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Suppression of murine experimental autoimmune encephalomyelitis development by 1,25-dihydroxyvitamin D3 with autophagy modulation
Journal of Neuroimmunology 280 (2015) 1–7
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Suppression of murine experimental autoimmune encephalomyelitis development by 1,25-dihydroxyvitamin D3 with autophagy modulation Chao Zhen a, Xuedan Feng a, Zhe Li b, Yabo Wang c, Bin Li a,d, Lin Li a, Moyuan Quan a, Gaoning Wang a, Li Guo a,d,⁎ a
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000 Hebei, China Department of Neurology, Hebei General Hospital, Shijiazhuang, 050051 Hebei, China Department of Orthopaedic Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051 Hebei, China d Key Laboratory of Hebei Neurology, Shijiazhuang, 050000 Hebei, China b c
a r t i c l e
i n f o
Article history: Received 13 December 2014 Accepted 28 January 2015 Available online xxxx Keywords: 1,25(OH)2D3 Experimental autoimmune encephalomyelitis Multiple sclerosis Autophagy Apoptosis
a b s t r a c t Multiple sclerosis (MS) has been associated with a history of sub-optimal exposure to ultraviolet light, implicating vitamin D3 as a possible protective agent. We evaluated whether 1,25(OH)2D3 attenuates the progression of experimental autoimmune encephalomyelitis (EAE), and explored its potential mechanisms. EAE was induced in C57BL/6 mice via immunization with MOG35–55, and some mice received 1,25(OH)2D3. 1,25(OH)2D3 inhibited EAE progression. Additionally, 1,25(OH)2D3 reduced inflammation, demyelination, and neuron loss in the spinal cord. The protective effect of 1,25(OH)2D3 was associated with significantly elevated expression of Beclin1, increased Bcl-2/Bax ratio, and decreased LC3-II accumulation. Thus, 1,25(OH)2D3 may represent a promising new MS treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). The significant correlation between MS incidence and global latitude indicates that a lack of sunlight is a significant risk factor for the disease (Simpson et al., 2011). Exposure to ultraviolet (UV) light stimulates the production of vitamin D in the skin, and represents the primary source of vitamin D in mammals. Thus, we naturally assume that vitamin D or its analogs might be a potential therapeutic agent for the prevention or treatment of MS; however, there is a lack of convincing evidence regarding this at present. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of vitamin D. Previous studies have shown that 1,25(OH)2D3 can effectively prevent and ameliorate the development of experimental autoimmune encephalomyelitis (EAE) (Lemire and Archer, 1991; Cantorna et al., 1996), an experimental animal model of MS, through regulation of the expression of immune factors such as IL-2, IFN-γ (Rigby et al., 1987) and IL-10 (Niino et al., 2014). Recently, several studies showed that 1,25(OH)2D3 may also induce autophagy in vivo (Demasters et al., 2006; Wang et al., 2008; Yuk et al., 2009; Wu and Sun, 2011). Autophagy (referred to as macroautophagy) is an evolutionarily conserved cellular catabolic process that degrades and recycles ⁎ Corresponding author at: Department of Neurology, The Second Hospital of Hebei Medical University, Key Laboratory of Hebei Neurology, 215 Heping West Road, Shijiazhuang, 050000 Hebei, China. E-mail address: [email protected] (L. Guo).
http://dx.doi.org/10.1016/j.jneuroim.2015.01.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
damaged organelles and long-lived cytoplasmic proteins (Chen and Karantza-Wadsworth, 2009). Autophagy plays an essential role in maintaining cellular and tissue homeostasis (Levine et al., 2011), pathogen defense (Deretic and Levine, 2009), and aging (Meléndez et al., 2003). At the molecular level, autophagy-related (Atg) genes are the primary controllers of autophagy machinery (Yang and Klionsky, 2010). During the formation of autophagosomes, LC3 is modified into LC3-I and LC3-II; the former is cytosolic, but the latter is localized to the membrane of autophagosomes and autolysosomes (Kabeya et al., 2000). Therefore, LC3-II is a prominent marker of autophagic flux (Weidberg et al., 2011). Additionally, Beclin1 together with hVps34 and p150 attaches to the class III phosphatidylinositol 3kinase complex (PI3K) (Liang et al., 1999),which induces the formation of autophagosomes. MS is characterized by demyelination, astrocytosis, and neurodegeneration. Moreover, neurodegeneration occurs in the early stages of MS and is assumed to mediate irreversible neurological disability in MS patients (Bjartmar et al., 2000). As early as 1868, Charcot noted that MS lesions were not only restricted to the white matter (WM), but also extended to the gray matter (GM) (Charcot, 1868). In the last decade, along with the appearance of new immunohistochemical staining techniques and modern imaging techniques, GM damage was gradually recognized as the other pathological hallmark of MS (Pirko et al., 2007; Hulst and Geurts, 2011). While the mechanisms of MS neurodegeneration remain uncertain, there is evidence implicating persistent CNS infection and an autoimmune-mediated attack against CNS components (Kurtzke, 1993). As neurons have polarized cell-bodies, dendrites and
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axons, proteins and organelles gather in cells to facilitate synaptic growth and activity; high quality control of these constituents is necessary for survival and highly dependent on autophagy (Tooze and Schiavo, 2008). Previous studies have shown that inhibition of autophagy causes neurodegeneration in mature neurons, which indicates that autophagy is a housekeeper of neuronal hemostasis (Hara et al., 2006; Komatsu et al., 2006); thus, we infer that autophagy may be involved in the development of MS. This study was designed to investigate the effects of 1,25(OH)2D3 by the MOG-induced EAE model in C57BL/6 mice. Moreover, we focused on the GM pathological changes in mice, and explored the potential mechanisms of 1,25(OH)2D3 in ameliorating EAE development. 2. Materials and methods 2.1. Animals All experimental protocols were approved by the Institutional Animal Care and Use Committee of Hebei Medical University. Female C57BL/6 mice (8–10 weeks of age, 18–20 g) were purchased from Vital River (Beijing, China). All mice were housed in the laboratory animal room and maintained in specific pathogen-free conditions. Mice had free access to food and water. 