Regulation of Intracellular Copper by Induction of Endogenous Metallothioneins Improves the Disease Course in a Mouse Model of Amyotrophic Lateral Sclerosis
Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease. The pathogenesis of the disease is poorly understood, but intracellular copper dyshomeostasis has been implicated as a key process in the disease. We recently observed that metallothioneins (MTs) are an excellent target for the modification of copper dyshomeostasis in a mouse model of ALS (SOD1G93A). Here, we offer a therapeutic strategy designed to increase the level of endogenous MTs. The upregulation of endogenous MTs by dexamethasone, a synthetic glucocorticoid, significantly improved the disease course and rescued motor neurons in SOD1G93A mice, even if the induction was initiated when peak body weight had decreased by 10 %. Neuroprotection was associated with the normalization of copper dyshomeostasis, as well as with decreased levels of SOD1G93A aggregates. Importantly, these benefits were clearly mediated in a MT-dependent manner, as dexamethasone did not provide any protection when endogenous MTs were abolished from SOD1G93A mice. In conclusion, the upregulation of endogenous MTs represents a promising strategy for the treatment of ALS linked to mutant SOD1.
KeywordsAmyotrophic lateral sclerosis Copper dyshomeostasis Dexamethasone Metallothioneins Superoxide dismutase-1
Amyotrophic lateral sclerosis (ALS) is the most common adult-onset form of motor neuron disease. Riluzole is the only available therapeutic option for ALS . However, its therapeutic effect is limited, extending survival by a few months without any improvement in muscle function . Thus, the development of new therapeutic strategies is urgently required. A better understanding of the disease mechanisms of ALS will provide important clues for the development of novel therapeutic targets . Our current knowledge regarding ALS pathogenesis mainly comes from evidence obtained using mouse models of ALS, in which mutated human SOD1 is overexpressed [3, 4].
Several mechanisms of how superoxide dismutase-1 (SOD1) mutants cause ALS have been proposed , including dyshomeostasis of intracellular copper. The expression of the copper uptake proteins, STEAP2 and CTR1, is increased, whereas that of the copper efflux protein, ATP7A, is decreased in the spinal cord of mutant SOD1 mice, regardless of their copper-binding ability . This impairment of the copper trafficking system results in the accumulation of copper ions in the spinal cord [6, 7, 8, 9, 10]. The therapeutic effects of copper-lowering strategies in mutant SOD1 mice, including pharmacological chelation [6, 11] or the restriction of copper intake , support a deleterious role of copper dyshomeostasis in ALS.
In addition to these copper-lowering therapies, metallothioneins (MTs) appear to be an excellent therapeutic target. MTs are a family of small, cysteine-rich proteins that bind copper ions through copper–thiolate clusters . Four MT isoforms exist in mammals, 2 of which, MT-I and MT-II (MT-I/-II), are major isoforms that exert neuroprotection against copper dyshomeostasis . Indeed, the overexpression of MT-I in SOD1G93A mice prolongs survival and restores copper dyshomeostasis . In contrast, the genetic ablation of Mt-I/-II in SOD1G93A mice exacerbates disease mortality and accelerates the death of motor neurons [16, 17]. Taken together, the functional potentiation of MT-I/-II represents a proof-of-concept strategy for the normalization of copper dyshomeostasis to improve the course of the disease.
The aim of this study was to investigate whether an alternative strategy focusing on MT-I/-II could ameliorate the disease course in SOD1G93A mice. Here, we show that the induction of MT-I/-II by dexamethasone (Dex), a synthetic glucocorticoid, extended survival and prevented motor neuron death, accompanied by the amelioration of copper dyshomeostasis. These benefits were completely blocked when MT-I/-II was abolished from SOD1G93A mice, indicating that MT-I/-II is required for the therapeutic effects of Dex in SOD1G93A mice.
