Research Article

Serotonin-1A Agonist 8-OH-DPAT Alleviates Motor Dysfunction and Motor Neuron Degeneration in a Model of Amyotrophic Lateral Sclerosis

Ikuko Miyazaki1*, Shinki Murakami1, Takashi Nakano2, Nao Torigoe2, Ryo Kikuoka2, Yoshihisa Kitamura2, Toshiaki Sendo2 and Masato Asanuma1
1Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2Department of Clinical Pharmacy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan


*Corresponding author: Ikuko Miyazaki, Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700- 8558, Japan


Published: 14 Nov, 2016
Cite this article as: Miyazaki I, Murakami S, Nakano T, Torigoe N, Kikuoka R, Kitamura Y, et al. Serotonin-1A Agonist 8-OH-DPAT Alleviates Motor Dysfunction and Motor Neuron Degeneration in a Model of Amyotrophic Lateral Sclerosis. Ann Pharmacol Pharm. 2016; 1(1): 1003.

Abstract

Glutaminergic excitotoxicity, oxidative stress, and inflammation are related to the pathogenesis of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by selective loss of upper and lower motor neurons, progressive paralysis, and muscle atrophy. We previously reported that 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), a serotonin 1A (5-HT1A) receptor full agonist, can induce astrocyte proliferation and upregulate antioxidative molecules such as metallothionein (MT) in astrocytes, and that the treatment with 8-OH-DPAT protected dopaminergic neurons in parkinsonian mice. In the present study, we examined whether 8-OH-DPAT shows neuroprotective effects in the mutant superoxide dismutase-1 (SOD1) transgenic ALS model mice (G93A–SOD1 mice). Treatment with 8-OH-DPAT attenuated motor neuronal loss in the spinal cord, and slowed progression of motor dysfunction, which was evaluated by the hanging test, rotarod test, and extension reflex test in G93A–SOD1 mice. Moreover, 8-OHDPAT administration markedly increased the MT expression in astrocytes in the ventral horn of spinal cord in G93A–SOD1 mice. These results suggest that the treatment with a 5-HT1A agonist, such as 8-OH-DPAT, is a possible therapeutic strategy against progressive neurodegeneration in ALS.
Keywords: Amyotrophic lateral sclerosis; Motor neurons; Serotonin 1A agonist; 8-OH-DPAT; Astrocyte; Metallothionein


Introduction

AAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, progressive paralysis, muscle atrophy, and death within 3-5 years after diagnosis [1]. Epidemiologically, 90-95% of ALS cases are classified as sporadic and 5-10% as familial cases, of which approximately 20% are caused by gene mutations of copper-zinc superoxide dismutase-1 (SOD1) [1]. In general, SOD can convert superoxide to hydrogen peroxide with less cytotoxicity. Mutations in SOD1 gene lead to mitochondrial dysfunction and result in the promotion of oxidative stress [2]. Transgenic mice expressing human SOD1 with the G93A mutation (G93A–SOD1 mice) develop a progressive motor impairment similar to clinical manifestation seen in ALS patients, and they have been used commonly as an experimental ALS model [3-5]. Although the underlying pathogenesis of ALS remains unclear, it has been proposed that glutamatergic excitotoxicity, oxidative stress, and inflammation are involved in the pathological process of ALS [1,6,7].
Astrocytes are the most abundant neuron-supporting glial cells in the central nervous system (CNS). There has been focus on the neuroprotective ability of astrocytes, such as production of antioxidants, release of neurotrophic factors, and the uptake of excess amounts of glutamate from the synaptic space through astrocyte-specific excitatory amino acid transporter 2 (EAAT2, GLT-1 in rodents) [6,8]. Early evidence showed that the expression of EAAT2 was reduced in the motor cortex and spinal cord of patients with sporadic ALS [9,10]. Numerous studies indicated that astrocyte dysfunctions, or loss-of-astrocytes, largely contribute to non-cell autonomous neurodegeneration and disease progression in ALS [6,8,11]. The transplantation of stem cell-derived astrocytes prevented loss of motor neurons and extended survival and disease duration by the promotion of axonal growth and production of neuroprotective molecules against oxidative or excitotoxic insults [12]. Moreover, several reports demonstrated the neuroprotective effects of cysteine-rich metal-binding protein metallothionein (MT), which exerts antioxidative property, in ALS models [13-15]. MT is expressed and secreted specifically by astrocytes [16-18]. We demonstrated previously that stimulation of serotonin 1A (5-HT1A) receptors in astrocytes by 8-OH-DPAT, a 5-HT1A receptor full agonist, enhanced astrocyte proliferation and upregulated MT expression in astrocytes, and that treatment with 8-OH-DPAT protected dopaminergic neurons against oxidative stress [19]. In the present study, we examined whether the treatment with 8-OH-DPAT can prevent the loss of motor neurons and ameliorate motor dysfunction in G93A mutant SOD1 mice.


