Activation of microglia, CNS resident immune cells, is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons. Despite evidence that microglia contribute to disease progression, the exact role of these cells in ALS pathology remains unknown. We immunomagnetically isolated microglia from different CNS regions of SOD1G93A rats at three different points in disease progression: presymptomatic, symptom onset and end-stage. We observed no differences in microglial number or phenotype in presymptomatic rats compared to wild-type controls. Although after disease onset there was no macrophage infiltration, there were significant increases in microglial numbers in the spinal cord, but not cortex. At disease end-stage, microglia were characterized by high expression of galectin-3, osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, BDNF and arginase-1. Flow cytometry revealed the presence of at least two phenotypically distinct microglial populations in the spinal cord. Immunohistochemistry showed that galectin-3/osteopontin positive microglia were restricted to the ventral horns of the spinal cord, regions with severe motor neuron degeneration. End-stage SOD1G93A microglia from the cortex, a less affected region, displayed similar gene expression profiles to microglia from wild-type rats, and displayed normal responses to systemic inflammation induced by LPS. On the other hand, end-stage SOD1G93A spinal microglia had blunted responses to systemic LPS suggesting that in addition to their phenotypic changes, they may also be functionally impaired. Thus, after disease onset, microglia acquired unique characteristics that do not conform to typical M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was observed only in the most affected CNS regions, suggesting that overexpression of mutated hSOD1 is not sufficient to trigger these changes in microglia. These novel observations suggest that microglial regional and phenotypic heterogeneity may be an important consideration when designing new therapeutic strategies targeting microglia and neuroinflammation in ALS.
microglia; gene expression; M1/M2 phenotype; lipopolysaccharide (LPS); ALS
Microglia have been implicated in amyotrophic lateral sclerosis (ALS) progression, but the underlying mechanisms are unclear. Mesci et al. reveal that suppression of system xC− (a cystine/glutamate transporter) reduces microglia-triggered glutamate excitotoxicity and switches microglia to a neuroprotective phenotype: this slows disease progression and increases neuronal survival in ALS mice.
Amyotrophic lateral sclerosis is the most common adult-onset motor neuron disease and evidence from mice expressing amyotrophic lateral sclerosis-causing SOD1 mutations suggest that neurodegeneration is a non-cell autonomous process where microglial cells influence disease progression. However, microglial-derived neurotoxic factors still remain largely unidentified in amyotrophic lateral sclerosis. With excitotoxicity being a major mechanism proposed to cause motor neuron death in amyotrophic lateral sclerosis, our hypothesis was that excessive glutamate release by activated microglia through their system xC− (a cystine/glutamate antiporter with the specific subunit xCT/Slc7a11) could contribute to neurodegeneration. Here we show that xCT expression is enriched in microglia compared to total mouse spinal cord and absent from motor neurons. Activated microglia induced xCT expression and during disease, xCT levels were increased in both spinal cord and isolated microglia from mutant SOD1 amyotrophic lateral sclerosis mice. Expression of xCT was also detectable in spinal cord post-mortem tissues of patients with amyotrophic lateral sclerosis and correlated with increased inflammation. Genetic deletion of xCT in mice demonstrated that activated microglia released glutamate mainly through system xC−. Interestingly, xCT deletion also led to decreased production of specific microglial pro-inflammatory/neurotoxic factors including nitric oxide, TNFa and IL6, whereas expression of anti-inflammatory/neuroprotective markers such as Ym1/Chil3 were increased, indicating that xCT regulates microglial functions. In amyotrophic lateral sclerosis mice, xCT deletion surprisingly led to earlier symptom onset but, importantly, this was followed by a significantly slowed progressive disease phase, which resulted in more surviving motor neurons. These results are consistent with a deleterious contribution of microglial-derived glutamate during symptomatic disease. Therefore, we show that system xC− participates in microglial reactivity and modulates amyotrophic lateral sclerosis motor neuron degeneration, revealing system xC− inactivation, as a potential approach to slow amyotrophic lateral sclerosis disease progression after onset of clinical symptoms.
ALS; neuroinflammation; excitotoxicity; motoneuron; xCT
Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression. Connexins (Cxs) form homotypic or heterotypic gap junctions (GJs) and allow direct intercellular communications among nervous tissue cells. The role of Cxs in motor neuron disease has never been investigated; therefore, we aimed to evaluate alterations of Cxs in mSOD1-transgenic (mSOD1-Tg) mice in comparison with their non-transgenic (non-Tg) littermates at the same ages.
We pathologically evaluated temporal changes to astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 immunoreactivities at presymptomatic, disease-progressive, and end stages, relative to aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-2 (EAAT2), myelin-oligodendrocyte glycoprotein (MOG), and Nogo-A immunoreactivities, and observed neuronal loss by NeuN and neurofilament immunostaining, and microglial response by Iba-1 immunostaining. We also performed quantitative immunoblotting and real-time PCR analyses for Cxs.