All the animal protocols were approved by the Institutional Animal Care and Use Committee of the School of Pharmacy, Nihon University, and adhered to our institutional animal guidelines. Transgenic mice expressing human SOD1G93A [strain name: B6SJL-Tg(SOD1-G93A)1Gur/J, stock number: 002726] were purchased from the Jackson Laboratory (Bar Harbor, MA, USA) . The transgenic line was maintained through hemizygotes by crossing transgenic males with F1 nontransgenic females on a B6SJL background.
Generation of SOD1G93A Mice with a Homozygous Deletion of Mt-I/-II
Knockout mice with a homozygous deletion of Mt-I and Mt-II (strain name: 129S7/SvEvBrd-Mt1tm1BriMt2tm1Bri/J, stock number: 002211) were purchased from the Jackson Laboratory . In the knockout mice, both the Mt-I and Mt-II genes are simultaneously disrupted using a vector that inserts inframe stop codons into the exons of the 2 genes . Homozygous Mt-I/-II (Mt–/–) knockout mice develop normally and show no apparent phenotypic abnormalities . The knockout mice were maintained as homozygotes on a 129Sv background.
To generate SOD1G93A mice with a homozygous deletion of Mt-I/-II (SOD1G93A/Mt–/–), a 2-step breeding strategy was performed to exclude differences caused by the strain background. First, male SOD1G93A mice were crossed with Mt–/– females. Next, male SOD1G93A/Mt+/– mice were intercrossed with female Mt+/– mice to obtain SOD1G93A/Mt–/– mice. The SOD1G93A/Mt–/– mice on a mixed background (B6SJL × 129Sv) were strictly compared with their simultaneously generated nontransgenic, Mt–/–, and SOD1G93A littermates.
To genotype offspring, genomic DNA was extracted from tail biopsies using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Genotypes were determined using polymerase chain reaction with primers according to the recommendations of the Jackson Laboratory.
Time Course of MT Isoform Induction After a Single Injection of Dex
C57BL/6 J mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and maintained in our animal institute. C57BL/6 J mice at 8 weeks of age were intraperitoneally injected with a single dose of Dex (2 mg/kg; Daiichi Sankyo, Tokyo, Japan). At different times after the injection (12–96 h), lumbar spinal cords were dissected from the injected mice (n = 3 per time point). The temporal expression patterns of Dex-induced MT-I/-II and MT-III were determined using Western blotting.
Preclinical Studies with Dex
The protocol for Dex treatment was designed according to criteria suggested for preclinical studies [19, 20]. The investigators were blind to the mouse genotype and treatment while performing all procedures. The copy number of the human SOD1 transgene greatly influences the disease course in SOD1G93A mice . Thus, the copy number was determined using a polymerase chain reaction assay according to instructions from the Jackson Laboratory, and mice with approximately 23 copies were selected.
For the therapeutic regimen in SOD1G93A/Mt–/– mice, we followed the same regimen as that used for the presymptomatic treatment with Dex in SOD1G93A mice. SOD1G93A/Mt–/– or Mt–/– mice were injected with Dex (2 mg/kg) or PBS, starting at the presymptomatic stage (30 days of age) and continuing until the clinical end stage. As a control for the preclinical study using SOD1G93A/Mt–/– mice, Mt–/– littermates on a mixed background (B6SJL × 129Sv) were used.
All clinical assessments were performed by those who were unaware of the genotype or treatment of the mice. The disease course was evaluated according to criteria based on body weight changes . Disease onset was regarded as the time when each mouse reached its peak weight before its weight began to decline. The end point was defined as the age at which a mouse was unable to right itself within 30 s after having been pushed onto its side . The disease duration was regarded as the period from disease onset until the end point. The motor coordination of mice was measured using a rota-rod apparatus (Muromachi Chemicals, Fukuoka, Japan), as described previously .
Dissection of Lumbar Spinal Cords
For the analysis of SOD1G93A mice treated with Dex or PBS from the day when the peak body weight of the mice had decreased by 10 %, the lumbar spinal cords were dissected from the injected mice at 120 days of age (20 days after the start of the injections, n = 3 per group), which is the mean lifespan of PBS-treated SOD1G93A mice. For the analysis of SOD1G93A/Mt–/– mice treated with Dex or PBS injections, the lumbar spinal cords were harvested from mice at 100 days of age (70 days after the start of the injetions, n = 3 per group), which is the mean lifespan of PBS-treated SOD1G93A/Mt–/– mice. The dissected spinal cords were used for the following biochemical and pathological experiments.