2.2. Induction of EAE Mice were injected subcutaneously into four flank sites with 250 μg MOG-35-55 peptide (Lysine Bio-system, XiAn, China), and then emulsified in an equivalent volume of complete Freund's adjuvant (CFA, Sigma, St Louis, MO, USA) supplemented with 1 mg/ml of heat-killed mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). Mice were injected intraperitoneally with 500 ng pertussis toxin (Alexis, San Diego, CA, USA) at 0 and 48 h post-immunization. 2.3. 1,25(OH)2D3 treatment Calcitriol (Roche Pharma Ltd, Schweiz) was diluted in normal castor oil in the concentration of 1 μg/ml 1,25(OH)2D3. Mice were randomly injected intraperitoneally with 0.1 ml 1,25(OH)2D3 or vehicle alone every three days since the day of immunization. Healthy mice in another control group were injected with the same dose of vehicle. 2.4. Clinical assessment All mice were weighed and examined daily for clinical signs of EAE and scored using the following scales: 0 = no paralysis; 1 = loss of tail tone; 2 = hindlimb weakness; 3 = hindlimb paralysis; 4 = hindlimb and forelimb paralysis; and 5 = moribund or dead. 2.5. Histology The intact spinal cords were removed from several mice in each group 20 days post-immunization, when they were directly sacrificed. After the lumbosacral enlargements were fixed with 4% (w/v) paraformaldehyde (Sigma), some of these tissues were embedded in paraffin. The spinal cords were sliced into 5 μm sections. The sections were stained with Luxol Fast Blue (LFB), hematoxylin & eosin (H&E), as well as Cresyl violet, to assess demyelination, inflammatory lesions, and changes in the number of Nissl bodies, respectively. Sections were sequentially analyzed by light microscopy. Semi-quantitative analysis was assessed by examining at least 3–6 sections from each mouse spinal cord under high magnification (× 400). Degree of inflammation was rated on the following scale: 0 = none; 1 = inflammatory cells are limited around blood vessels and meninges; 2 = 1–10 inflammatory cell infiltrated foci within the spinal cord; 3 = 11–100 inflammatory cell infiltrated foci within the spinal cord; and 4 = more than 100
inflammatory cells infiltrated foci within the spinal cord. For demyelination, the following scale was used: 0 = none; 1 = rare foci; 2 = a few areas of demyelination; and 3 = large areas of demyelination (Zhang et al., 2003). For Nissl staining, the data represented the number of staining cells in each field. 2.6. Immunohistochemistry Immunohistochemistry for NeuN was performed to assess the changes in the number and morphology of neurons in the spinal cord tissue sections. The sections were blocked by 3% H2O2 at room temperature for 15 min, and then incubated in 10% normal goat serum for 1 h at room temperature. Sections were incubated with anti-NEUN (1:200, Millipore) overnight at 4 °C. After the primary antibody was washed off, the sections were incubated with goat anti-mouse biotinylated secondary antibodies at 37 °C for 30 min via the ABC kit, and stained with diaminobenzidine. The immunostained sections were examined under a light microscope. 2.7. Confocal microscopy The brains and spinal cords were removed from the sacrificed mice, as above. After fixing the lateral ventricle and lumbosacral enlargements with 4% (w/v) paraformaldehyde (Sigma), tissues were saturated in 30% (w/v) sucrose in PBS. The crystal sections (30 μm) were washed in 0.01 M PBS three times, each for 10 min, and then permeabilized with 0.3% (v/v) Triton X-100 in PBS for about 30 min at room temperature, followed by an incubation in 10% (v/v) equine serum in PBS for 30 min. Subsequently, the brain sections were co-incubated with NeuN (Millipore, 1:200) and anti-LC3B (SIGMA, 1:200). Parts of the spinal cord sections were stained with NeuN (Millipore, 1:200), while others were double-stained with NeuN (Millipore, 1:200) and Beclin1 (Proteintech, 1:150) overnight at 4 °C with gentle shake to achieve co-localization. This procedure was followed by incubation with a fluorochrome-conjugated secondary antibody (Alexa Flour 488 or 594, Zhongshan Golden Bridge Bio-technology, Beijing) in TBS for 1 h in the dark at room temperature. Sections were then stained with Hoechst 33258 (Beyotime) for 5 min. Images were obtained using a confocal laser scanning biological microscope (FV500, Olympus, Japan). 2.8. Western blot analysis On day 20 post-immunization, some mice were directly euthanized, and the brain tissue surrounding the lateral ventricle was removed and immediately frozen via submersion in liquid nitrogen. The cytosol and nuclear proteins were extracted using a nuclear-cytosol extraction kit (Applygen, Beijing, China), according to its manufacturer's instructions. Protein concentrations were measured using a BCA protein assay reagent kit (Novagen). The same amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and followed by transference onto polyvinylidene fluoride membranes (PVDF, Millipore). The membranes were blocked with 5% (w/v) non-fat dry milk in TBST for 1 h at room temperature with continuous shaking, and then incubated with primary antibodies against Beclin1 (1:800, Proteintech), LC3 (1:2000, Sigma), Bcl-2 (1:500, Bioworld), Bax (1:200, Bioworld), and control GAPDH (1:1000, Millipore) overnight at 4 °C. After being washed with TBS-T, corresponding secondary antibodies were blocked to detect the bound antibodies. The expressed levels of target protein were measured by densitometric scanning using an Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA). 2.9. Statistical analysis Data are presented as mean ± SD. The onset rates of EAE between EAE and VD3 [referred to as 1,25(OH)2D3] groups were analyzed by