The mSOD1-Tg mice showed neuronal and axonal loss in the anterior horns of the lumbar spinal cord accompanied by increased activation of microglia compared with non-Tg mice at the disease-progressive and end stages. Expression patterns of Cxs were not different between mSOD1-Tg and non-Tg mice at the presymptomatic stage, but immunoreactivities for GFAP, Cx43, Cx30 and AQP4 were increased in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. By contrast, Cx47 and Cx32 immunoreactivities were markedly diminished in Nogo-A-positive oligodendrocytes in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages, especially in oligodendrocytes showing SOD1 accumulation. EAAT2 immunoreactivity was also diminished in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. Quantitative immunoblotting revealed a significant reduction in Cx47 and Cx32 protein levels in mSOD1-Tg mice at the disease-progressive and end stages. The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.
Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.
amyotrophic lateral sclerosis; astrocyte; connexin; gap junction; oligodendrocyte; superoxide dismutase 1
The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials, and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model, and then validating the findings in human tissues. Ventral roots from SOD1G93A “ALS” mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with matched samples from non-transgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1WT). Out of 1299 proteins, statistical inference of the data in the three groups identified 14 proteins that were dramatically altered in the ALS mice compared with the two control groups. The protein galectin-3 emerged as a lead biomarker candidate based on its differential expression as assessed by immunoblot and immunocytochemistry in SOD1G93A mice as compared to controls, and because it is a secreted protein that could potentially be measured in human biofluids. Spinal cord tissue from ALS patients also showed increased levels of galectin-3 when compared to controls. Further measurement of galectin-3 in cerebrospinal fluid samples showed that ALS patients had approximately twice as much galectin-3 as normal and disease controls. These results provide the proof of principle that biomarker identification in relevant and well-controlled animal models can be translated to human disease. The challenge is to validate our biomarker candidate proteins as true biomarkers for ALS that will be useful for diagnosis and/or monitoring disease activity in future clinical trials.
Motor neuron disease; Galectin-3; human; biomarkers
Non-neuronal cells, such as microglia and lymphocytes, are thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Previous studies have demonstrated neuroprotective effects of lymphocytes at the end stage of ALS, partly through induction of alternatively activated microglia (M2 microglia), which are neuroprotective. In this study, we investigated the role of lymphocytes in the early stage of the disease using an animal model of inherited ALS.
We established a transgenic mouse line overexpressing the familial ALS-associated G93A-SOD1 mutation (harboring a single amino acid substitution of glycine to alanine at codon 93) with depletion of the Rag2 gene (mSOD1/RAG2-/- mice), an animal model of inherited ALS lacking mature lymphocytes. Body weights, clinical scores and motor performance (hanging wire test) of mSOD1/RAG2-/- mice were compared to those of mutant human SOD1 transgenic mice (mSOD1/RAG2+/+ mice). Activation of glial cells in the spinal cords of these mice was determined immunohistochemically, and the expression of mRNA for various inflammatory and anti-inflammatory molecules was evaluated.
Clinical onset in mSOD1/RAG2-/- mice was significantly delayed, and the number of lectin-positive cells in spinal cord was increased at the early stage of disease when compared to mSOD1/RAG2+/+ mice. Quantitative RT-PCR confirmed that mRNA for Ym1, an M2 microglial-related molecule, was significantly increased in mSOD1/RAG2-/- mouse spinal cords at the early disease stage.
Compared with mSOD1/RAG2+/+ mice, mSOD1/RAG2-/- mice displayed delayed onset and increased M2 microglial activation at the early stage of disease. Thus, lymphocytes at the early pathological phase of ALS display a deleterious effect via inhibition of M2 microglial activation.
Aberrant neuroinflammation is suspected to contribute to the pathogenesis of myriad neurological diseases. As such, determining the pathways that promote or inhibit glial activation is of interest. Activation of the surface glycoprotein T-cell immunoglobulin and mucin-domain containing protein 3 (Tim-3) by the lectin galectin-9 has been implicated in promoting innate immune cell activation by potentiating or synergizing toll-like receptor (TLR) signaling. In the present study we examined the role of the Tim-3/galectin-9 pathway in glial activation in vitro.
Primary monocultures of microglia or astrocytes, co-cultures containing microglia and astrocytes, and mixed glial cultures consisting of microglia, astrocytes and oligodendrocytes were stimulated with poly(I:C) or LPS, and galectin-9 up-regulation was determined. The effect of endogenous galectin-9 production on microglial activation was examined using cultures from wild-type and Lgals9 null mice. The ability for recombinant galectin-9 to promote microglia activation was also assessed. Tim-3 expression on microglia and BV2 cells was examined by qPCR and flow cytometry and its necessity in transducing the galectin-9 signal was determined using a Tim-3 specific neutralizing antibody or recombinant soluble Tim-3.