Figure 1

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Figure 1
Effects of repeated injections of 8-OH-DPAT for 6 weeks on motor function of G93A–SOD1 mice. (A) Schematic illustration of the experimental schedule. Motor function was assessed by the hanging test (B), extension reflex test (C), and rotarod test (D). Each data was expressed as the mean ± SEM. Data were analyzed by one-way ANOVA, followed by post hoc Scheffe’s t-test (n=3-7 animals). *p < 0.05, **p < 0.01, ***p < 0.001 vs. pre-injection (pre). #p < 0.05 vs. vehicletreated G93A mice.

Figure 2

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Figure 2
Effects of repeated injection of 8-OH-DPAT for 3 weeks on MT-1/2 expression in astrocytes in the ventral horn of cervical (A-C) or lumbar (D-F) spinal cords of WT or G93A–SOD1 mice. Representative microphotographs of MT-1/-2 (red) and GFAP (green) double immunostaining in the cervical spinal cord (A) or lumbar spinal cord (D) of WT mice or G93A mice 3 weeks after repeated injections of 8-OH-DPAT (0.1 mg/kg/day). Scale bar = 20 μm. (B, C, E, F) Quantitation of the MT-1/-2 and GFAP expression in the cervical spinal cords (B, C) or lumbar spinal cords (E, F) of WT or G93A mice after repeated injections of 8-OH-DPAT. (B, E) Number of immunopositive cells, (C, F) proportion of MT-1/-2-positive cells/GFAP-positive cells. Data are mean ± SEM (n=7-9). *p < 0.05, **p < 0.01 vs. vehicle-treated WT mice. #p < 0.05, ##p < 0.01 vs. vehicle-treated G93A mice.

Figure 3

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Figure 3
Effects of repeated injection of 8-OH-DPAT for 6 weeks on MT-1/2 expression in astrocytes in the ventral horn of cervical (A-C) or lumbar (D-F) spinal cords of WT or G93A–SOD1 mice. Representative microphotographs of MT-1/-2 (red) and GFAP (green) double immunostaining in the cervical spinal cord (A) or lumbar spinal cord (D) of WT mice or G93A mice 6 weeks after repeated injections of 8-OH-DPAT (0.1 mg/kg/day). Scale bar = 20 μm. (B, C, E, F) Quantitation of the MT-1/-2 and GFAP expression in the cervical spinal cords (B, C) or lumbar spinal cords (E, F) of WT or G93A mice after repeated injections of 8-OH-DPAT. (B, E) Number of immunopositive cells, (C, F) proportion of MT-1/-2-positive cells/GFAP-positive cells. Data are mean ± SEM (n=7-9). *p < 0.05, **p < 0.01 vs. vehicle-treated WT mice. #p < 0.05, ##p < 0.01 vs. vehicle-treated G93A mice.