Western Blot Analysis
Western blot analyses were performed as described previously . For the detection of MT isoforms, protein extracts were chemically modified with 20 mM monobromobimane (Molecular Probes, Eugene, OR, USA) to block the artificial formation of disulfide bonds in MTs during gel electrophoresis [15, 24]. For the detection of MT-I/-II in Mt–/– mice, recombinant MT-I protein was used as a positive control (Alexa Biochemicals, Lausen, Switzerland). The following primary antibodies were used: mouse anti-MT-I/-II (1:100; clone E9; Dako, Glostrup, Denmark), and mouse anti-MT-III (1:1,000) . Anti-β-tubulin (1:10,000; clone TUB 2.1; Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.
Immunohistochemistry was performed as described previously . Lumbar spinal cord sections (6-μm thick) were immunostained with a mouse anti-MT-I/-II antibody (1:400; clone E9; Dako). Signals were detected using the Histofine Mouse Stain Kit (Nichirei Biosciences Inc., Tokyo, Japan) and 3,3′-diaminobenzidine (Nichirei Biosciences Inc.) as the chromogen. The sections were mounted in Malinole (Muto Pure Chemicals Co., Tokyo, Japan) and imaged using a light microscope (DP70; Olympus, Tokyo, Japan).
Immunofluorescence was performed as described elsewhere . Lumbar spinal cord sections (6-μm thick) were immunostained using the following primary antibodies: mouse anti-MT-I/-II (1:50; clone E9; Dako), mouse anti-NeuN (1:400; clone A60; Chemicon, Temecula, CA, USA), biotin-conjugated anti-NeuN (1:400; clone A60; Chemicon), goat anti-glial fibrillary acidic protein (GFAP) (1:50, sc-6170; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-CD11b (1:50, sc-6614; Santa Cruz Biotechnology), mouse anti-human SOD1 [1:100 (raised against full-length human SOD1) sc-101523; Santa Cruz Biotechnology], goat antihuman SOD1 [1:100 (raised against peptides corresponding to residues surrounding the N-terminus of human SOD1) sc-8636; Santa Cruz Biotechnology], or rabbit antiubiquitin (1:100; Z0458; Dako).
The pathological features of SOD1G93A mice were evaluated as described previously . All measurements were performed by an observer who was blind to the genotype and treatment.
For the quantification of α-motor neurons, every fifth section of the lumbar spinal cord (L4-L5) was immunostained with a mouse anti-NeuN antibody (1:1000; clone A60; Chemicon). α-Motor neurons were molecularly and morphologically identified as described elsewhere . The number of α-motor neurons in the ventral horn was counted in 10 sections per mouse.
For axon counting, the ventral roots were dissected from the lumbar spinal cord (L5) and were immunostained with a mouse anti-SMI-312 antibody (1:2000; ab24574; Abcam, Cambridge, UK). The number of large axons (>4 μm in diameter) was counted using Image J software (National Institutes of Health, Bethesda, MD, USA).
To quantify fluorescence intensity of astrocytes, microglia, human SOD1, or ubiquitin, lumbar spinal cord sections were immunostained with mouse monoclonal anti-GFAP cocktail (1:200, 556330; BD Biosciences, San Jose, CA, USA), goat polyclonal anti-CD11b (1:50, sc-6614; Santa Cruz Biotechnology), mouse antihuman SOD1 [1:100 (raised against full-length human SOD1) sc-101523; Santa Cruz Biotechnology], or rabbit antiubiquitin (1:100, Z0458; Dako) antibodies, respectively. Fluorescence images were acquired using a confocal microscope (Laser Scanning System Z510; Carl Zeiss, Jena, Germany). The fluorescence intensity in the ventral horn was measured using Image J software (National Institutes of Health).