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Fisher's exact test. The difference in clinical scores between groups was examined by Mann–Whitney U-test. The differences in inflammation and demyelination between the two groups were examined by Student's t-test, All other statistical comparisons among groups were tested by One-Way ANOVA, followed by the SNK-q test or the Dunnett's Multiple Comparison test. A P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Treatment with 1,25(OH)2D3 significantly alleviates clinical symptoms in EAE mice To assess the effect of prevention and treatment with 1,25(OH)2D3 on EAE, this study used preventive medication. The incidence of EAE was calculated for both groups: it was 11.1% in the VD3 group and 100% in the EAE group (given vehicle alone), and there was a significant difference between these groups (P b 0.01). The mean clinical scores in each group are shown in Fig. 1. 1,25(OH)2D3 was found to significantly prevent the development of EAE. 3.2. Treatment with 1,25(OH)2D3 mitigates inflammation and demyelination in EAE mice On the 20th day after immunization, the spinal cords of mice from each group were removed and made into paraffin sections. After staining, they were examined under a light microscope. Inflammatory infiltrates and severe demyelination were observed in the EAE mice. By contrast, there were few inflammatory cells or signs of demyelination in the control and VD3 mice (Fig. 2A–F). 3.3. Treatment with 1,25(OH)2D3 prevents neurodegeneration in GM of EAE To demonstrate whether EAE mice exhibit neuronal damage and loss in GM, spinal cord sections were evaluated using Nissl staining. Compared to controls, there was an obvious decrease in neuronal number in EAE mice (P b 0.01, Fig. 3C), and the neurons present showed signs of damage, such as significant shrinkage with condensed nuclei and few Nissl bodies. In the VD3 group, there were more neurons and Nissl bodies (P b 0.01, Fig. 3C). Moreover, the neurons had larger cell bodies and milder condensed nuclei than did EAE mice (Fig. 3A–B). Considering that neuronal nuclear protein (NeuN) is a neuronspecific marker, the expression of NeuN in consecutive sections was examined through immunohistochemistry and confocal microscopy in our study. A markedly decreased number of GM neurons occurred in EAE mice compared with controls (Fig. 3D–E). In addition, small
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neurons were hardly seen in EAE mice (Fig. 3D), which were more abundant and observed more clearly in the VD3 group (Fig. 3D–E). 3.4. Autophagic flux is impaired in EAE mice and 1,25(OH)2D3 influences autophagy in vivo Considering their importance to autophagosome formation, we measured the expression of Beclin1 and LC3 by western blot. We found markedly decreased expression of Beclin1 in acute EAE mice compared with the age- and gender-matched controls (P b 0.01, Fig. 4B), as well as with VD3 mice (P b 0.05, Fig. 4B). However, results showed a higher level of LC3-II expression in EAE mice relative to controls (P b 0.05, Fig. 4C) and VD3-treated ones (P b 0.01, Fig. 4C), which displayed an “opposite” result. This pattern of inhibited Beclin1 accompanied by increased LC3-II protein expression has also been reported in Miapaca2 pancreatic cancer cells (Li et al., 2013). LC3-II level reflects the number of autophagosomes, and elevated LC3-II may result from two conditions: one is an increase of autophagosome formation, and the other is a blockage of autophagosome degradation. In recent years, autophagic flux assays have been used to evaluate the amount of LC3-II delivered to lysosomes by treating with or without lysosomal protease inhibitors (Mizushima and Yoshimori, 2007). To address these possibilities, we used a LC3 turnover assay, and results indicated that LC3-II level remained unchanged in chloroquine (lysosome inhibitor)-treated EAE mice compared to vehicle-treated EAE mice (P b 0.01, Fig. 4E). Thus, our findings indicate that the autophagosome accumulation that occurred in EAE mice was due to the blockage of autophagosome degradation. Beclin1 and LC3 expression was evaluated via confocal microscopy. Beclin1 and NeuN, and LC3 and NeuN were double-stained to measure the number of neurons undergoing autophagy in the different groups of mice (Fig. 4F–G). Similar to the western blot results, we found reduced expression of Beclin1 in EAE, and an accumulation of LC3, especially in neurons; this pattern was minimally observable in control mice. In the VD3 group, Beclin1 expression was elevated, and LC3 accumulation was decreased in neurons. 3.5. 1,25(OH)2D3 modulated the expression of apoptotic proteins in EAE mice To evaluate the effect of 1,25(OH)2D3 treatment on neuronal damage, proteins linked with neuronal apoptosis in the cortices of the mice were analyzed by western blot. Bcl-2 (an anti-apoptotic protein) and Bax (a member of the Bcl-2 family) were measured. Bax expression was enhanced, while Bcl-2 was inhibited in EAE mice. Further, the ratio of Bcl-2 to Bax was prominently down-regulated (P b 0.01, Fig. 4D). This result is consistent with those of other studies (He et al., 2014). On the contrary, the Bcl-2/Bax ratio in VD3 mice was elevated compared with that of EAE mice (P b 0.05, Fig. 4D). 4. Discussion
Fig. 1. VD3 alleviates clinical symptoms of EAE mice. Mice were immunized with MOG peptide, and were respectively injected with VD3 or vehicle alone every three days since the day of immunization. Data represent the mean ± SD of the clinical scores of mice in each group (n = 9 per group). *P b 0.05 or †P b 0.01 vs. the EAE + VD3 group.
In the present study, the effects of 1,25(OH)2D3 treatment were evaluated in EAE mice. 1,25(OH)2D3 significantly decreased the incidence and attenuated the progression of EAE in mice. Clinical scores, inflammatory infiltration, demyelination, as well as neuron loss and damage were markedly reduced in 1,25(OH)2D3-treated mice. In addition, impaired autophagic flux was observed in EAE mice, which could be effectively remedied by 1,25(OH)2D3. GM lesions in MS patients and EAE mice include mild demyelination, neuron loss, synaptic stripping, axonal sparing, activation of astrocytes and microglia, and fewer perivascular cuffs than WM damage. The diverse pathological changes delineated above were observed in varying degrees in different GM regions, such as the neocortex, hippocampus, basal ganglia, thalamus, hypothalamus, cerebellum, and spinal cord (Bö et al., 2006; Geurts et al., 2007; Calabrese et al., 2009; Ziehn et al., 2010; Ceccarelli et al., 2012; Vigeveno et al., 2012). In
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Fig. 2. VD3 mitigates inflammation and demyelination in EAE mice. On the 20th day post-immunization, the spinal cords from the mice of each group were removed, made into paraffin sections, and stained with H&E (A, B) or LFB (C, D). Subsequently, the degree of inflammation and demyelination was semi-quantitatively measured. (n = 6 per group in H&E, n = 3 per group in LFB). The boxed areas in A and C are enlarged in B and D. E, F, the pathological scores of inflammation and demyelination in EAE and VD3 mice. Data represent the mean ± SD. ** P b 0.01 vs. the EAE group.
the present study, damaged neurons were observed in the spinal cord GM of EAE mice: some neurons showed wizened cell bodies, some had fewer Nissl bodies, and some showed shrinkage with condensed nuclei.