Materials and Methods

Animals
Transgenic mice expressing human SOD1 with the G93A mutation (B6SJL-Tg(SOD1*G93A)1Gur/J) (G93A–SOD1 mice) or human wild-type SOD1 (B6SJLF1/J) (WT mice) were purchased from Jackson Laboratory (Bar Harbor, ME, U.S.A.). Mice were acclimated to and maintained at 23°C under a 12-h light/dark cycle (lights on, 08:00-20:00 h), and were housed in standard laboratory cages with free access to food and water throughout the study period. All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Experimental Animals and the Guideline for Animal Experiments of Okayama University Advanced Science Research Center, and were approved by the Animal Care and Use Committee of Okayama University Advanced Science Research Center. Special care was taken to minimize the number of animals used in this research.
Genotyping
Mice were genotyped within 2 weeks after birth by direct polymerase chain reaction (PCR) methods using Thermo Scientific Phire Animal Tissue Direct PCR kit (Pierce Biotechnology, Rockford, IL, USA) according to the manual. The DNA extracts from ear or tail tissue (5 mm) were used for the PCR reaction by TaKaRa PCR Thermal Cycler Dice Gradient (Takara Bio Inc., Shiga, Japan). The following primers were used: Internal positive standard, forward: CTA GGC CAC AGA ATT GAA AGA TCT, reverse: GTA GGT GGA AAT TCT AGC ATC ATC C, hSOD1 G93A; primer, forward: CGC GAC TAA CAA TCA AAG TGA, reverse: CAT CAG CCC TAA TCC ATC TGA. The amplified PCR products were electrophoresed on 1.5% agarose gel; the specific bands were reacted with ethidium bromide and detected by UV irradiation (CSF-AF20, Cosmo Bio Co., Ltd., Tokyo, Japan).
Treatment of mice with 8-OH-DPAT
G93A–SOD1 mice (n=13) or WT mice (n=11, 10-week-old) were injected intraperitoneally with (R)-(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), a 5-HT1A receptor full agonist (Sigma-Aldrich, St. Louis, MO, USA) (0.1 mg/kg) dissolved in saline every day for 3 or 6 weeks. One day after the final administration of the drug, the mice were perfused transcardially with fixative under deep sodium pentobarbital anesthesia (80 mg/kg, i.p.) for immunohistochemical analysis.
Behavioral tests
Animal weighing and all behavioral data collection began at 10 weeks of age, and were conducted twice weekly for 6 weeks. Motor functions were evaluated by the hanging test, extension reflex, and rotarod test. Mice were trained before testing measurements commenced. The hanging test was performed for use as the index of phenotype and grip strength. Mice gripped a grid upside down (score: 0; without any problems, can keep hanging with the grip, 1; be likely to fall, 2; fall after a while, 3; cannot hang). For evaluation of the α motor nerves function, the extension reflex test was performed on the mice; the extension reflex test measures the abduction of the hind legs that appears when the mouse is lifted by the tail (score: 0; open leg without any problems, 1; open leg with convulsions, 2; cannot open, 3; cannot react and atrophy). The rotarod test for evaluation of motor function disorder was performed at a constant speed of 26 rpm for 5 min and holding time on the rotating rod was measured.
Immunohistochemistry
For preparation of brain slices, mice were perfused transcardially with ice-cold saline followed by ice-cold 4% paraformaldehyde (PFA) with 0.35% glutaraldehyde in 0.1 M phosphate buffered (PB; pH 7.4). The spinal cords were corrected and post-fixed using 4% PFA for 18 h at 4°C. Following cryoprotection in 15% sucrose in PB for 48 h, the spinal cord tissues were divided into cervical, thoracic, and lumbar spinal cord, and then frozen sections were cut at 20-µm thicknesses using a cryostat. For staining motor neurons, the sections were completely dried and incubated in 0.1% cresyl violet (Chroma-Gesellschaft, Schmid and Company, Köngen, Germany) solution for 10 min at 37°C. For the immunohistochemistry, the sections were blocked with 1% normal goat serum in phosphate–buffered saline with 0.2% Triton X-100 (PBS-T) for 30 min, and reacted with mouse anti-MT-1/-2 monoclonal antibody (1:100, DakoCytomation, Glostrup, Denmark) and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:5000, DakoCytomation) for 18 h at 4°C. After washing, the sections were reacted with the appropriate secondary antibody of goat anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 594 or anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000, Molecular Probes, Eugene, OR, U.S.A.), for 2 h at room temperature. Observation and counts of cresyl violet-positive cells or MT- and GFAP-positive cells in spinal cords were performed using a microscope (Olympus BX50-FLA, Olympus Co., Tokyo, Japan) and an image analysis system (NIH ImageJ 1.44K, National Institutes of Health, Bethesda, MD, U.S.A.).
Statistical analysis
Each value was expressed as mean ± SEM. Difference among groups was examined by two-way ANOVA with Student’s t-test, or Friedman test followed by Scheffe post-hoc test (J STAT software). A p value less than 0.05 was considered a significant difference.