Quantification of Copper Ions
The total amount of copper ions in lumbar spinal cords was measured using inductively coupled plasma–mass spectrometry (Agilent 7500; Yokogawa Analytical Systems, Tokyo, USA), as described previously . The values are expressed as μg/g wet tissue.
Measurement of SOD1 Enzymatic Activity
SOD1 enzymatic activity was measured using a SOD1 Assay Kit-WST (Dojindo Molecular Technology Inc., Kumamoto, Japan), as described previously . One unit of SOD1 activity was defined as a 50 % inhibition of the rate of water-soluble tetrazolium salt reduction. The values are presented as units/μg spinal cord protein.
Measurement of Lipid Peroxides
The concentration of lipid peroxides was determined by measurement of malondialdehyde according to the thiobarbituric acid method as previously described . The malondialdehyde chromophore in lumbar spinal cords was detected using a spectrofluorometer (excitation: 515 nm; emission: 553 nm, FP 6200; JASCO Corporation, Hachioji, Tokyo). The levels of lipid peroxides were estimated using 1,3,3-tetraetoxypropane as a reference standard. The results are expressed as nanomoles of malondialdehyde reactive substances/g wet lumbar spinal cord.
Measurement of Human SOD1 Protein Level
Human SOD1 protein level was determined as described previously . Briefly, lumbar spinal cords were homogenized in 20 volumes (w/v) of ice-cold lysis buffer containing 1 % (v/v) Nonidet P-40 and cOmplete, EDTA-free (Roche Applied Science, Indianapolis, IN, USA) protease inhibitor in PBS (pH 7.0). The homogenates were sonicated, producing a whole homogenate fraction. To prepare the detergent-insoluble fraction, the sonicated homogenate was centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant was collected as the detergent-soluble fraction. The pellets were dissolved in lysis buffer containing 2 % (w/v) sodium dodecyl sulfate. Equal amounts of protein (5 μg) were analyzed by Western blotting with a mouse antihuman SOD1 antibody [1:1000 (raised against full-length human SOD1) sc-101523; Santa Cruz Biotechnology).
The results are expressed as the mean ± SD. All statistical tests were performed using Statcel 3 software (OMS Publishing Inc., Tokorozawa, Saitama, Japan). Disease onset and survival were compared using Kaplan–Meier analysis with the log-rank test. Motor coordination was compared using a repeated-measures analysis of variance. After the determination of normality and homoscedasticity, 2 group comparisons were analyzed using a two-tailed Student’s t test. Multiple group comparisons were performed using a one-way analysis of variance followed by the Tukey–Kramer post hoc test. Statistical significance was defined as p < 0.05. The “n” values indicate the number of individual animals, but not replicate measures of one sample.
Single Injection of Dex Induces MT-I/-II but not MT-III Protein in the Spinal Cords of Nontransgenic Mice
The peripheral injection of Dex induces MT-I/-II protein in the brains of rats because of the presence of glucocorticoid response elements in Mt-I/Mt-II [26, 27]. First, we determined the temporal changes in MT-I/-II protein levels in the lumbar spinal cord after a single injection of Dex. C57BL/6 J mice were intraperitoneally injected with a single dose of Dex (2 mg/kg). At different times after the injection (12–96 h), the level of MT-I/-II in the spinal cord was quantified using Western blotting (Fig. 1a). The induction of MT-I/-II reached a peak level at 24 h after the injection, and this level persisted for a 48-h period (Fig. 1b). The single injection of Dex did not affect the protein level of MT-III, another MT isoform in the central nervous system (Supplementary Fig. 1a, b).