Astrocytes potentiated TNF production from microglia following TLR stimulation. Poly(I:C) stimulation increased galectin-9 expression in microglia and microglial-derived factors promoted galectin-9 up-regulation in astrocytes. Astrocyte-derived galectin-9 in turn enhanced microglial TNF production. Similarly, recombinant galectin-9 enhanced poly(I:C)-induced microglial TNF and IL-6 production. Inhibition of Tim-3 did not alter TNF production in mixed glial cultures stimulated with poly(I:C).
Galectin-9 functions as an astrocyte-microglia communication signal and promotes cytokine production from microglia in a Tim-3 independent manner. Activation of CNS galectin-9 likely modulates neuroinflammatory processes in which TNF and IL-6 contribute to either pathology or reparation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0144-0) contains supplementary material, which is available to authorized users.
Galectin-9; TNFα; Astrocytes; Microglia activation; Poly(I:C); Neuroinflammation
Atherosclerosis is a major risk factor for cardiovascular disease (CVD) and stroke. Galectin-3 is a carbohydrate-binding lectin implicated in the pathophysiology of CVD and is highly expressed within atherosclerotic lesions in mice and humans. The object of this present study was to use genetic deletion and pharmacological inhibition in a well-characterized mouse model of atherosclerosis to determine the role of galectin-3 in plaque development. Apolipoprotein-E/galectin-3 knockout mice were generated and fed a high-cholesterol “western” diet. Galectin-3 deletion had no consistent effect on the serum lipid profile but halved atherosclerotic lesion formation in the thoracic aorta (57% reduction), the aortic arch (50% reduction) and the brachiocephalic arteries. The aortic plaques were smaller, with reduced lipid core and less collagen. In apolipoprotein E-deficient (ApoE−/−) mice, there was a switch from high inducible nitric oxide expression in early lesions (6 weeks) to arginase-1 expression in later lesions (20 weeks), which was reversed in ApoE−/−/gal-3−/− mice. Administration of modified citrus pectin, an inhibitor of galectin-3, during the latter stage of the disease reduced plaque volume. We conclude that inhibiting galectin-3 causes decreased atherosclerosis. Strategies to inhibit galectin-3 function may reduce plaque progression and potentially represent a novel therapeutic strategy in the treatment of atherosclerotic disease.
atherosclerosis; galectin-3; inflammation; macrophages; plaque
Parkinson’s disease (PD) is the most prevalent neurodegenerative motor disorder. The neuropathology is characterized by intraneuronal protein aggregates of α-synuclein and progressive degeneration of dopaminergic neurons within the substantia nigra. Previous studies have shown that extracellular α-synuclein aggregates can activate microglial cells, induce inflammation and contribute to the neurodegenerative process in PD. However, the signaling pathways involved in α-synuclein-mediated microglia activation are poorly understood. Galectin-3 is a member of a carbohydrate-binding protein family involved in cell activation and inflammation. Therefore, we investigated whether galectin-3 is involved in the microglia activation triggered by α-synuclein.
We cultured microglial (BV2) cells and induced cell activation by addition of exogenous α-synuclein monomers or aggregates to the cell culture medium. This treatment induced a significant increase in the levels of proinflammatory mediators including the inducible Nitric Oxide Synthase (iNOS), interleukin 1 Beta (IL-1β) and Interleukin-12 (IL-12). We then reduced the levels of galectin-3 expression using siRNA or pharmacologically targeting galectin-3 activity using bis-(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)-sulfane. Both approaches led to a significant reduction in the observed inflammatory response induced by α-synuclein. We confirmed these findings using primary microglial cells obtained from wild-type and galectin-3 null mutant mice. Finally, we performed injections of α-synuclein in the olfactory bulb of wild type mice and observed that some of the α-synuclein was taken up by activated microglia that were immunopositive for galectin-3.
We show that α-synuclein aggregates induce microglial activation and demonstrate for the first time that galectin-3 plays a significant role in microglia activation induced by α-synuclein. These results suggest that genetic down-regulation or pharmacological inhibition of galectin-3 might constitute a novel therapeutic target in PD and other synucleinopathies.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0156-0) contains supplementary material, which is available to authorized users.
Microglia; Galectin-3; Neuroinflammation; α-synuclein; Parkinson’s disease
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression.
We examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1G93A) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1G93A and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene’s test showed that the variance in WT and SOD1G93A measurements was substantially different.
Disease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1G93A mice. The ventral horn of the SOD1G93A spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1G93A mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1G93A mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1G93A mice, suggesting that Arg1 may have a neuroprotective function.
This study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.