To our knowledge, few studies have examined autophagic levels in MS patients or EAE mice. Beclin1 (the ortholog of yeast Atg6) is one of the first identified autophagy proteins in mammals, and is a core part of the PI3-kinase complex, which functions in the nucleation of the
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Fig. 3. 1,25(OH)2D3 is neuroprotective in EAE. The spinal cords from mice of each group were removed on the 20th day post-immunization, and evaluated by Nissl staining to assess neuronal damage and loss in EAE mice and the effects of 1,25(OH)2D3 treatment (n = 6 per group). The boxed areas in A are presented enlarged in B. C, the staining cell numbers in mice of each group were counted. Data represent the mean ± SD. In later experiments, NeuN expression was examined through immunohistochemistry (D) and confocal microscopy (E). **P b 0.01 vs. the control group, ††P b 0.01 vs. the EAE group.
isolation membrane, as well as in autophagosome and endosome maturation (Cecconi and Levine, 2008). In addition, Beclin1 has a conserved BH3 domain, which can bind to the anti-apoptotic family members Bcl-2 and Bcl-XL (Maiuri et al., 2007a, 2007b; Wang, 2008). Therefore, Beclin1 bridges autophagy and apoptosis. Studies have shown that
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Fig. 4. Autophagic flux is impaired in EAE mice and VD3 influences autophagy and apoptosis in vivo. The brain cortices from mice of each group were removed on the 20th day postimmunization, and autophagic- and apoptotic- associated proteins were evaluated by western blot (A). B–D, gray intensity analysis of Beclin1, LC3-II, and Bcl-2/Bax was measured. Data represent the mean ± SD. LC3 turnover assay was conducted in our subsequent experiment (E), and indicated that the LC3-II level remained unchanged in chloroquine-treated EAE mice compared to vehicle-treated EAE mice. Beclin1 and LC3 expression was also evaluated via confocal microscopy. **P b 0.01 or *P b 0.05 vs. controls, ††P b 0.01 or †P b 0.05 vs. EAE group.
when autophagy is blocked by depletion of Beclin1 at an early stage, cells do not contain autophagic vacuoles, but still exhibit the typical morphological characteristics of apoptosis (a typical type I cell death),
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which suggests that Beclin1 plays a central role in incipient autophagy, as well as in the interaction between autophagy and apoptosis. By contrast, when lysosomal inhibitors (e.g., chloroquine, which was used in this study) are added to block the fusion of autophagosomes and lysosomes, an accumulation of autophagic vacuoles is observed in the cells, which also exhibit type I morphology prior to death (Boya et al., 2005; González-Polo et al., 2005; González-Polo et al., 2005). We found decreased Beclin1 and increased LC3-II expression in the neurons of EAE mice, and results of the turnover assay indicated an inhibition of autophagic flux in EAE mice. Given the Zhong et al. finding that knocking down Beclin1 impairs autophagy-mediated clearance of LC3II (Zhong et al., 2009), we inferred that in the peak stage of EAE development, a lower level of Beclin1 could not balance autophagy and apoptosis in neurons. This would presumably lead to an accumulation of autophagic vacuoles, blocking the clearance of damaged organelles and long-lived cytoplasmic proteins in neurons and resulting in neuronal death, as described above. Evidence suggests that when etiologic factors of the disease impair the efficacy of autophagy, neurodegeneration and neuronal death subsequently occur (Boland and Nixon, 2006), as observed in the EAE mice in our study. Alteration of autophagy may be involved in the pathogenesis of EAE mice; therefore, this study focused on the peak stage of the disease and on neurons. In subsequent experiments, we will concentrate on different stages and different cell types such as astrocytes and oligodendrocytes. The previous studies described above indicate that blocking any stage of the autophagy process could potentially trigger apoptosis (Maiuri et al., 2007a, 2007b). The Bcl-2/Bax ratio in the EAE mice was decreased compared with controls in our study, indicating that Baxdependent apoptosis was triggered in EAE, which is consistent with previous findings (Dasgupta et al., 2013; He et al., 2014). Under stress, the Bcl-2–Beclin1 complex is disrupted by pro-apoptotic BH3-only proteins, leading to displacement of Beclin1 from Bcl-2 to induce autophagy (Miller et al., 2007). Only ER (endoplasmic reticulum)-localized Bcl-2 or Bcl-XL inhibits autophagy by reducing the pro-autophagy activity of Beclin1 (Maiuri et al., 2007a), resulting in an inability of Beclin1 to neutralize the anti-apoptotic activity of Bcl-2 (Maiuri et al., 2007b). The role of Beclin1 on autophagy and apoptosis remains controversial, as does the interaction between Beclin1 and Bcl-2. Currently, there are many therapeutic agents available that reduce inflammatory and immune responses in MS, and these show a modest effect on GM damage and permanent disability (Miller et al., 2007). However, to effectively act on GM lesions or prevent permanent disability in MS, neuroprotective therapies are necessary. In this study, 1,25(OH)2D3 was found to effectively inhibit neurodegeneration. 1,25(OH)2D3 treatment was associated with a reduction in neuronal loss and EAE damage, reduced inflammatory infiltration in both GM and WM, and mitigated demyelination in WM. Previous studies demonstrated that 1,25(OH)2D3 prevents the development of EAE by mediating the immune response (Rigby et al., 1987; Lemire and Archer, 1991; Cantorna et al., 1996; Correale et al., 2009; Niino et al., 2014), and our study verified that 1,25(OH)2D3 increases Beclin1 and the Bcl-2/Bax ratio, and leads to a reduction in LC3-II in the brains of EAE mice. Taken together, these results suggest that the increase in Beclin1 leads to a level of autophagic activity sufficient to effectively degrade and recycle the damaged organelles and long-lived cytoplasmic proteins, and that Bax-dependent apoptosis was inhibited. However, the interactions of autophagy and immunology with 1,25(OH)2D3 on EAE remains to be explored, which will be the precise goal of our subsequent experiments. Additionally, by double-staining NeuN and Beclin1, and NeuN and LC3, we found a certain amount of Beclin1 in normal neurons, whereas LC3-II was hardly detectable in healthy neurons, which is consistent with previous findings (Nixon et al., 2000; Mizushima et al., 2004). One possibility may be that a low level of autophagic activity is maintained in normal neurons (Boland et al., 2008); another possibility is that autophagic degradation is so efficient in healthy neurons LC3-II cannot accumulate detectably (Furuta et al., 2010).
5. Conclusion In summary, 1,25(OH)2D3 administration alleviated the development and progression of EAE by modulating multiple aspects of MS pathogenesis, including neuroinflammation, demyelination, autophagic activity, and neuroapoptosis. In addition, our data suggest that impaired autophagy is involved in MS, and that 1,25(OH)2D3 can mitigate GM lesions. Taken together, our findings indicate that 1,25(OH)2D3 might represent a promising new treatment for MS.
