Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4(+)CD25(High) T lymphocytes (Tregs) and cytotoxic CD4(+)CD25(-) T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4(+)CD25(High)IL-4(+), CD4(+)CD25(High)IL-10(+) and CD4(+)CD25(High)TGF-β(+) Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.     

Extracellular mutant SOD1 induces microglial‐mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1^G93A or mSOD1^G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1^G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1^G93A or mSOD1^G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1^G93A injure motoneurons. The microglial activation mediated by mSOD1^G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1^G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS. © 2009 Wiley‐Liss, Inc.     

Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS

Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1(G93A) motoneuron death through the Fas/NO pathway. In SOD1(G93A) mice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1(G93A) motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.     

Origin and distribution of bone marrow-derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.     

Impaired recruitment of neuroprotective microglia and T cells during acute neuronal injury coincides with increased neuronal vulnerability in an amyotrophic lateral sclerosis model

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 microglia and T cells play significant roles in disease progression. However, it remains unknown whether these cells are toxic or protective. The present study aimed to clarify the developmental age-related alterations of neuronal, glial and T cell responses to acute neuron injury in non-transgenic (N-Tg) mice, and the in vivo effects of mSOD1 on these changes by studying N-Tg and mSOD1-Tg mice subjected to unilateral hypoglossal nerve axotomy at young (8 weeks) and adult (17 weeks) ages. Adult N-Tg mice showed increased neuronal viability on day 21 after axotomy and trends toward increased numbers of recruited microglia on day 3 and T cells on day 7, in the hypoglossal nucleus, compared with young N-Tg mice. Quantitative comparisons between mSOD1-Tg and N-Tg mice at the same ages, on day 3 after axotomy, showed that microglial recruitment was significantly lower in mSOD1-Tg mice than in 17-week-old N-Tg mice (the disease progression stage), but the same difference was not seen in 8-week-old mice (the presymptomatic stage), despite good preservation of hypoglossal neurons. Infiltration of CD3-positive T cells, mostly CD4-positive, on day 7 and the viability rate of hypoglossal neurons on the operated side compared with the contralateral side on day 21 were significantly decreased in mSOD1-Tg mice compared with N-Tg mice aged 17 weeks, but the same difference was not seen in mice aged 8 weeks. On day 3 after axotomy, expression levels of IGF-1 mRNA in the operated hypoglossal nucleus were significantly lower in mSOD1-Tg mice than N-Tg mice at 17 weeks of age. The observation that depressed microglial and T cell responses and expression of neurotrophic factors coincided with reduced neuronal viability in adult mSOD1-Tg mice suggests that diminished neuroprotective functions of mSOD1 microglia and T cells may contribute to exaggerated neuronal death.     

Resveratrol Improves Motoneuron Function and Extends Survival in SOD1G93A ALS Mice

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that causes progressive paralysis and death due to degeneration of motoneurons in spinal cord, brainstem and motor cortex. Nowadays, there is no effective therapy and patients die 2–5 years after diagnosis. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural polyphenol found in grapes, with promising neuroprotective effects since it induces expression and activation of several neuroprotective pathways involving Sirtuin1 and AMPK. The objective of this work was to assess the effect of resveratrol administration on SOD1^G93A ALS mice. We determined the onset of symptoms by rotarod test and evaluated upper and lower motoneuron function using electrophysiological tests. We assessed the survival of the animals and determined the number of spinal motoneurons. Finally, we further investigated resveratrol mechanism of action by means of western blot and immunohistochemical analysis. Resveratrol treatment from 8 weeks of age significantly delayed disease onset and preserved lower and upper motoneuron function in female and male animals. Moreover, resveratrol significantly extended SOD1^G93A mice lifespan and promoted survival of spinal motoneurons. Delayed resveratrol administration from 12 weeks of age also improved spinal motoneuron function preservation and survival. Further experiments revealed that resveratrol protective effects were associated with increased expression and activation of Sirtuin 1 and AMPK in the ventral spinal cord. Both mediators promoted normalization of the autophagic flux and, more importantly, increased mitochondrial biogenesis in the SOD1^G93A spinal cord. Taken together, our findings suggest that resveratrol may represent a promising therapy for ALS.     

Ovariectomy and 17beta-estradiol modulate disease progression of a mouse model of ALS

The incidence of amyotrophic lateral sclerosis (ALS) is higher among men than women but rises in women after the menopause. Estrogens may play a protective role. Treatment with estrogens has been shown to be neuroprotective in models of several neurodegenerative diseases. We therefore determined the effect of ovariectomy on female G93A mSOD1 transgenic mice, and the effect of subsequent treatment with 17beta-estradiol (E2). Ovariectomy led to a significant acceleration of disease progression of the mice, and high-dose E2 treatment significantly delayed disease progression of ovariectomized G93A mSOD1 transgenic mice. We conclude that treatment with E2 may also delay disease progression of post-menopausal women with ALS.     