Amyotrophic lateral sclerosis; Microglia; Inducible nitric oxide synthase; Arginase1; Motor neurons; Lumbar spinal cord; Cervical spinal cord; Neuroinflammation
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with autocrine neuroprotective and paracrine anti-inflammatory properties in various models of acute neuronal damage and neurodegenerative diseases. Therefore, we examined a possible beneficial role of endogenous PACAP in the superoxide dismutase 1, SOD1(G93A), mouse model of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease particularly affecting somatomotor neurons. In wild-type mice, somatomotor and visceromotor neurons in brain stem and spinal cord were found to express the PACAP specific receptor PAC1, but only visceromotor neurons expressed PACAP as a potential autocrine source of regulation of these receptors. In SOD1(G93A) mice, only a small subset of the surviving somatomotor neurons showed induction of PACAP mRNA, and somatomotor neuron degeneration was unchanged in PACAP-deficient SOD1(G93A) mice. Pre-ganglionic sympathetic visceromotor neurons were found to be resistant in SOD1(G93A) mice, while pre-ganglionic parasympathetic neurons degenerated during ALS disease progression in this mouse model. PACAP-deficient SOD1(G93A) mice showed even greater pre-ganglionic parasympathetic neuron loss compared to SOD1(G93A) mice, and additional degeneration of pre-ganglionic sympathetic neurons. Thus, constitutive expression of PACAP and PAC1 may confer neuroprotection to central visceromotor neurons in SOD1(G93A) mice via autocrine pathways. Regarding the progression of neuroinflammation, the switch from amoeboid to hypertrophic microglial phenotype observed in SOD1(G93A) mice was absent in PACAP-deficient SOD1(G93A) mice. Thus, endogenous PACAP may promote microglial cytodestructive functions thought to drive ALS disease progression. This hypothesis was consistent with prolongation of life expectancy and preserved tongue motor function in PACAP-deficient SOD1(G93A) mice, compared to SOD1(G93A) mice. Given the protective role of PACAP expression in visceromotor neurons and the opposing effect on microglial function in SOD1(G93A) mice, both PACAP agonism and antagonism may be promising therapeutic tools for ALS treatment, if stage of disease progression and targeting the specific auto- and paracrine signaling pathways are carefully considered.
Amyotrophic lateral sclerosis; Microglia; Neuroinflammation; Neuroprotection; Neuropeptide; Parasympathetic; Sympathetic
Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1G93A mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1G93A mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1G93A mice. Systemic MB-treatment of SOD1G93A mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1G93A mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.
Nonalcoholic fatty liver disease (NAFLD) is increasingly recognized as a condition in which excess fat accumulates in hepatocytes. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD in which inflammation and fibrosis in the liver are noted, may eventually progress to end-stage liver disease. Galectin-3, a β-galactoside-binding animal lectin, is a multifunctional protein. This protein is involved in inflammatory responses and carcinogenesis. We investigated whether galectin-3 is involved in the development of NASH by comparing galectin-3 knockout (gal3−/−) mice and wild-type (gal3+/+) mice with choline-deficient L-amino-acid-defined (CDAA) diet-induced NAFLD/NASH. Hepatic injury was significantly more severe in the gal3−/− male mice, as compared to the gal3+/+ mice. Data generated by microarray analysis of gene expression suggested that galectin-3 deficiency causes alterations in the expression of various genes associated with carcinogenesis and lipid metabolism. Through canonical pathway analysis, involvement of PDGF and IL-6 signaling pathways was suggested in galectin-3 deficiency. Significant increase of CD14, Fos, and Jun, those that were related to lipopolysaccharide-mediated signaling, was candidate to promote hepatocellular damages in galectin-3 deficiency. In conclusion, galectin-3 deficiency in CDAA diet promotes NAFLD features. It may be caused by alterations in the expression profiles of various hepatic genes including lipopolysaccharide-mediated inflammation.
The peripheral immune system is implicated in modulating microglial activation, neurodegeneration and disease progression in amyotrophic lateral sclerosis (ALS). Specifically, there is reduced thymic function and regulatory T cell (Treg) number in ALS patients and mutant superoxide dismutase 1 (SOD1) mice, while passive transfer of Tregs ameliorates disease in mutant SOD1 mice. Here, we assessed the effects of augmenting endogenous CD4+ T cell number by stimulating the thymus using surgical castration on the phenotype of transgenic SOD1G93A mice.
Male SOD1G93A mice were castrated or sham operated, and weight loss, disease onset and progression were examined. Thymus atrophy and blood CD4+, CD8+ and CD4+ FoxP3+ T cell numbers were determined by fluorescence activated cell sorting (FACS). Motor neuron counts, glial cell activation and androgen receptor (AR) expression in the spinal cord were investigated using immunohistochemistry and Western blotting. Differences between castrated and sham mice were analysed using an unpaired t test or one-way ANOVA.