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Suppression of murine experimental autoimmune encephalomyelitis development by 1,25-dihydroxyvitamin D3 with autophagy modulation Chao Zhen a, Xuedan Feng a, Zhe Li b, Yabo Wang c, Bin Li a,d, Lin Li a, Moyuan Quan a, Gaoning Wang a, Li Guo a,d,⁎ a
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000 Hebei, China Department of Neurology, Hebei General Hospital, Shijiazhuang, 050051 Hebei, China Department of Orthopaedic Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051 Hebei, China d Key Laboratory of Hebei Neurology, Shijiazhuang, 050000 Hebei, China b c
a r t i c l e
i n f o
Article history: Received 13 December 2014 Accepted 28 January 2015 Available online xxxx Keywords: 1,25(OH)2D3 Experimental autoimmune encephalomyelitis Multiple sclerosis Autophagy Apoptosis
a b s t r a c t Multiple sclerosis (MS) has been associated with a history of sub-optimal exposure to ultraviolet light, implicating vitamin D3 as a possible protective agent. We evaluated whether 1,25(OH)2D3 attenuates the progression of experimental autoimmune encephalomyelitis (EAE), and explored its potential mechanisms. EAE was induced in C57BL/6 mice via immunization with MOG35–55, and some mice received 1,25(OH)2D3. 1,25(OH)2D3 inhibited EAE progression. Additionally, 1,25(OH)2D3 reduced inflammation, demyelination, and neuron loss in the spinal cord. The protective effect of 1,25(OH)2D3 was associated with significantly elevated expression of Beclin1, increased Bcl-2/Bax ratio, and decreased LC3-II accumulation. Thus, 1,25(OH)2D3 may represent a promising new MS treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). The significant correlation between MS incidence and global latitude indicates that a lack of sunlight is a significant risk factor for the disease (Simpson et al., 2011). Exposure to ultraviolet (UV) light stimulates the production of vitamin D in the skin, and represents the primary source of vitamin D in mammals. Thus, we naturally assume that vitamin D or its analogs might be a potential therapeutic agent for the prevention or treatment of MS; however, there is a lack of convincing evidence regarding this at present. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of vitamin D. Previous studies have shown that 1,25(OH)2D3 can effectively prevent and ameliorate the development of experimental autoimmune encephalomyelitis (EAE) (Lemire and Archer, 1991; Cantorna et al., 1996), an experimental animal model of MS, through regulation of the expression of immune factors such as IL-2, IFN-γ (Rigby et al., 1987) and IL-10 (Niino et al., 2014). Recently, several studies showed that 1,25(OH)2D3 may also induce autophagy in vivo (Demasters et al., 2006; Wang et al., 2008; Yuk et al., 2009; Wu and Sun, 2011). Autophagy (referred to as macroautophagy) is an evolutionarily conserved cellular catabolic process that degrades and recycles ⁎ Corresponding author at: Department of Neurology, The Second Hospital of Hebei Medical University, Key Laboratory of Hebei Neurology, 215 Heping West Road, Shijiazhuang, 050000 Hebei, China. E-mail address: [email protected] (L. Guo).
http://dx.doi.org/10.1016/j.jneuroim.2015.01.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
damaged organelles and long-lived cytoplasmic proteins (Chen and Karantza-Wadsworth, 2009). Autophagy plays an essential role in maintaining cellular and tissue homeostasis (Levine et al., 2011), pathogen defense (Deretic and Levine, 2009), and aging (Meléndez et al., 2003). At the molecular level, autophagy-related (Atg) genes are the primary controllers of autophagy machinery (Yang and Klionsky, 2010). During the formation of autophagosomes, LC3 is modified into LC3-I and LC3-II; the former is cytosolic, but the latter is localized to the membrane of autophagosomes and autolysosomes (Kabeya et al., 2000). Therefore, LC3-II is a prominent marker of autophagic flux (Weidberg et al., 2011). Additionally, Beclin1 together with hVps34 and p150 attaches to the class III phosphatidylinositol 3kinase complex (PI3K) (Liang et al., 1999),which induces the formation of autophagosomes. MS is characterized by demyelination, astrocytosis, and neurodegeneration. Moreover, neurodegeneration occurs in the early stages of MS and is assumed to mediate irreversible neurological disability in MS patients (Bjartmar et al., 2000). As early as 1868, Charcot noted that MS lesions were not only restricted to the white matter (WM), but also extended to the gray matter (GM) (Charcot, 1868). In the last decade, along with the appearance of new immunohistochemical staining techniques and modern imaging techniques, GM damage was gradually recognized as the other pathological hallmark of MS (Pirko et al., 2007; Hulst and Geurts, 2011). While the mechanisms of MS neurodegeneration remain uncertain, there is evidence implicating persistent CNS infection and an autoimmune-mediated attack against CNS components (Kurtzke, 1993). As neurons have polarized cell-bodies, dendrites and
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axons, proteins and organelles gather in cells to facilitate synaptic growth and activity; high quality control of these constituents is necessary for survival and highly dependent on autophagy (Tooze and Schiavo, 2008). Previous studies have shown that inhibition of autophagy causes neurodegeneration in mature neurons, which indicates that autophagy is a housekeeper of neuronal hemostasis (Hara et al., 2006; Komatsu et al., 2006); thus, we infer that autophagy may be involved in the development of MS. This study was designed to investigate the effects of 1,25(OH)2D3 by the MOG-induced EAE model in C57BL/6 mice. Moreover, we focused on the GM pathological changes in mice, and explored the potential mechanisms of 1,25(OH)2D3 in ameliorating EAE development. 2. Materials and methods 2.1. Animals All experimental protocols were approved by the Institutional Animal Care and Use Committee of Hebei Medical University. Female C57BL/6 mice (8–10 weeks of age, 18–20 g) were purchased from Vital River (Beijing, China). All mice were housed in the laboratory animal room and maintained in specific pathogen-free conditions. Mice had free access to food and water. 