, were kindly provided by Dr Josep Esquerda (Lleida, Spain). The pEGFP expression vector was used for mock transfection.
High resolution respirometry
Fresh spinal cords were rinsed with ice-cold normal saline and cut into slices with a tissue chopper adjusted to a cut width of 300 μm. O2 consumption of sets of 5 lumbar spinal cord slices (LSCS) or suspensions of N2A cells (100,000 cells/mL) was measured at 37 °C with high-resolution respirometry, both in routine setup (without additional substrates or effectors) and in permeabilized conditions (including substrates and inhibitors of specific respiratory complexes) using an Oxygraph-2 k (Oroboros Instruments, Innsbruck, Austria) with chamber volumes set at 2 mL as detailed in the Additional file 1.
Western blot analysis
The content of specific mitochondrial respiratory chain complexes was estimated in Neuro-2A cells using Western blot analysis. Equal amounts of protein (10–25 μg) were separated by SDS-PAGE gels. Proteins were transferred using a Mini Trans-Blot Transfer Cell (BioRad) to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Immunodetection was performed using specific antibodies for the 39 kDa (NDUFA9; CI) subunit of complex I (1:1000), 70 kDa subunit (Flavoprotein) of complex II (1:500), 29 kDa (Rieske iron-sulfur protein; CIII) subunit of complex III (1:1000), and COXI subunit of complex IV (1:1000) (ref. A21344, A11142, A21346, and A6403, respectively; Molecular Probes, Eugene, OR).
Expression of mutant and wild type human SOD1 in transfected N2A cells was immunodetected using a monoclonal antibody specific against human SOD1 (Abcam AB52950). Antibodies to porin (1:5000, A31855, Molecular Probes) and β-actin (1:5000, AB20272, Abcam, Cambridge, MA), as a control for total mitochondrial mass or total protein charge, were also used in order to determine the proportion of protein levels to total mitochondrial mass or total protein content. Appropriate peroxidase-coupled secondary anti-rabbit antibodies (1:40000, Pierce, Rockford, IL, USA) or anti-mouse antibodies (1:10000, GE Healthcare, UK) and chemiluminescence horseradish peroxidase substrate (Millipore, Billerica, MA) were used for primary antibody detection. Signal quantification and recording was performed with a Chemi-Doc unit (Bio-Rad Laboratories, Inc.). Protein concentration was determined with the Bradford method. Data were expressed as arbitrary units.
Oxidation-derived protein damage marker measurements by gas chromatography coupled to mass spectrometry (GC/MS) and fatty acid analyses
Glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε- (carboxyethyl)-lysine (CEL), Nε-(carboxymethyl)-lysine (CML), and Nε-(malondialdehyde)-lysine (MDAL) concentrations in total proteins from spinal cord homogenates were measured with GC/MS as previously described  using deuterated internal standards for each protein oxidative modification adduct as further described in the Additional file 1.
Cytotoxicity assay and ATP content
Viability of N2A cells was assessed with a lactate dehydrogenase (LDH)Cytotoxicity Assay Kit (Promega, Madison, WI) according to manufacturer’s instructions. Briefly, the amount of LDH released into the culture medium is measured using an enzymatic reaction that results in a red formazan product that can be measured spectrophotometrically. Cell viability was evaluated relative to the total LDH from whole cell lysate and the results were expressed as the percentage of viability versus treated with vehicle and/or mock transfected cells.
ATP content in N2A cell homogenates was measured with the ATP Bioluminiscent Assay Kit (Sigma) according to the manufacturer’s instructions. Results are expressed as nanomole ATP per milligram protein.
All statistical analyses were performed using the SPSS software (SPSS Inc., Chicago, IL) or the Prism software (GraphPad Software). Differences between groups were analyzed with the Student’s t tests or ANOVA with Post-Hoc analyses, after normality of variable distribution was ensured by Kolmogorov-Smirnov test. Correlations between variables were evaluated with the Pearson’s statistic. For multivariate analysis of fatty acid composition, the Metaboanalyst platform was used after autoscaling and log transformation of the relative abundances of their composition . The 0.05 level was selected as the point of minimal statistical significance in every comparison.