Castration significantly increased thymus weight and total CD4+ T cell numbers in SOD1G93A mice, although Tregs levels were not affected. Despite this, disease onset and progression were similar in castrated and sham SOD1G93A mice. Castration did not affect motor neuron loss or astrocytic activation in spinal cords of SOD1G93A mice; however, microglial activation was reduced, specifically M1 microglia. We also show that AR is principally expressed in spinal motor neurons and progressively downregulated in spinal cords of SOD1G93A mice from disease onset which is further enhanced by castration.
These results demonstrate that increasing thymic function and CD4+ T cell number by castration confers no clinical benefit in mutant SOD1 mice, which may reflect an inability to stimulate neuroprotective Tregs. Nonetheless, castration decreases M1 microglial activation in the spinal cord without any clinical improvement and motor neuron rescue, in contrast to other approaches to suppress microglia in mutant SOD1 mice. Lastly, diminished AR expression in spinal motor neurons, which links to another motor neuron disorder, spinal bulbar muscular atrophy (SBMA), may contribute to ALS pathogenesis and suggests a common disease pathway in ALS and SBMA mediated by disruption of AR signalling in motor neurons.
ALS; SBMA; SOD1; T cells; Tregs; Androgen receptor
Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and to a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analyzed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 mice at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences were also considered. No changes of HSPB8, BAG3, and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analyzed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cord. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded proteins accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.
amyotrophic lateral sclerosis; motoneurons; autophagy; HSPB8; BAG3; BAG1; protein quality control
Background and Objectives
Existing data on the spatiotemporal expression patterns of a variety of galectins in murine atherosclerosis are limited. We investigated the expression levels of galectins, and their in vivo spatiotemporal expression patterns and statin responsiveness in the inflamed atherosclerotic plaques of apolipoprotein E (apoE)-/- mice.
Materials and Methods
Galectins expression patterns in aortic atherosclerotic plaques and serum galectin-3 levels were investigated in 26-week-old apoE-/- (n=6) and C57BL/6 mice (n=9). To investigate the spatial and temporal patterns of galectin-1 and galectin-3 in plaques, high-cholesterol diet-fed 26-week-old (n=12) and 36-week-old apoE-/- mice (n=6) were sacrificed and their aortas were examined for galectins' expression using immunoblot analysis and immunohistochemical stain. 36-week-old apoE-/- mice were treated with atorvastatin (n=3, 0.57 mg/kg/day) for the evaluation of its effect on aortic galectins' expression.
Immunoblot analyses showed that galectin-1 and galectin-3 were the predominant galectins expressed in murine atherosclerosis. The serum galectin-3 level was significantly higher in apoE-/- mice (p<0.001). While galectin-1 was weakly expressed in both intimal plaques and the media of atherosclerotic aortas, galectin-3 was heavily and exclusively accumulated in intimal plaques. Galectin-3 distribution was colocalized with plaque macrophages' distribution (r=0.66). As the degree of plaque extent and inflammation increased, the intraplaque galectin-3 expression levels proportionally elevated (p<0.01 vs. baseline), whereas galectin-1 expression had not elevated (p=0.14 vs. baseline). Atorvastatin treatment markedly reduced intraplaque galectin-3 and macrophage signals (p<0.001 vs. baseline), whereas it failed to reduce galectin-1 expression in the aortas.
Galectin-3 is the predominant gal and is colocalized with macrophages within atherosclerotic plaques. Intraplaque galectin-3 expression reflects the degree of plaque inflammation.
Galectin 1; Galectin 3; Atherosclerosis; Macrophages
Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery.
Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro.
Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS.
GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.
Amyotrophic lateral sclerosis; GCSF; pegfilgrastim; inflammation; monocytes; cytokines
Microglial neuroinflammation is thought to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). The purpose of this study was to provide a histopathological evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A transgenic rat.
Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptomatic, early symptomatic (onset), and late symptomatic (end stage), using immuno- and lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin B4.
Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early as the presymptomatic stage. During the symptomatic stages there was prominent formation of multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red nucleus of the midbrain. Other brain regions, including substantia nigra, cranial nerve nuclei, hippocampus and cortex showed normal appearing microglia. In animals during end stage disease at 4–5 months of age virtually all microglia in the spinal cord gray matter showed extensive fragmentation of their cytoplasm (cytorrhexis), indicative of widespread microglial degeneration. Few microglia exhibiting nuclear fragmentation (karyorrhexis) indicative of apoptosis were identified at any stage.
The current findings demonstrate the occurrence of severe abnormalities in microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant SOD1 in these cells. The microglial changes observed are different from those that accompany normal microglial activation, and they demonstrate that aberrant activation and degeneration of microglia is part of the pathogenesis of motor neuron disease.
Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection.
Methodology and Principal Findings
Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain.
Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.