Figure 4

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Figure 4
Effects of repeated injection of 8-OH-DPAT for 3 weeks on motor neuron survival in the ventral horn of cervical (A) or lumbar (B) spinal cords of WT or G93A–SOD1 mice. Upper panels are representative photomicrographs of Nissl staining using spinal cord sections from WT or G93A mice with/ without 8-OH-DPAT treatment for 3 weeks. Scale bar = 20 μm. Lower panels show the number of motor neurons in the ventral horn. Data are mean ± SEM (n=7-9). **p < 0.01 vs. vehicle-treated WT mice. #p < 0.05, ##p < 0.01 vs. vehicle-treated G93A mice.

Figure 5

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Figure 5
Effects of repeated injection of 8-OH-DPAT for 6 weeks on motor neuron survival in the ventral horn of cervical (A) or lumbar (B) spinal cords of WT or G93A–SOD1 mice. Upper panels are representative photomicrographs of Nissl staining using spinal cord sections from WT or G93A mice with/ without 8-OH-DPAT treatment for 6 weeks. Scale bar = 20 μm. Lower panels show the number of motor neurons in the ventral horn. Data are mean ± SEM (n=7-9). **p < 0.01 vs. vehicle-treated WT mice. #p < 0.05 vs. vehicle-treated G93A mice.