Results and discussion
Disease onset, clinical evolution and survival in G93A mice show gender dimorphism with a less severe phenotype in female mice
Gender appears to modify the course of disease and lifespan in other animal models of ALS with mutations in the hSOD1 gene, with an earlier disease onset in male transgenic rats and mice overexpressing hSOD1-G93A [34, 35], although gender appears to have no consistent effect on survival of ALS patients . To ascertain whether this was due to different expression of hSOD1-G93A protein expression, this parameter was evaluated in spinal cord with western blot analysis, using a monoclonal antibody specific against human SOD1, which has been shown to recognize distinct mutated human SOD1 forms, including hSOD1-G93A . Additional file 2: Figure S2 shows the western blot analysis of transgenic male and female mice at 30 and 60 days, leading to the expression of a protein with an apparent MW of 16 kDa, whereas, as expected, the protein was not detected in control mice. These results provide evidence that while transgenic protein levels, at 30 days, were higher in males, at 60 days this difference was reversed. Thus, at 30 days we cannot rule out different levels of aggregated or aberrant SOD1 activity between male and female transgenic mice as the primary origin of gender differences in survival. However, maximum protein levels of hSOD1-G93A coincide temporally with the late pre-symptomatic stage and the disease onset.
Oxygen consumption of mouse lumbar spinal cord slices shows early mitochondrial impairment in males linked to complex I dysfunction
When comparing the effect of transgene hSOD1-G93A, overexpression only decreased routine O2 consumption at the later stages of the disease (control vs. G93A: for males, 37.0 ± 3.8 vs. 22.5 ± 2.7 pmol O2/min/mg protein, P < 0.01; for females, 40.1 ± 1.5 vs. 31.9 ± 2.8 pmol O2/min/mg protein, p < 0.01; Additional file 2: Figure S2A and S2B). This may be attributed to the terminal status of the animals and general atrophy of spinal cords at this stage, while no gender differences appeared in control mice (Additional file 2: Figure S3A).
However, considering that this difference was only present at later stages of the disease, we wanted to explore whether any specific respiratory complex was impaired earlier or showed gender related differences. For this reason we studied the sensitivity to complex inhibitors, a validated technique for molecular disection of mitochondrial function, as described in the Materials and methods section. Complex I respiration in G93A females was greater than in males at 60 (20.3 %, p = 0.076), 90 (31.4 %, p < 0.05), and 120 days (25.6 %, p < 0.05) (Fig. 2b). Reinforcing feminine mitochondrial resilience to hSOD1-G93A-linked complex I decay, control-transgenic comparison offered significant differences in complex I function in male mice at all ages studied, whereas transgene only affected complex I activity significantly at later stages of the disease in females (Additional file 2: Figure S2C and S2D). Interestingly, gender-related differences were found in control animals at the earliest ages analyzed. Complex I respiration was 39.6 % higher (p < 0.01) in 30-day-old males, whereas in older animals the values were similar in both genders, lacking significant differences (Additional file 2: Figure S3B).
Regarding complex II, no gender differences were found for G93A animals (Fig. 2c) along disease progression. Residual oxygen consumption is due to oxidative side reactions remaining after application of mitochondrial chain complex III inhibitors, such as antimycin A. This has been related to reactive oxygen species (ROS) production. Interestingly, no gender-related differences were found in G93A mice (Fig. 2d). Antimycin A-resistant respiration was higher in control than in G93A of both genders both at 30 days and at 120 days (Additional file 2: Figure S2E and S2F). Interestingly, the opposite results were obtained in residual respiration at 60 days and 90 days, displaying higher rates in male G93A mice at 60 days (Additional file 2: Figure S2E) and in female G93A mice at 90 days (Additional file 2: Figure S2F).
After cell permeabilization for direct evaluation of mitochondrial function no difference was found for endogenous respiration between genders in G93A, supporting the notion that concentration of metabolites of mitochondria do not differ between genders (Fig. 2e). However, complex I-related measures showed higher capacities for females at early stages: state 2 respiration for complex I was higher in females than in males in G93A mice, at a presymptomatic stage (30 days, Fig. 2f). Interestingly, O2 consumption of complex I state 3 was significantly higher in females compared with males all along disease progression (Fig. 2g). Similarly complexI/II state 3 measurements show increased consumption in G93A females in comparison with males (Fig. 2h).
Our results are consistent with previous reports  showing that routine (intact tissue, without any inhibitor) respiration only shows evident reduction in G93A animals at the end stage of the disease. Although the routine respiration of intact LSCS was not significantly different in males and female G93A mice (only at endpoint), the higher rotenone resistant respiration in males at 60 days suggests a lower complex I contribution to overall respiration, as calculated by the difference with respect to routine respiration.