Galectins are a family of endogenous lectins defined by a well-conserved carbohydrate recognition domain (CRD) that recognizes β-galactoside-related glycans presented by several glycoconjugates. Up to now, fifteen galectins have been identified in a variety of cells and tissues and proposed to be crucial in diverse biological processes. Galectin–1 (Gal–1), a prototype member of the galectin family, plays key roles in pathogen recognition and in the modulation of innate and adaptive host immune responses. Following infection with the intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, Gal–1 was found to be up-regulated in cardiac tissue from patients with chronic Chagas cardiomyopathy. In the present study, we identified a protective role of Gal–1 in T. cruzi infection of cardiac cells, highlighting the ability of this parasite to control the glycophenotype of these cells. Our data also disclose the relevance of parasite strain-dependent differences in Gal-1-mediated control of T. cruzi infection in vivo. The findings presented here will contribute to delineate the role of Gal-1-glycan interactions during T. cruzi infection, particularly in the context of heart tissue injury, with critical implications in Chagas disease.
Hallmarks of CNS inflammation, including microglial and astrocyte activation, are prominent features in post-mortem tissue from amyotrophic lateral sclerosis (ALS) patients and in mice overexpressing mutant superoxide dismutase-1 (SOD1G93A). Administration of non-targeted glucocorticoids does not significantly alter disease progression, but this may reflect poor CNS delivery. Here, we sought to discover whether CNS-targeted, liposomal encapsulated glucocorticoid would inhibit the CNS inflammatory response and reduce motor neuron loss. SOD1G93A mice were treated with saline, free methylprednisolone (MP, 10 mg/kg/week) or glutathione PEGylated liposomal MP (2B3-201, 10 mg/kg/week) and compared to saline treated wild-type animals. Animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. Weights and motor performance were monitored during this period. At the end of the experimental period (116 days) mice were imaged using T2-weighted MRI for brainstem pathology; brain and spinal cord tissue were then collected for histological analysis.
All SOD1G93A groups showed a significant decrease in motor performance, compared to baseline, from ~100 days. SOD1G93A animals showed a significant increase in signal intensity on T2 weighted MR images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. Treatment with 2B3-201, but not free MP, significantly reduced T2 hyperintensity observed in SOD1G93A mice. Compared to saline-treated and free-MP-treated SOD1G93A mice, those animals given 2B3-201 displayed significantly improved histopathological outcomes in brainstem motor nuclei, which included reduced gliosis and neuronal loss.
In contrast to previous reports that employed free steroid preparations, CNS-targeted anti-inflammatory agent 2B3-201 (liposomal methylprednisolone) has therapeutic potential, reducing brainstem pathology in the SOD1G93A mouse model of ALS. 2B3-201 reduced neuronal loss and vacuolation in brainstem nuclei, and reduced activation preferentially in astrocytes compared with microglia. These data also suggest that other previously ineffective therapies could be of therapeutic value if delivered specifically to the CNS.
SOD1G93A; Steroids; MRI; Astrocytes; Vacuolation
Components of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS); however, a comprehensive examination of complement expression in this disease has not been performed. This study therefore aimed to determine the expression of complement components (C1qB, C4, factor B, C3/C3b, C5 and CD88) and regulators (CD55 and CD59a) in the lumbar spinal cord of hSOD1G93A mice during defined disease stages.
hSOD1G93A and wild-type mice were examined at four different ages of disease progression. mRNA and protein expression of complement components and regulators were examined using quantitative PCR, western blotting and ELISA. Localisation of complement components within lumbar spinal cord was investigated using immunohistochemistry. Statistical differences between hSOD1G93A and wild-type mice were analysed using a two-tailed t-test at each stage of disease progression.
We found several early complement factors increased as disease progressed, whilst complement regulators decreased; suggesting overall increased complement activation through the classical or alternative pathways in hSOD1G93A mice. CD88 was also increased during disease progression, with immunolocalisation demonstrating expression on motor neurons and increasing expression on microglia surrounding the regions of motor neuron death.
These results indicate that local complement activation and increased expression of CD88 may contribute to motor neuron death and ALS pathology in the hSOD1G93A mouse. Hence, reducing complement-induced inflammation could be an important therapeutic strategy to treat ALS.
C1q; C4; Factor B; C3; C5; CD55; CD88; Motor neuron disease; Neuroinflammation
Retinal ischemia results in a progressive degeneration of neurons and a pathological activation of glial cells, resulting in vision loss. In the brain, progressive damage after ischemic insult has been correlated to neuroinflammatory processes involving microglia. Galectin-3 has been shown to mediate microglial responses to ischemic injury in the brain. Therefore, we wanted to explore the contribution of Galectin-3 (Gal-3) to hypoperfusion-induced retinal degeneration in mice.