Results

Effects of 8-OH-DPAT on motor dysfunction in G93A–SOD1 mice
Treatment with 8-OH-DPAT caused no significant change in the body weight in WT or G93A–SOD1 mice (data not shown). WT mice did not show any motor dysfunctions when evaluated by the hanging test, extension reflex, and rotarod test with/without 8-OH-DPAT (data not shown). Conversely, G93A–SOD1 mice displayed motor dysfunction from 10 weeks of age, which became more severe time-dependently, indicating disease progression (Figure. 1). Treatment with 8-OH-DPAT for 5 weeks or longer significantly ameliorated motor dysfunction of G93A mice, i.e., reduction of score in the hanging test (Figure 1B) and extension reflex (Figure 1C), and extension of time in the rotarod test (Figure 1D).
Effects of 8-OH-DPAT on MT expression in astrocytes
We previously demonstrated upregulation of MT in the striatal astrocytes after 8-OH-DPAT administration [19]. Therefore, we examined MT expression in astrocytes in the ventral horn of spinal cords after 8-OH-DPAT treatment. In G93A–SOD1 mice, MT expression in GFAP-positive astrocytes was upregulated in the ventral horn of cervical (Figure 2A, B, 3A and B) and lumbar (Figure 2D, E, 3D and E) spinal cords at 13- and 16-weeks of age (i.e., after the treatment with vehicle for 3 and 6 weeks, respectively). The 3-week treatments with 8-OH-DPAT slightly enhanced increases in the number of GFAP-positive astrocytes in the ventral horn of both cervical and lumbar spinal cords of G93A mice (Figure 2B, E), but the treatments showed no further effects on astrogliosis in both regions at 16-weeks of age (Figure 3B, E). Administration of 8-OH-DPAT for 3 or 6 weeks significantly increased the number of MT-positive astrocytes in both cervical and lumbar spinal cords of G93A mice (Figure 2 and 3). Interestingly, both 3-week and 6-week treatment with 8-OH-DPAT significantly increased the MT-positive cells/GFAP-positive cells ratio in the cervical (Figure 2C and 3C) and lumbar (Figure 2F and 3F) spinal cords of G93A mice. However, 8-OH-DPAT did not upregulate astrocyte MT expression in the spinal cord of WT mice either after 3- or 6-week treatment (Figure 2 and 3).
Effects of 8-OH-DPAT on motor neuron cell death
Finally, we examined whether administration of 8-OH-DPAT could ameliorate loss of motor neurons in the spinal cord of G93A–SOD1 mice (Figure 4 and 5). The cell bodies of motor neurons were detected using Nissl staining. In G93A mice, the number of motor neurons in the ventral horn of cervical or lumbar spinal cord was significantly decreased compared to that in WT mice at 13- or 16-weeks of age (i.e., after 3- or 6-week treatment with vehicle, respectively) (Figure 4 and 5). Treatment with 8-OH-DPAT (0.1 mg/kg) for 3 or 6 weeks significantly prevented the reduction of motor neurons either in the cervical or lumbar spinal cord. Although motor neurons showed a tendency to decrease in number in the thoracic spinal cord of G93A–SOD1 mice, the treatment with the 5-HT1A agonist had no apparent effect on the loss of motor neurons (data not shown). The neuroprotective effect of 8-OH-DPAT treatment on the loss of lumbar motor neurons in G93A mice was less than that on cervical motor neurons. Administration of 8-OH-DPAT could ameliorate reduction in the number of motor neurons in the cervical spinal cord of G93A mice at 16-weeks of age (Figure 5A), but not in the lumbar spinal cord (Figure 5B).