Focusing on the G93A animals, females showed greater complex I oxygen consumption than males from 60 days. Nevertheless, this advantage was only significant for 90-day and endpoint animals. Since rotenone-mediated cytotoxicity was proved to be alleviated in female neuron primary cultures , this decreased respiration could contribute to the survival demise in males. Studies from  showed that, in this mouse model, motor neuron abnormalities begin at 44–60 days. However, cytosol vacuolization and swollen and mega-mitochondria were evident before (30 days, even P7). In line with this we also show early complex I dysfunction in males. This gender dimorphism can be partially explained by previous data  showing a protective mechanism of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master metabolic regulator in mammals. As PGC-1α could enhance the transcriptional activities of sex hormone receptors—e.g., androgen receptor  or estrogen receptors —this may result in a feed-back loop, enhancing (in the case of estrogens) survival.
Oxidative stress markers and fatty acid composition of spinal cord also show gender dimorphism
Univariant general lineal model linking specific fatty acid amount with gender, G93A-Transgen status and age, as well as its interactions
Fatty acid composition (% of abundance) spinal cord (30 days)
2,086 ± 0,005
1,961 ± 0,077
23,045 ± 0,182
22,985 ± 0,152
1,433 ± 0,043
1,325 ± 0,041
17,324 ± 0,026
16,808 ± 0,331
35,341 ± 0,041
34,552 ± 0,219
1,141 ± 0,063
1,907 ± 0,653
0,075 ± 0,001
0,067 ± 0,002
0,060 ± 0,001
0,058 ± 0,004
0,102 ± 0,003
0,101 ± 0,004
1,912 ± 0,058
1,658 ± 0,035
0,774 ± 0,033
0,778 ± 0,017
0,766 ± 0,048
0,779 ± 0,046
5,944 ± 0,040
6,183 ± 0,128
0,025 ± 0,001
0,022 ± 0,001
0,058 ± 0,003
0,056 ± 0,003
2,277 ± 0,069
2,246 ± 0,040
0,333 ± 0,013
0,307 ± 0,035
0,149 ± 0,013
0,160 ± 0,008
0,090 ± 0,001
0,135 ± 0,018
6,823 ± 0,090
7,687 ± 0,157
0,095 ± 0,007
0,107 ± 0,015
0,136 ± 0,006
0,107 ± 0,008
Fatty acid composition (% of abundance) spinal cord (60 days)
1,571 ± 0,050
1,502 ± 0,050
20,183 ± 0,106
19,943 ± 0,117
1,3876 ± 0,091
1,257 ± 0,063
17,072 ± 0,107
17,458 ± 0,187
38,651 ± 0,196
37,692 ± 0,230
2,059 ± 0,070
1,980 ± 0,032
0,082 ± 0,002
0,085 ± 0,005
0,205 ± 0,040
0,251 ± 0,007
0,4981 ± 0,040
0,454 ± 0,024
1,846 ± 0,043
1,802 ± 0,043
0,744 ± 0,036
0,764 ± 0,026
0,648 ± 0,012
0,666 ± 0,025
5,137 ± 0,072
5,411 ± 0,150
0,048 ± 0,007
0,275 ± 0,002
0,028 ± 0,004
0,019 ± 0,001
2,175 ± 0,051
2,338 ± 0,056
0,208 ± 0,014
0,239 ± 0,077
0,195 ± 0,014
0,190 ± 0,009
0,314 ± 0,028
0,295 ± 0,011
6,785 ± 0,139
7,475 ± 0,132
0,082 ± 0,005
0,058 ± 0,001
0,078 ± 0,006
0,082 ± 0,002
Fatty acid composition (% of abundance) spinal cord (90 days)
1,362 ± 0,036
1,395 ± 0,058
19,191 ± 0,133
18,902 ± 0,249
1,219 ± 0,055
1,317 ± 0,034
17,866 ± 0,127
17,735 ± 0,187
38,326 ± 0,258
39,209 ± 0,349
2,239 ± 0,056
1,903 ± 0,082
0,128 ± 0,021
0,111 ± 0,005
0,134 ± 0,021
0,102 ± 0,011
0,418 ± 0,017
0,478 ± 0,026
1,888 ± 0,037
1,782 ± 0,015
0,694 ± 0,014
0,605 ± 0,010
0,598 ± 0,011
0,533 ± 0,006
5,538 ± 0,112
5,466 ± 0,080
0,038 ± 0,002
0,051 ± 0,006
0,017 ± 0,001
0,038 ± 0,006
2,357 ± 0,086
2,353 ± 0,042
0,296 ± 0,015
0,385 ± 0,059
0,202 ± 0,004
0,259 ± 0,011
0,295 ± 0,018
0,409 ± 0,021
7,039 ± 0,107
6,808 ± 0,067
0,060 ± 0,001
0,084 ± 0,008
0,090 ± 0,005
0,124 ± 0,012
Fatty acid composition (% of abundance) spinal cord (120 days)
1,128 ± 0,243
1,461 ± 0,107
19,346 ± 0,314
19,983 ± 0,380
1,193 ± 0,178
1,338 ± 0,193
17,274 ± 0,243
17,452 ± 0,206
40,311 ± 0,559
39,427 ± 0,454
2,076 ± 0,071
1,968 ± 0,120
0,149 ± 0,005
0,146 ± 0,006
0,214 ± 0,010
0,207 ± 0,014
0,429 ± 0,020
0,497 ± 0,038
1,644 ± 0,018
1,675 ± 0,064
0,502 ± 0,022
0,478 ± 0,017
0,619 ± 0,029
0,591 ± 0,023
6,101 ± 0,112
5,758 ± 0,171
0,078 ± 0,016
0,090 ± 0,012
0,069 ± 0,005
0,095 ± 0,007
2,138 ± 0,068
1,902 ± 0,059
0,193 ± 0,017
0,226 ± 0,033
0,244 ± 0,007
0,247 ± 0,011
0,584 ± 0,023
0,659 ± 0,039
5,533 ± 0,055
5,612 ± 0,100
0,061 ± 0,008
0,062 ± 0,004
0,106 ± 0,012
0,116 ± 0,014