Gal-3 knockout (Gal-3 KO) and wildtype (WT) C57BL/6 mice were subjected to chronic cerebral hypoperfusion by bilateral narrowing of the common carotid arteries using metal coils resulting in a 30% reduction of blood flow. Sham operated mice served as controls. After 17 weeks, the mice were sacrificed and the eyes were analyzed for retinal architecture, neuronal cell survival, and glial reactivity using morphological staining and immunohistochemistry.
Hypoperfusion caused a strong increase in Gal-3 expression and microglial activation in WT mice, coupled with severe degenerative damage to all retinal neuronal subtypes, remodeling of the retinal lamination and Müller cell gliosis. In contrast, hypoperfused Gal-3 KO mice displayed a retained laminar architecture, a significant preservation of photoreceptors and ganglion cell neurons, and an attenuation of microglial and Müller cell activation.
Moderate cerebral blood flow reduction in the mouse results in severe retinal degenerative damage. In mice lacking Gal-3 expression, pathological changes are significantly attenuated. Gal-3 is thereby a potential target for treatment and prevention of hypoperfusion-induced retinal degeneration and a strong candidate for further research as a factor behind retinal degenerative disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-015-0312-x) contains supplementary material, which is available to authorized users.
Retina; Microglia; Galectin 3; Müller cells; Neuroinflammation; Gliosis; Ischemia
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs). The molecular pathogenesis of ALS is not understood, thus effective therapies for this disease are lacking. Some forms of ALS are inherited by mutations in the superoxide dismutase-1 (SOD1) gene. Transgenic mice expressing human Gly93 → Ala (G93A) mutant SOD1 (mSOD1) develop severe MN disease, oxidative and nitrative damage, and mitochondrial pathology that appears to involve nitric oxide-mediated mechanisms. We used G93A-mSOD1 mice to test the hypothesis that the degeneration of MNs is associated with an aberrant up-regulation of the inducible form of nitric oxide synthase (iNOS or NOS2) activity within MNs. Western blotting and immunoprecipitation showed that iNOS protein levels in mitochondrial-enriched membrane fractions of spinal cord are increased significantly in mSOD1 mice at pre-symptomatic stages of disease. The catalytic activity of iNOS was also increased significantly in mitochondrial-enriched membrane fractions of mSOD1 mouse spinal cord at pre-symptomatic stages of disease. Reverse transcription-PCR showed that iNOS mRNA was present in the spinal cord and brainstem MN regions in mice and was increased in pre-symptomatic and early symptomatic mice. Immunohistochemistry showed that iNOS immunoreactivty was up-regulated first in spinal cord and brainstem MNs in pre-symptomatic and early symptomatic mice and then later in the course of disease in numerous microglia and few astrocytes. iNOS accumulated in the mitochondria in mSOD1 mouse MNs. iNOS immunoreactivity was also up-regulated in Schwann cells of peripheral nerves and was enriched particularly at the paranodal regions of the nodes of Ranvier. Drug inhibitors of iNOS delayed disease onset and significantly extended the lifespan of G93A-mSOD1 mice. This work identifies two new potential early mechanisms for MN degeneration in mouse ALS involving iNOS at MN mitochondria and Schwann cells and suggests that therapies targeting iNOS might be beneficial in treating human ALS.
Apoptosis-necrosis cell death continuum; Mitochondrial permeability transition pore; Mutant SOD1; Nitration; Node of Ranvier; Schwann cell
Lung cancer is a major cause of mortality and morbidity worldwide. Galectin-3 is multifunctional protein, which is involved in regulation of cell growth, cell adhesion, cell proliferation, angiogenesis and apoptosis. Cyclin D1 together with other cyclin plays an important role in cell cycle control. Cyclin D1 regulates the G1-to-S phase transition. The aim of this study was the evaluation of correlations between clinicopathological findings and cyclin D1 and galectin-3 expression in non-small cell lung cancer (NSCLC). We wanted also to analyze the prognostic value of cyclin D1 and galectin-3 expression. Moreover we tried to evaluate the correlations between galectin-3 and cyclin D1 expression in tumor tissue.
Materials and methods
We used the immunochemistry method to investigate the expression of galectin-3 and cyclin D1 in the paraffin-embedded tumor tissue of 47 patients (32 men and 15 women; mean age 59.34 ± 8.90). years. We used monoclonal antibodies to cyclin D1 (NCL-L-cyclin D1-GM clone P2D11F11 NOVO CASTRA) and to galectin-3 (mouse monoclonal antibody NCL-GAL3 NOVO CASTRA).