2.2. Induction of EAE Mice were injected subcutaneously into four flank sites with 250 μg MOG-35-55 peptide (Lysine Bio-system, XiAn, China), and then emulsified in an equivalent volume of complete Freund's adjuvant (CFA, Sigma, St Louis, MO, USA) supplemented with 1 mg/ml of heat-killed mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). Mice were injected intraperitoneally with 500 ng pertussis toxin (Alexis, San Diego, CA, USA) at 0 and 48 h post-immunization. 2.3. 1,25(OH)2D3 treatment Calcitriol (Roche Pharma Ltd, Schweiz) was diluted in normal castor oil in the concentration of 1 μg/ml 1,25(OH)2D3. Mice were randomly injected intraperitoneally with 0.1 ml 1,25(OH)2D3 or vehicle alone every three days since the day of immunization. Healthy mice in another control group were injected with the same dose of vehicle. 2.4. Clinical assessment All mice were weighed and examined daily for clinical signs of EAE and scored using the following scales: 0 = no paralysis; 1 = loss of tail tone; 2 = hindlimb weakness; 3 = hindlimb paralysis; 4 = hindlimb and forelimb paralysis; and 5 = moribund or dead. 2.5. Histology The intact spinal cords were removed from several mice in each group 20 days post-immunization, when they were directly sacrificed. After the lumbosacral enlargements were fixed with 4% (w/v) paraformaldehyde (Sigma), some of these tissues were embedded in paraffin. The spinal cords were sliced into 5 μm sections. The sections were stained with Luxol Fast Blue (LFB), hematoxylin & eosin (H&E), as well as Cresyl violet, to assess demyelination, inflammatory lesions, and changes in the number of Nissl bodies, respectively. Sections were sequentially analyzed by light microscopy. Semi-quantitative analysis was assessed by examining at least 3–6 sections from each mouse spinal cord under high magnification (× 400). Degree of inflammation was rated on the following scale: 0 = none; 1 = inflammatory cells are limited around blood vessels and meninges; 2 = 1–10 inflammatory cell infiltrated foci within the spinal cord; 3 = 11–100 inflammatory cell infiltrated foci within the spinal cord; and 4 = more than 100
inflammatory cells infiltrated foci within the spinal cord. For demyelination, the following scale was used: 0 = none; 1 = rare foci; 2 = a few areas of demyelination; and 3 = large areas of demyelination (Zhang et al., 2003). For Nissl staining, the data represented the number of staining cells in each field. 2.6. Immunohistochemistry Immunohistochemistry for NeuN was performed to assess the changes in the number and morphology of neurons in the spinal cord tissue sections. The sections were blocked by 3% H2O2 at room temperature for 15 min, and then incubated in 10% normal goat serum for 1 h at room temperature. Sections were incubated with anti-NEUN (1:200, Millipore) overnight at 4 °C. After the primary antibody was washed off, the sections were incubated with goat anti-mouse biotinylated secondary antibodies at 37 °C for 30 min via the ABC kit, and stained with diaminobenzidine. The immunostained sections were examined under a light microscope. 2.7. Confocal microscopy The brains and spinal cords were removed from the sacrificed mice, as above. After fixing the lateral ventricle and lumbosacral enlargements with 4% (w/v) paraformaldehyde (Sigma), tissues were saturated in 30% (w/v) sucrose in PBS. The crystal sections (30 μm) were washed in 0.01 M PBS three times, each for 10 min, and then permeabilized with 0.3% (v/v) Triton X-100 in PBS for about 30 min at room temperature, followed by an incubation in 10% (v/v) equine serum in PBS for 30 min. Subsequently, the brain sections were co-incubated with NeuN (Millipore, 1:200) and anti-LC3B (SIGMA, 1:200). Parts of the spinal cord sections were stained with NeuN (Millipore, 1:200), while others were double-stained with NeuN (Millipore, 1:200) and Beclin1 (Proteintech, 1:150) overnight at 4 °C with gentle shake to achieve co-localization. This procedure was followed by incubation with a fluorochrome-conjugated secondary antibody (Alexa Flour 488 or 594, Zhongshan Golden Bridge Bio-technology, Beijing) in TBS for 1 h in the dark at room temperature. Sections were then stained with Hoechst 33258 (Beyotime) for 5 min. Images were obtained using a confocal laser scanning biological microscope (FV500, Olympus, Japan). 2.8. Western blot analysis On day 20 post-immunization, some mice were directly euthanized, and the brain tissue surrounding the lateral ventricle was removed and immediately frozen via submersion in liquid nitrogen. The cytosol and nuclear proteins were extracted using a nuclear-cytosol extraction kit (Applygen, Beijing, China), according to its manufacturer's instructions. Protein concentrations were measured using a BCA protein assay reagent kit (Novagen). The same amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and followed by transference onto polyvinylidene fluoride membranes (PVDF, Millipore). The membranes were blocked with 5% (w/v) non-fat dry milk in TBST for 1 h at room temperature with continuous shaking, and then incubated with primary antibodies against Beclin1 (1:800, Proteintech), LC3 (1:2000, Sigma), Bcl-2 (1:500, Bioworld), Bax (1:200, Bioworld), and control GAPDH (1:1000, Millipore) overnight at 4 °C. After being washed with TBS-T, corresponding secondary antibodies were blocked to detect the bound antibodies. The expressed levels of target protein were measured by densitometric scanning using an Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA). 2.9. Statistical analysis Data are presented as mean ± SD. The onset rates of EAE between EAE and VD3 [referred to as 1,25(OH)2D3] groups were analyzed by
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Fisher's exact test. The difference in clinical scores between groups was examined by Mann–Whitney U-test. The differences in inflammation and demyelination between the two groups were examined by Student's t-test, All other statistical comparisons among groups were tested by One-Way ANOVA, followed by the SNK-q test or the Dunnett's Multiple Comparison test. A P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Treatment with 1,25(OH)2D3 significantly alleviates clinical symptoms in EAE mice To assess the effect of prevention and treatment with 1,25(OH)2D3 on EAE, this study used preventive medication. The incidence of EAE was calculated for both groups: it was 11.1% in the VD3 group and 100% in the EAE group (given vehicle alone), and there was a significant difference between these groups (P b 0.01). The mean clinical scores in each group are shown in Fig. 1. 