Dex Induces MT-I/-II in Spinal Cord Astrocytes and Microglia in SOD1G93A Mice
We determined whether Dex could induce MT-I/-II in the lumbar spinal cord of SOD1G93A mice harboring a high transgene copy number (Fig. 1c–h). SOD1G93A mice on a B6SJL background and nontransgenic littermates were intraperitoneally injected with Dex (2 mg/kg) or PBS, starting from the time when the peak body weight of the mice had decreased by 10 % . Western blot analyses showed that the level of MT-I/-II in the lumbar spinal cords of PBS-treated SOD1G93A mice at the end stage of the disease (120 days of age) was significantly higher compared with that in PBS-treated nontransgenic B6SJL littermates. Dex induced MT-I/-II protein in SOD1G93A mice at 120 days of age, representing a 20 % increase relative to that in PBS-treated SOD1G93A mice (Fig. 1c, d).
As shown in Fig. 1(e), the distribution pattern of MT-I/-II protein in the lumbar spinal cord of Dex-treated SOD1G93A mice differed considerably from that of PBS-treated SOD1G93A mice. Immunohistochemical studies revealed that MT-I/-II in PBS-treated SOD1G93A mice was distributed not only in the white matter, but also in the entire gray matter, as previously reported by ourselves and others [15, 16, 28]. Notably, Dex predominantly induced MT-I/-II in the ventral horn (Fig. 1e, right), but not the dorsal horn (data not shown), of SOD1G93A mice. To identify the cell types expressing MT-I/-II in the lumbar spinal cord, we performed dual immunofluorescence using antibodies against NeuN, GFAP, and CD11b, which are markers for neurons, astrocytes, and microglia, respectively (Fig. 1f–h). The cell types expressing MT-I/-II in Dex-treated SOD1G93A mice were similar to those in PBS-treated SOD1G93A mice, demonstrating that Dex-induced MT-I/-II was localized in astrocytes and microglia but not in motor neurons.
We examined the effects of Dex on the level of MT-III in SOD1G93A mice (Supplementary Fig. 1c, d). Consistent with our earlier findings , the level of MT-III protein in the lumbar spinal cord of PBS-treated SOD1G93A mice at the end stage of the disease was significantly increased compared with that in PBS-treated B6SJL mice. However, Dex did not further upregulate MT-III in SOD1G93A mice.
Dex Ameliorates the Disease Course in SOD1G93A Mice, Even When Induction is Started at a Symptomatic Stage
The therapeutic effect of Dex on the disease course was also evident when the treatment was initiated on the day when peak body weight had decreased by 10 % (Fig. 2d–f) . Dex significantly prolonged the lifespan by 15 days from 129 ± 6.7 to 144 ± 6.4 days (Fig. 2d), as well as the disease duration by 16 days from 18 ± 1.9 to 34 ± 5.1 days (Fig. 2e). Interestingly, 1 week after the start of treatment, motor coordination in the SOD1G93A mice was temporally restored, and the decline in motor coordination was significantly delayed (Fig. 2f).
Dex Does Not Provide Therapeutic Benefits to SOD1G93A/Mt–/– Mice
Before determining the role of MT-I/-II in Dex treatment, we first characterized how the complete deletion of MT-I/-II influences the disease course in SOD1G93A mice (Supplementary Fig. 2). The deletion of MT-I/-II significantly exacerbated the disease course, including disease onset (88 ± 2.9 days in SOD1G93Avs 73 ± 2.4 days in SOD1G93A/Mt–/–), survival (130 ± 3.2 vs 100 ± 3.0 days), and disease duration (41 ± 0.8 vs 27 ± 1.2 days).
For a preclinical study using SOD1G93A/Mt-–/– mice, we followed the same regimen as that used for presymptomatic treatment with Dex in SOD1G93A mice. Sex-balanced SOD1G93A/Mt–/– mice were intraperitoneally injected with Dex (2 mg/kg) or PBS, starting at 30 days of age until the end stage of the disease (Fig. 3b–d). Interestingly, when MT-I/-II was absent from the SOD1G93A mice, Dex did not have any therapeutic effects on the disease course, including disease onset (75 ± 3.9 days in PBS vs 77 ± 4.3 days in Dex), lifespan (102 ± 5.1 days vs 105 ± 3.9 days), and disease duration (27 ± 3.0 days vs 28 ± 3.1 days). Also, Dex did not ameliorate the impaired motor coordination in SOD1G93A/Mt–/– mice. These results strongly indicate that MT-I/-II is required for the therapeutic effects of Dex on SOD1G93A mice.