Concerning the potential mechanisms connecting enhanced DHA amount and female resilience to ALS development, it should be recalled that DHA is a precursor of endogenous modulators of neuroinflammation . One of these modulators, resolvin D1 shows a relevant role as inhibitor of inflammatory cytokine secretion in the same model we have examined .
Overexpression of HSOD1-G93A in Neuro 2A cells leads to a loss in complex I function which can be prevented by estradiol
As limitations of this work we acknowledge that measurements performed in total spinal cord, either fatty acid composition, oxidative damage or mitochondrial function, do not allow to specify the cell involved. Thus, we do not know whether the in vivo effect of gender is located in neurons, glia or both. Also, important differences in oxidative metabolism in skeletal muscle between males and females cannot be neglected , and it may be very relevant in ALS development. Further, a recent metaanalysis review indicates that some of the gender related differences in survival present in this model may be related to the genetic background . This indicates that the relationship between gender, disease development and mitochondrial function is complex. Furthermore, mitochondrial vacuolisation and degeneration is a dominant and early phenomenon in the mouse used in this study, a fact that may be attributed to excessive SOD1 overexpression. However, the in vitro experiments demonstrating loss of complex I function, without cell death, and the preventive role of estrogens in this phenomenon, point out that even subtle endocrine effects on mitochondrial function could be relevant at long term. The mechanisms behind the gender and background sparing effects on the pathogenic burden of overexpression of mutant SOD1 merit further exploration, but our data show that this should comprise evaluation of mitochondrial function and fatty acid composition.
Even accounthing these limitations, we have described an early mitochondrial defect during the presymptomatic stage associated with the expression of G93A-mutated human SOD1 that appears at a younger age in male mice than in females, correlating with the delay in the clinical features of ALS-like phenotype.
Our data support a role for dysfunctional mitochondria in ALS pathogenesis and, in particular, the gender bias towards males observed in ALS. This is added to changes in fatty acid composition and oxidative stress markers. These factors could be disease modifiers, affecting the phenotypic differences among ALS patients. Furhter, mitochondrial targets such as complex I may represent a viable focus for novel treatments of a range of disorders affecting motor systems. The presented data, finally, underlines the importance of further studies directed at establishing the role of sex hormones and related molecules in an ALS cure.
amyotrophic lateral sclerosis
butylated hydroxyl toluene
enhanced green fluorescent protein
gas-chromatography coupled to mass spectrometry
Cu,Zn superoxide dismutase with the G93A mutation
lumbar spinal cord slices
reactive oxygen species
We wish to thank T. Yohannan for editorial assistance, and I. Sala for support in this work. We also thank Dr J. Esquerda aa nd L. Piedrafita for constructive comments and hSOD1 plasmids.
This study was funded by the Spanish Ministry of Health, Institute Carlos III: FIS grants PI14/00757, PI14/00328, PI 14/01115. Financed by the European Union, program European Regional Development Fund “A way to build Europe”. Supported by the Generalitat de Catalunya (2014SGR168 and predoctoral fellows for OR-N and PT), by FUNDELA (C100013), “RedELA Investigación” platform and by the Fundació Miquel Valls (Jack Van den Hoek donation for ALS research). These funding bodies do not have any role in the design of the study and collection, analysis, and interpretation of data nor in writing the manuscript.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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