Galectin-3 expression was positive in 18 cases (38.29%) and cyclin D1 in 39 (82.97%). We showed only weak trend, that galectin-3 expression was lower in patients without lymph node involvement (p = 0.07) and cyclin D1 expression was higher in this group (p = 0.080). We didn't reveal differences in cyclin D1 and galectin-3 expression in SCC and adenocarcinoma patients. We didn't demonstrated also differences in galectin-3 and cyclin D1 expression depending on disease stage. Moreover we analyzed the prognostic value of cyclin D1 expression and galectin-3 in all examinated patients and separately in SCC and in adenocarcinoma and in all stages, but we didn't find any statistical differences. We demonstrated that in galectin-3 positive tumors cyclin D1 expression was higher (96.55% vs 61.11%, Chi2 Yatesa 7.53, p = 0.0061) and we revealed negative correlation between cyclin D1 and galectin-3 expression (R Spearman -0.458, p = 0.0011). In squamous cell lung cancer we didn't observed correlations between these both examinated markers (R = -0.158, p = 0.460), and in adenocarcinoma the negative correlation was very strong (R = -0.829 p = 0.000132).
We didn't reveal any important correlations between clinicopathological findings and galectin-3 and cyclin D1 expression and in non small cell lung cancer. We didn't observed also prognostic value of cyclin D1 or galectin-3 expression. But we showed higher cyclin D1 expression in galectin-3 negative tumor tissues. We revealed also differences in correlations between galectin-3 and cyclin D1 expression in two main histopathological types of NSCLC.
galectin-3; cyclin D1; non-small cell lung cancer; prognostic factor
galectin-1 has been implicated in tumor invasion and metastasis and is frequently over-expressed in epithelial ovarian cancer (EOC), but its potential as a biomarker remains unclear. In this novel study, we have explored the possible use of galectin-1 as a biomarker for EOC.
galectin-1 in sera was evaluated by ELISA in a pilot panel of EOC patients, healthy volunteers, patients with benign gynecologic tumors or other gynecologic malignancies. We examined galectin-1 expression in EOC tumor samples by Western Blot, qRT-PCR and immunohistochemistry. In vitro experiments were conducted to elucidate the biologic role of galectin-1 in EOC progression using over-expression of galectin-1 in OVCAR-3 cells. We also looked for the association of galectin-1 expression with clinic pathological variables and survival outcomes in EOC.
A significant difference was detected in serum galectin-1 between EOC patients with non-metastatic and those with metastatic disease, but not between EOC patients and healthy volunteers. It increased in recurrent cases and decreased after debulking surgery. Both of galectin-1 mRNA and protein levels were increased in 90 % of the examined EOC tissue samples, compared with a wedge resection of a normal ovary. High galectin-1 in peritumor stroma was primarily detected in advanced stages of EOC. Over expression of galectin-1 significantly increased the ability of OVCAR-3 cells’ migration and invasion.
Our results suggest that galectin-1 might play a role in tumor progression and be associated with poor outcome in EOC. It could be a novel prognostic and progression biomarker in EOC patients.
galectin-1; epithelial ovarian cancer; bio-marker; progression; serum
The role of neuroinflammation in motor neuron death of amyotrophic lateral sclerosis (ALS) is unclear. The human mutant superoxide dismutase-1 (hmSOD1)-expressing murine transgenic model of ALS has provided some insight into changes in microglia activity during disease progression. The purpose of this study was to gain further knowledge by characterizing the immunological changes during disease progression in the spinal cord and peripheral nerve using the more recently developed hmSOD1 rat transgenic model of ALS.
Using immunohistochemistry, the extent and intensity of tissue CD11b expression in spinal cord, lumbar nerve roots, and sciatic nerve were evaluated in hmSOD1 rats that were pre-clinical, at clinical onset, and near disease end-stage. Changes in CD11b expression were compared to the detection of MHC class II and CD68 microglial activation markers in the ventral horn of the spinal cord, as well as to the changes in astrocytic GFAP expression.
Our study reveals an accumulation of microglia/macrophages both in the spinal cord and peripheral nerve prior to clinical onset based on CD11b tissue expression. The microglia formed focal aggregates in the ventral horn and became more widespread as the disease progressed. Hypertrophic astrocytes were not prominent in the ventral horn until after clinical onset, and the enhancement of GFAP did not have a strong correlation to increased CD11b expression. Detection of MHC class II and CD68 expression was found in the ventral horn only after clinical onset. The macrophages in the ventral nerve root and sciatic nerve of hmSOD1 rats were observed encircling axons.
These findings describe for the first time in the hmSOD1 rat transgenic model of ALS that enhancement of microglia/macrophage activity occurs pre-clinically both in the peripheral nerve and in the spinal cord. CD11b expression is shown to be a superior indicator for early immunological changes compared to other microglia activation markers and astrogliosis. Furthermore, we suggest that the early activity of microglia/macrophages is involved in the early phase of motor neuron degeneration and propose that studies involving immunomodulation in hmSOD1transgenic models need to consider effects on macrophages in peripheral nerves as well as to microglia in the spinal cord.