MT-I/-II is Necessary for the Therapeutic Benefits of Dex on the Pathological Changes Observed in SOD1G93A Mice
Normalization of Copper Dyshomeostasis and Lipid Peroxidation by Dex is Mediated via MT-I/-II
Dex-induced MT-I/-II Decreases the Amount of Insoluble SOD1G93A Aggregates
MT-I/-II was also required for the modification of insoluble SOD1 aggregates. Dex did not decrease the amount of insoluble mutant SOD1 (Fig. 7c, d) or SOD1 aggregates (Fig. 7g, h) in the lumbar spinal cord of SOD1G93A/Mt–/– mice.
Dex-induced MT-I/-II Effectively Interacts with SOD1G93A Aggregates Within Astrocytes and Microglia
Given that Dex-induced MT-I/-II was distributed in the astrocytes and microglia of SOD1G93A mice (Fig. 1g, h), we hypothesized that Dex-induced MT-I/-II may contribute to the reduction in SOD1G93A aggregates within these cells. Double immunofluorescence showed that Dex-induced MT-I/-II remarkably decreased the number of SOD1G93A aggregates in astrocytes (Fig. 8b) and microglia (data not shown) compared with PBS-treated SOD1G93A mice.
Once again, MT-I/-II appears to be a key molecule in the reduction of SOD1G93A aggregates during Dex treatment. Dex did not influence the amount of SOD1 aggregates within astrocytes (Fig. 8c) and microglia (data not shown) in SOD1G93A/Mt–/– mice.
MT-I/-II Interacts with Ubiquitinated Protein aggregates and Dex-induced MT-I/-II Decreases the Amount of These Aggregates
We have shown that the induction of MT-I/-II by Dex restored copper dyshomeostasis and modified the disease course observed in SOD1G93A mice. We have also shown that MT-I/-II was essential for these effects because Dex did not rescue the ALS-like phenotype of SOD1G93A/Mt–/– mice. These results are consistent with our recent findings, which demonstrated that overexpression of MT-I in SOD1G93A mice ameliorates the disease course through the normalization of copper dyshomeostasis . Thus, the functional potentiation of MT-I/-II is a promising strategy for the treatment of mutant SOD1-linked ALS.
What are the therapeutic advantages of MT-I/-II induction? Three main benefits may exist. First, despite the striking induction of MT-I/-II during the disease process [8, 15, 28], MT-I/-II was further induced by Dex in the spinal cords of SOD1G93A mice. This highly inducible property may be advantageous for its clinical use in ALS, even though MT-I/-II levels are already increased in the spinal cords of patients with sporadic ALS . Second, the therapeutic benefits of MT-I/-II required only a modest increase in the protein level, equivalent to a 20 % increase. The degree of MT-I/-II induction observed in Dex-treated SOD1G93A mice corresponded to that observed in MT-I overexpressing SOD1G93A mice . Thus, MT-I/-II exerted neuroprotection against SOD1G93A toxicity, even though the induction was largely restricted. Finally, MT-I/-II induction significantly prolonged the survival and prevented the death of motor neurons, even when the induction was started on the day when peak body weight had decreased by 10 %, which is probably equivalent to an advanced stage in a clinical setting.
Treatment involving MT-I/-II induction might also be capable of influencing the pathogenesis of ALS by suppressing the expression of SOD1 mutants and their pathogenetic effects. We consider there are 4 rationales for suggesting MT-I/-II as a treatment target. First, MT-I/-II directly interacted with SOD1G93A aggregates in the spinal cord , and Dex-induced MT-I/-II effectively recognized these aggregates. Second, the genetic or pharmacological induction of MT-I/-II significantly decreased the amount of insoluble SOD1G93A . Third, the ablation of Mt-I/-II enhanced the accumulation of insoluble SOD1G93A. Finally, MT-I/-II induction significantly decreased SOD1G93A expression in astrocytes and microglia. The expression of SOD1 mutants in astrocytes and microglia is a well-known determinant of the disease duration [23, 34, 35, 36]. This is consistent with MT-I/-II induction having the ability to significantly prolong survival even when induction was initiated on the day when peak body weight of the mice had decreased by 10 %.