1,25(OH)2D3 was found to significantly prevent the development of EAE. 3.2. Treatment with 1,25(OH)2D3 mitigates inflammation and demyelination in EAE mice On the 20th day after immunization, the spinal cords of mice from each group were removed and made into paraffin sections. After staining, they were examined under a light microscope. Inflammatory infiltrates and severe demyelination were observed in the EAE mice. By contrast, there were few inflammatory cells or signs of demyelination in the control and VD3 mice (Fig. 2A–F). 3.3. Treatment with 1,25(OH)2D3 prevents neurodegeneration in GM of EAE To demonstrate whether EAE mice exhibit neuronal damage and loss in GM, spinal cord sections were evaluated using Nissl staining. Compared to controls, there was an obvious decrease in neuronal number in EAE mice (P b 0.01, Fig. 3C), and the neurons present showed signs of damage, such as significant shrinkage with condensed nuclei and few Nissl bodies. In the VD3 group, there were more neurons and Nissl bodies (P b 0.01, Fig. 3C). Moreover, the neurons had larger cell bodies and milder condensed nuclei than did EAE mice (Fig. 3A–B). Considering that neuronal nuclear protein (NeuN) is a neuronspecific marker, the expression of NeuN in consecutive sections was examined through immunohistochemistry and confocal microscopy in our study. A markedly decreased number of GM neurons occurred in EAE mice compared with controls (Fig. 3D–E). In addition, small
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neurons were hardly seen in EAE mice (Fig. 3D), which were more abundant and observed more clearly in the VD3 group (Fig. 3D–E). 3.4. Autophagic flux is impaired in EAE mice and 1,25(OH)2D3 influences autophagy in vivo Considering their importance to autophagosome formation, we measured the expression of Beclin1 and LC3 by western blot. We found markedly decreased expression of Beclin1 in acute EAE mice compared with the age- and gender-matched controls (P b 0.01, Fig. 4B), as well as with VD3 mice (P b 0.05, Fig. 4B). However, results showed a higher level of LC3-II expression in EAE mice relative to controls (P b 0.05, Fig. 4C) and VD3-treated ones (P b 0.01, Fig. 4C), which displayed an “opposite” result. This pattern of inhibited Beclin1 accompanied by increased LC3-II protein expression has also been reported in Miapaca2 pancreatic cancer cells (Li et al., 2013). LC3-II level reflects the number of autophagosomes, and elevated LC3-II may result from two conditions: one is an increase of autophagosome formation, and the other is a blockage of autophagosome degradation. In recent years, autophagic flux assays have been used to evaluate the amount of LC3-II delivered to lysosomes by treating with or without lysosomal protease inhibitors (Mizushima and Yoshimori, 2007). To address these possibilities, we used a LC3 turnover assay, and results indicated that LC3-II level remained unchanged in chloroquine (lysosome inhibitor)-treated EAE mice compared to vehicle-treated EAE mice (P b 0.01, Fig. 4E). Thus, our findings indicate that the autophagosome accumulation that occurred in EAE mice was due to the blockage of autophagosome degradation. Beclin1 and LC3 expression was evaluated via confocal microscopy. Beclin1 and NeuN, and LC3 and NeuN were double-stained to measure the number of neurons undergoing autophagy in the different groups of mice (Fig. 4F–G). Similar to the western blot results, we found reduced expression of Beclin1 in EAE, and an accumulation of LC3, especially in neurons; this pattern was minimally observable in control mice. In the VD3 group, Beclin1 expression was elevated, and LC3 accumulation was decreased in neurons. 3.5. 1,25(OH)2D3 modulated the expression of apoptotic proteins in EAE mice To evaluate the effect of 1,25(OH)2D3 treatment on neuronal damage, proteins linked with neuronal apoptosis in the cortices of the mice were analyzed by western blot. Bcl-2 (an anti-apoptotic protein) and Bax (a member of the Bcl-2 family) were measured. Bax expression was enhanced, while Bcl-2 was inhibited in EAE mice. Further, the ratio of Bcl-2 to Bax was prominently down-regulated (P b 0.01, Fig. 4D). This result is consistent with those of other studies (He et al., 2014). On the contrary, the Bcl-2/Bax ratio in VD3 mice was elevated compared with that of EAE mice (P b 0.05, Fig. 4D). 4. Discussion
Fig. 1. VD3 alleviates clinical symptoms of EAE mice. Mice were immunized with MOG peptide, and were respectively injected with VD3 or vehicle alone every three days since the day of immunization. Data represent the mean ± SD of the clinical scores of mice in each group (n = 9 per group). *P b 0.05 or †P b 0.01 vs. the EAE + VD3 group.
In the present study, the effects of 1,25(OH)2D3 treatment were evaluated in EAE mice. 1,25(OH)2D3 significantly decreased the incidence and attenuated the progression of EAE in mice. Clinical scores, inflammatory infiltration, demyelination, as well as neuron loss and damage were markedly reduced in 1,25(OH)2D3-treated mice. In addition, impaired autophagic flux was observed in EAE mice, which could be effectively remedied by 1,25(OH)2D3. GM lesions in MS patients and EAE mice include mild demyelination, neuron loss, synaptic stripping, axonal sparing, activation of astrocytes and microglia, and fewer perivascular cuffs than WM damage. The diverse pathological changes delineated above were observed in varying degrees in different GM regions, such as the neocortex, hippocampus, basal ganglia, thalamus, hypothalamus, cerebellum, and spinal cord (Bö et al., 2006; Geurts et al., 2007; Calabrese et al., 2009; Ziehn et al., 2010; Ceccarelli et al., 2012; Vigeveno et al., 2012). In
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Fig. 2. VD3 mitigates inflammation and demyelination in EAE mice. On the 20th day post-immunization, the spinal cords from the mice of each group were removed, made into paraffin sections, and stained with H&E (A, B) or LFB (C, D). Subsequently, the degree of inflammation and demyelination was semi-quantitatively measured. (n = 6 per group in H&E, n = 3 per group in LFB). The boxed areas in A and C are enlarged in B and D. E, F, the pathological scores of inflammation and demyelination in EAE and VD3 mice. Data represent the mean ± SD. ** P b 0.01 vs. the EAE group.
the present study, damaged neurons were observed in the spinal cord GM of EAE mice: some neurons showed wizened cell bodies, some had fewer Nissl bodies, and some showed shrinkage with condensed nuclei.