Discussion

The present study demonstrated that 5-HT1A agonist 8-OH-DPAT improved motor dysfunction and prevented loss of motor neurons in the spinal cord of ALS model mice. In this study, G93A–SOD1 mice were injected with 8-OH-DPAT or vehicle from 10 weeks of age, when almost all G93A mice begin to exhibit motor symptoms. In addition, the motor dysfunction of G93A mice became more severe time-dependently, which denotes disease progression. Administration of 8-OH-DPAT improved motor dysfunction of ALS mice especially in the advanced stage. Furthermore, 8-OH-DPAT significantly and dramatically ameliorated loss of motor neurons in the spinal cords of ALS model mice. These results suggest that 8-OH-DPAT is effective in the treatment of ALS.
In this study, we demonstrated upregulation of MT in astrocytes in the ventral horn of spinal cords after 8-OH-DPAT treatment. MTs are low molecular weight and cysteine-rich proteins with antioxidative, anti-apoptotic, and anti-inflammatory properties. MTs bind to metals such as zinc, copper, and cadmium to function in metal homeostasis and detoxification [20]. MTs have radical-scavenging property based on their abundant thiol groups, which form metal-thiolate clusters [20-22]. Therefore, MTs play an important role in the regulation of metal homeostasis in the brain and neuroprotection in various pathological and inflammatory states [18,20]. Astrocytes are known to protect neurons by synthesis and secretion of various antioxidants or detoxification enzymes [23]. Nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master transcription factor, produces phase II drug-metabolizing enzymes including MT, by binding to the antioxidant response element (ARE) [24,25]. Nrf2 is mainly expressed in astrocytes. We reported previously that 8-OH-DPAT increased the binding activity of Nrf2 to ARE of MT-1 promoter region, which is followed by upregulation of MTs in striatal astrocytes and MT secretion from astrocytes [19]. We also showed neuroprotective effects of 8-OH-DPAT against dopaminergic neurodegeneration via MTs secreted from astrocytes in parkinsonian models [19]. Various studies reported neuroprotective effects of extracellular MTs against brain pathology including neuronal damage caused by brain injury, inflammation, neurotoxins, and oxidative stress [17,18,24,26,27].
Previous studies showed that activation or upregulation of Nrf2 in astrocytes and implantation of normally functional astrocytes prevented loss of motor neurons, delayed onset of motor dysfunction, and extended survival in G93A–SOD1 mice [12,28]. In addition, deficit of MT expression shortened the time until onset, as well as the lifespan in ALS model mice [13,14]. Over expression of MT-1 in G93A–SOD1 mice significantly extended the lifespan and slowed disease progression [15]. The administration of 8-OH-DPAT could prevent the loss of spinal motor neurons and slow motor dysfunction by augmenting the anti-oxidative function of astrocytes, including increase in MT expression. The preventative effect of the 8-OH-DPAT injections against motor neuronal loss in the lumbar cord at 3 weeks after the treatment was more prominent than that after 6-week treatment, suggesting that the degree of progression of motor neuron death might overwhelm the preventative effect of the drug in the advanced stage. The 8-OH-DPAT treatment showed significant protective effects against the loss of motor neurons in the cervical and lumbar spinal cords but not in the thoracic cord. The differences in the degree of drug effect among spinal cords may be due to the differences in expression level of 5-HT1A receptors or distribution of 8-OH-DPAT, which is influenced by blood flow.
Taken together with the evidence of neuroprotection by focal replacement of normal astrocytes against astrocyte dysfunction in mutant SOD1 models, enhancement of astrocyte proliferation seems to be a therapeutic strategy for ALS. However, neurotoxic properties of reactive astrocytes in mutant SOD1 ALS model mice are involved in non-cell autonomous neurodegeneration and disease progression [6,8,29-31]. Several studies reported that reactive astrocytes surrounded motor neurons in ALS patients, and mutant SOD1 transgenic mice showed astrogliosis and motor symptoms at the same time; thus, reactive astrocytes have been considered to participate in the manifestation and pathology of ALS [32-35]. Furthermore, it has been reported that astrocytes with mutant SOD1 contribute to the progression of pathology, and that astrocytes that abolish the mutation of SOD1 delay the onset and progression in ALS model mice [31,36]. Toxic molecules released from abnormal astrocytes in mutant SOD1 mice are related to degeneration of motor neurons and disease progression. Endo et al. [37], showed that transforming growth factor-1 (TGF-1) from reactive astrocytes in G93A mice accelerated disease progression by inhibiting neuroprotective functions of microglia and T cells [37]. Hydrogen sulfide released as inflammatory gas from astrocytes is increased in the spinal cords in G93A mice [38]. Therefore, the neurotoxic factors from dysregulated astrocytes or mutant SOD1-expressing astrocytes may cause non-cell autonomous degeneration of motor neurons in ALS models [6,8,11,29-31,36,39]. Therefore, simply promoting the proliferation of reactive astrocytes with mutant SOD1 may exacerbate degeneration of motor neurons. Although our previous study showed that 8-OH-DPAT induced astrocyte proliferation and increased MTs in cultured astrocytes and the striatum of mice [19], in the present study, repeated injection of 8-OH-DPAT markedly induced MT expression without apparent increases in the number of reactive astrocytes (increased the MT-positive astrocytes/GFAP-positive astrocytes ratio) in the cervical and lumbar spinal cords of G93A mice (Figure 2 and 3). It may be a good disease-modifying strategy for ALS to compensate astrocyte dysfunction by enhancing anti-oxidative and anti-excitotoxic positive profiles of astrocytes without promoting neurotoxic negative properties of astrocytes by affecting less the astrocyte proliferation surrounding motor neurons.
In conclusion, the present study demonstrated that administration of 8-OH-DPAT alleviates motor symptoms and protects motor neurons in ALS model mice, suggesting a possible disease-modifying strategy of neuroprotection against progressive motor neurodegeneration associated with ALS.