Given the function of MT-I/-II as a copper regulator, our data strongly suggest that MT-I/-II modifies SOD1 aggregates through the normalization of copper dyshomeostasis. How does MT-I/-II modulate SOD1 aggregates? We speculate that MT-I/-II attenuates the impairment of the ubiquitin-proteasome system, which is probably triggered by copper dyshomeostasis, promoting the degradation of SOD1 aggregates by the system. This speculation is supported by the following three observations. First, copper ions inhibited the proteolytic activities of the proteasome in the bovine central nervous system . Second, the coordination of copper ions to ubiquitin compromised the structural stability of the protein, leading to the aggregation of ubiquitin . Finally, increased levels of copper ions in the spinal cords of SOD1G93A rats were concomitant with the inhibition of proteasome activity and significantly shortened survival . Thus, MT-I/-II may remove aberrant copper ions from ubiquitinated proteins through a direct interaction with ubiquitin, facilitating proteolytic activity of the proteasome.
Apart from the normalization of intracellular copper dyshomeostasis, another possible mechanism by which Dex-induced MT-I/-II ameliorates the disease course should be discussed. MT-I/-II is a well-known antioxidant, and its neuroprotective effects may be mediated through the suppression of oxidative stress . Oxidative stress has been implicated in the pathogenesis of mutant SOD1-linked ALS , and is exacerbated by intracellular copper dyshomeostasis. In the present study, we showed that the endogenous MT-I/-II level was correlated with oxidative damage in the spinal cords of SOD1G93A mice. These observations indicate that the upregulation of MT-I/-II by Dex treatment significantly decreased the amount of lipid peroxides in SOD1G93A mice, whereas the absence of Mt-I/-II in SOD1G93A mice resulted in a further increase in the level of lipid peroxides.
The therapeutic benefits observed in the SOD1G93A mice in the present study might be partly mediated through Dex itself. A Dex dose of 2 mg/kg in mice corresponds to a dose of 500 mg/kg in humans, which approximates the dose used in pulsed steroid therapy (three consecutive administrations of methyl prednisolone at a dose of 500–1000 mg/kg). We suspect that the effects of adrenal glucocorticoids were negligible in the present study because the dose of Dex was sufficient to suppress the effects of endogenous adrenal glucocorticoids. Nevertheless, our observation that treatment with Dex in SOD1G93A/Mt–/– mice did not result in a good outcome means that the possible therapeutic contribution of glucocorticoids cannot be ruled out. The possible cooperation of intrinsic glucocorticoids with or without MT-I/-II should be thoroughly considered. In fact, glucocorticoid levels increase constantly in patients with ALS, whereas their levels exhibit a circadian rhythm in healthy humans . Moreover, the serum glucocorticoid levels were significantly higher in SOD1G93A mice at a terminal stage of illness and were negatively correlated with both disease onset and survival . Additionally, wobbler mice, another rodent model for ALS , become vulnerable after adrenalectomy (surgical removal of the adrenal glands, which produce intrinsic glucocorticoids) . Although these changes might be secondary rather than primary effects, further studies investigating how alterations in endogenous glucocorticoids after adrenalectomy influence the disease course in SOD1G93A mice with or without Mt-I/-II are needed.
This work was supported by a Grant-in-Aid for a research fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists from JSPS (08J10542 to E.T.), by a Grant-in-Aid for Exploratory Research from JSPS (21659222 to S.O.), and by an Academic Frontier Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan (years 2007–2009 to S.O.). We thank Drs. Shin-ichi Miyairi (Laboratory of Organic Chemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, Japan) and Akira Naganuma (Laboratory of Molecular and Biochemical Toxicology, School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan) for kindly providing the anti-MT-III antibody.
Conflict of Interest
The authors declare that they have no conflict of interest.
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