To our knowledge, few studies have examined autophagic levels in MS patients or EAE mice. Beclin1 (the ortholog of yeast Atg6) is one of the first identified autophagy proteins in mammals, and is a core part of the PI3-kinase complex, which functions in the nucleation of the
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Fig. 3. 1,25(OH)2D3 is neuroprotective in EAE. The spinal cords from mice of each group were removed on the 20th day post-immunization, and evaluated by Nissl staining to assess neuronal damage and loss in EAE mice and the effects of 1,25(OH)2D3 treatment (n = 6 per group). The boxed areas in A are presented enlarged in B. C, the staining cell numbers in mice of each group were counted. Data represent the mean ± SD. In later experiments, NeuN expression was examined through immunohistochemistry (D) and confocal microscopy (E). **P b 0.01 vs. the control group, ††P b 0.01 vs. the EAE group.
isolation membrane, as well as in autophagosome and endosome maturation (Cecconi and Levine, 2008). In addition, Beclin1 has a conserved BH3 domain, which can bind to the anti-apoptotic family members Bcl-2 and Bcl-XL (Maiuri et al., 2007a, 2007b; Wang, 2008). Therefore, Beclin1 bridges autophagy and apoptosis. Studies have shown that
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Fig. 4. Autophagic flux is impaired in EAE mice and VD3 influences autophagy and apoptosis in vivo. The brain cortices from mice of each group were removed on the 20th day postimmunization, and autophagic- and apoptotic- associated proteins were evaluated by western blot (A). B–D, gray intensity analysis of Beclin1, LC3-II, and Bcl-2/Bax was measured. Data represent the mean ± SD. LC3 turnover assay was conducted in our subsequent experiment (E), and indicated that the LC3-II level remained unchanged in chloroquine-treated EAE mice compared to vehicle-treated EAE mice. Beclin1 and LC3 expression was also evaluated via confocal microscopy. **P b 0.01 or *P b 0.05 vs. controls, ††P b 0.01 or †P b 0.05 vs. EAE group.
when autophagy is blocked by depletion of Beclin1 at an early stage, cells do not contain autophagic vacuoles, but still exhibit the typical morphological characteristics of apoptosis (a typical type I cell death),
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which suggests that Beclin1 plays a central role in incipient autophagy, as well as in the interaction between autophagy and apoptosis. By contrast, when lysosomal inhibitors (e.g., chloroquine, which was used in this study) are added to block the fusion of autophagosomes and lysosomes, an accumulation of autophagic vacuoles is observed in the cells, which also exhibit type I morphology prior to death (Boya et al., 2005; González-Polo et al., 2005; González-Polo et al., 2005). We found decreased Beclin1 and increased LC3-II expression in the neurons of EAE mice, and results of the turnover assay indicated an inhibition of autophagic flux in EAE mice. Given the Zhong et al. finding that knocking down Beclin1 impairs autophagy-mediated clearance of LC3II (Zhong et al., 2009), we inferred that in the peak stage of EAE development, a lower level of Beclin1 could not balance autophagy and apoptosis in neurons. This would presumably lead to an accumulation of autophagic vacuoles, blocking the clearance of damaged organelles and long-lived cytoplasmic proteins in neurons and resulting in neuronal death, as described above. Evidence suggests that when etiologic factors of the disease impair the efficacy of autophagy, neurodegeneration and neuronal death subsequently occur (Boland and Nixon, 2006), as observed in the EAE mice in our study. Alteration of autophagy may be involved in the pathogenesis of EAE mice; therefore, this study focused on the peak stage of the disease and on neurons. In subsequent experiments, we will concentrate on different stages and different cell types such as astrocytes and oligodendrocytes. The previous studies described above indicate that blocking any stage of the autophagy process could potentially trigger apoptosis (Maiuri et al., 2007a, 2007b). The Bcl-2/Bax ratio in the EAE mice was decreased compared with controls in our study, indicating that Baxdependent apoptosis was triggered in EAE, which is consistent with previous findings (Dasgupta et al., 2013; He et al., 2014). Under stress, the Bcl-2–Beclin1 complex is disrupted by pro-apoptotic BH3-only proteins, leading to displacement of Beclin1 from Bcl-2 to induce autophagy (Miller et al., 2007). Only ER (endoplasmic reticulum)-localized Bcl-2 or Bcl-XL inhibits autophagy by reducing the pro-autophagy activity of Beclin1 (Maiuri et al., 2007a), resulting in an inability of Beclin1 to neutralize the anti-apoptotic activity of Bcl-2 (Maiuri et al., 2007b). The role of Beclin1 on autophagy and apoptosis remains controversial, as does the interaction between Beclin1 and Bcl-2. Currently, there are many therapeutic agents available that reduce inflammatory and immune responses in MS, and these show a modest effect on GM damage and permanent disability (Miller et al., 2007). However, to effectively act on GM lesions or prevent permanent disability in MS, neuroprotective therapies are necessary. In this study, 1,25(OH)2D3 was found to effectively inhibit neurodegeneration. 1,25(OH)2D3 treatment was associated with a reduction in neuronal loss and EAE damage, reduced inflammatory infiltration in both GM and WM, and mitigated demyelination in WM. Previous studies demonstrated that 1,25(OH)2D3 prevents the development of EAE by mediating the immune response (Rigby et al., 1987; Lemire and Archer, 1991; Cantorna et al., 1996; Correale et al., 2009; Niino et al., 2014), and our study verified that 1,25(OH)2D3 increases Beclin1 and the Bcl-2/Bax ratio, and leads to a reduction in LC3-II in the brains of EAE mice. Taken together, these results suggest that the increase in Beclin1 leads to a level of autophagic activity sufficient to effectively degrade and recycle the damaged organelles and long-lived cytoplasmic proteins, and that Bax-dependent apoptosis was inhibited. However, the interactions of autophagy and immunology with 1,25(OH)2D3 on EAE remains to be explored, which will be the precise goal of our subsequent experiments. Additionally, by double-staining NeuN and Beclin1, and NeuN and LC3, we found a certain amount of Beclin1 in normal neurons, whereas LC3-II was hardly detectable in healthy neurons, which is consistent with previous findings (Nixon et al., 2000; Mizushima et al., 2004). One possibility may be that a low level of autophagic activity is maintained in normal neurons (Boland et al., 2008); another possibility is that autophagic degradation is so efficient in healthy neurons LC3-II cannot accumulate detectably (Furuta et al., 2010).
5. Conclusion In summary, 1,25(OH)2D3 administration alleviated the development and progression of EAE by modulating multiple aspects of MS pathogenesis, including neuroinflammation, demyelination, autophagic activity, and neuroapoptosis. In addition, our data suggest that impaired autophagy is involved in MS, and that 1,25(OH)2D3 can mitigate GM lesions. Taken together, our findings indicate that 1,25(OH)2D3 might represent a promising new treatment for MS.
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