Acknowledgment

This work was supported by Grants-in-Aid for Challenging Exploratory Research (grant number: 24659431 to M.A.), for Scientific Research (C) (grant number: 22590934 and 25461279 to I.M.) from Japan Society for the Promotion of Science, by Grant-in Aid for Scientific Research on Innovative Areas "Brain Environment" from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (grant number: 24111533 to M.A.), by the Okayama Medical Foundation, Kawasaki Foundation for Medical Science & Medical Welfare (to I.M.), and by a grant from Setsurou Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research (to M.A.).


References

  1. Boillee S, Vande Velde C, Cleveland DW. ALS: a disease of motor neurons and their non neuronal neighbors. Neuron. 2006; 52: 39-59.
  2. Kong J, Xu Z. Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J Neurosci. 1998; 18: 3241-3250.
  3. Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science. 1994; 264: 1772-1775.
  4. Tu PH, Raju P, Robinson KA, Gurney ME, Trojanowski JQ, Lee VM. Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions. Proc Natl Acad Sci USA. 1996; 93: 3155-3160.
  5. Turner BJ, Talbot K. Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS. Prog Neurobiol. 2008; 85: 94-134.
  6. Allaman I, Belanger M, Magistretti PJ. Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci. 2011; 34: 76-87.
  7. Philips T, Robberecht W. Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease. Lancet Neurol. 2011; 10: 253-263.
  8. Seifert G, Schilling K, Steinhauser C. Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat Rev Neurosci. 2006; 7: 194-206.
  9. Rothstein JD, Martin LJ, Kuncl RW. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med. 1992; 326: 1464-1468.
  10. Rothstein JD, Van Kammen M, Levey AI, Martin LJ, Kuncl RW. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol. 1995; 38: 73-84.
  11. Rothstein JD. Current hypotheses for the underlying biology of amyotrophic lateral sclerosis. Ann Neurol. 2009; 1: S3-9.
  12. Lepore AC, Rauck B, Dejea C, Pardo AC, Rao MS, Rothstein JD, et al. Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease. Nat Neurosci. 2008; 11: 1294-1301.
  13. Nagano S, Satoh M, Sumi H, Fujimura H, Tohyama C, Yanagihara T, et al. Reduction of metallothioneins promotes the disease expression of familial amyotrophic lateral sclerosis mice in a dose-dependent manner. Eur J Neurosci. 2001; 13: 1363-1370.
  14. Puttaparthi K, Gitomer WL, Krishnan U, Son M, Rajendran B, Elliott JL. Disease progression in a transgenic model of familial amyotrophic lateral sclerosis is dependent on both neuronal and non-neuronal zinc binding proteins. J Neurosci. 2002; 22: 8790-8796.
  15. Tokuda E, Okawa E, Watanabe S, Ono S. Over expression of metallothionein-I, a copper-regulating protein, attenuates intracellular copper dyshomeostasis and extends lifespan in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase-1. Hum Mol Genet. 2014; 23: 1271-1285.
  16. Chung RS, Vickers JC, Chuah MI, West AK. Metallothionein-IIA promotes initial neurite elongation and post injury reactive neurite growth and facilitates healing after focal cortical brain injury. J Neurosci. 2003; 23: 3336-3342.
  17. Chung RS, West AK. A role for extracellular metallothioneins in CNS injury and repair. Neuroscience. 2004; 123: 595-599.
  18. Penkowa M. Metallothioneins are multipurpose neuro protectants during brain pathology. FEBS J. 2006; 273: 1857-1870.
  19. Miyazaki I, Asanuma M, Murakami S, Takeshima M, Torigoe N, Kitamura Y, et al. Targeting 5-HT1A receptors in astrocytes to protect dopaminergic neurons in parkinsonian models. Neurobiol Dis. 2013; 59244-256.
  20. Aschner M. Metallothionein (MT) isoforms in the central nervous system (CNS): regional and cell-specific distribution and potential functions as an antioxidant. Neurotoxicology. 1998; 19: 653-660.
  21. Hussain S, Slikker W, Ali SF. Role of metallothionein and other antioxidants in scavenging superoxide radicals and their possible role in neuroprotection. Neurochem Int. 1996; 29: 145-152.
  22. Thornalley PJ, Vasak M. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta. 1985; 827: 36-44.
  23. Lee JM, Calkins MJ, Chan K, Kan YW, Johnson JA. Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem. 2003; 278: 12029-12038.
  24. Miyazaki I, Asanuma M, Kikkawa Y, Takeshima M, Murakami S, Miyoshi K, et al. Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity. Glia. 2011; 59: 435-451.
  25. Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, et al. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003; 23: 3394-3406.
  26. Chung RS, Penkowa M, Dittmann J, King CE, Bartlett C, Asmussen JW, et al. Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury. J Biol Chem. 2008; 283: 15349-15358.
  27. Kohler LB, Berezin V, Bock E, Penkowa M. The role of metallothionein II in neuronal differentiation and survival. Brain Res. 2003; 992: 128-136.
  28. Vargas MR, Johnson DA, Sirkis DW, Messing A, Johnson JA. Nrf2 activation in astrocytes protects against neurodegeneration in mouse models of familial amyotrophic lateral sclerosis. J Neurosci. 2008; 28: 13574-13581.
  29. Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol. 2009; 187: 761-772.
  30. Lobsiger CS, Cleveland DW. Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci. 2007; 10: 1355-1360.
  31. Yamanaka K, Chun SJ, Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH, et al. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008; 11: 251-253.
  32. Kushner PD, Stephenson DT, Wright S. Reactive astrogliosis is widespread in the subcortical white matter of amyotrophic lateral sclerosis brain. J Neuropathol Exp Neurol. 1991; 50: 263-277.
  33. Nagy D, Kato T, Kushner PD. Reactive astrocytes are widespread in the cortical gray matter of amyotrophic lateral sclerosis. J Neurosci Res. 1994; 38: 336-347.
  34. O'Reilly SA, Roedica J, Nagy D, Hallewell RA, Alderson K, Marklund SL, et al. Motor neuron-astrocyte interactions and levels of Cu,Zn superoxide dismutase in sporadic amyotrophic lateral sclerosis. Exp Neurol. 1995; 131: 203-210.
  35. Schiffer D, Cordera S, Cavalla P, Migheli A. Reactive astrogliosis of the spinal cord in amyotrophic lateral sclerosis. J Neurol Sci. 1996; 139: 27-33.
  36. Wang L, Gutmann DH, Roos RP. Astrocyte loss of mutant SOD1 delays ALS disease onset and progression in G85R transgenic mice. Hum Mol Genet. 2011; 20: 286-293.
  37. Endo F, Komine O, Fujimori-Tonou N, Katsuno M, Jin S, Watanabe S, et al. Astrocyte-derived TGF-beta1 accelerates disease progression in ALS mice by interfering with the neuroprotective functions of microglia and T cells. Cell Rep. 2015; 11: 592-604.
  38. Davoli A, Greco V, Spalloni A, Guatteo E, Neri C, Rizzo GR. Evidence of hydrogen sulfide involvement in amyotrophic lateral sclerosis. Ann Neurol. 2015; 77: 697-709.
  39. Sasaki S, Komori T, Iwata M. Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis. Acta Neuropathol. 2000; 100: 138-144.