Local GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in SOD1(G93A) transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are associated with mutations in the cytosolic Cu/Zn superoxide dismutase 1 (SOD1) gene. Transgenic SOD1 mice constitute useful models of ALS to screen therapeutical approaches. Glial cell line-derived neurotrophic factor (GDNF) holds promises for the treatment of motoneuron disease. In the present study, GDNF expression in motoneurons of SOD1(G93A) transgenic mice was assessed by facial nucleus or intraspinal injection of lentiviral vectors (LV) encoding GDNF. We show that lentiviral vectors allow the expression for at least 12 weeks of GDNF that was clearly detected in motoneurons. This robust intraspinal expression did, however, not prevent the loss of motoneurons and muscle denervation of transgenic mice. In contrast, LV-GDNF induced a significant rescue of motoneurons in the facial nucleus and prevented motoneuron atrophy. The differential effect of GDNF on facial nucleus versus spinal motoneurons suggests different vulnerability of motoneurons in ALS.     

Increased expression of the pro-inflammatory enzyme cyclooxygenase-2 in amyotrophic lateral sclerosis

Mutations in the copper/zinc superoxide dismutase (mSOD1) gene are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Recent observations suggest a role for inflammatory-related events in the progression and propagation of the neurodegenerative process in ALS. Consistent with this view, the present study demonstrates that, during the course of the disease, the expression of cyclooxygenase type 2 (Cox-2), a key enzyme in the synthesis of prostanoids, which are potent mediators of inflammation, is dramatically increased. In both early symptomatic and end-stage transgenic mSOD1 mice, neurons and, to a lesser extent, glial cells in the anterior horn of the spinal cord exhibit robust Cox-2 immunoreactivity. Cox-2 mRNA and protein levels and catalytic activity are also significantly increased in the spinal cord of the transgenic mSOD1 mice. The time course of the spinal cord Cox-2 upregulation parallels that of motor neuronal loss in transgenic mSOD1 mice. We also show that Cox-2 activity is dramatically increased in postmortem spinal cord samples from sporadic ALS patients. We speculate that Cox-2 upregulation, through its pivotal role in inflammation, is instrumental in the ALS neurodegenerative process and that Cox-2 inhibition may be a valuable therapeutic avenue for the treatment of ALS.     

Involvement of activated microglia in increased vulnerability of motoneurons after facial nerve avulsion in presymptomatic amyotrophic lateral sclerosis model rats

Activated microglia are observed in various neurodegenerative diseases and are thought to be involved in the processes of neuronal cell death. Motoneuron damage in the facial nuclei after facial nerve avulsion is accelerated in presymptomatic transgenic rats expressing human mutant Cu(2+) /Zn(2+) superoxide dismutase 1 (SOD1), compared with that in wild-type rats. To reveal the functional role of microglia in motoneuronal death, we investigated the microglial response after facial nerve avulsion in presymptomatic mutant SOD1(H46R) (mSOD1(H46R) ) rats. At 3 days after avulsion, microglial clusters were observed in the facial nuclei of both wild-type and mSOD1(H46R) rats. The numbers of microglial clusters, proliferating microglia, and microglial attachments to motoneurons were significantly higher in mSOD1(H46R) rats, compared with those in wild-type rats. Immunopositive signals for the phagocytic marker ED1 were significantly stronger in mSOD1(H46R) rats, compared with that in wild-type rats, at 2 weeks after avulsion. Furthermore, primary microglia prepared from mSOD1(H46R) rats showed enhanced phagocytic activity, compared with that in wild-type rats. The expression of P2Y(12) mRNA was higher in the facial nuclei of mSOD1(H46R) rats, compared with that in wild-type rats. A laser microdissection system revealed that the expression of ATF3 mRNA was higher in the motoneurons of mSOD1(H46R) rats, compared with that in wild-type rats, at 2 days after avulsion. These results indicate that microglial activation in response to early neuronal damage increased in mSOD1(H46R) rats and suggest that the enhanced activation of microglia may lead to an increase in the vulnerability of motoneurons after avulsion in mSOD1(H46R) rats.     

Cellular changes in motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis as revealed by monoclonal antibody Py.

Transgenic mice (G93A) carrying the human amyotrophic lateral sclerosis (ALS) linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. The affected motoneurons are characterized by the presence of cellular alterations. The antigen recognized by the monoclonal antibody Py is suggested to be associated with the neurofilamentous and microtubular elements of the cytoskeleton of specific neuron populations including the spinal motoneurons. The aim of the present study was to measure changes in the relative Py-immunoreactivity per identified Choline-Acetyl-Transferase (ChAT)-immunoreactive motoneuron during the disease progression. The relative Py-immunoreactivity of identified spinal motoneurons was measured on double stained (Py and ChAT) motoneurons using a digital imaging system coupled to an inverse microscope. A significant decrease of Py-immunoreactivity was already noted in the pre-symptomatic stages of the disease even before the onset of massive motoneuron degeneration. It is concluded that the Py-antibody detects early intracellular abnormalities related to neurodegenerative changes in spinal motoneurons of transgenic SOD1-(G93A) mice.     

Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.     

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu²⁺/Zn²⁺ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1^H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage. © 2010 Wiley‐Liss, Inc.     

Malonate-induced cortico-motoneuron death is attenuated by NT-4, but not by BDNF or NT-3

Neurotrophins are promising candidates to slow the progression of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease in which spinal and cortical motoneurons selectively degenerate. In a long-term in vitro model, malonate-induced toxicity and cell death of motoneurons have been demonstrated. Here we studied the neuroprotective effect of BDNF, NT-3, and NT-4 on the cell death of cortical motoneurons in an organotypic culture model after chronic mitochondrial inhibition with malonate. Our data show that NT-4 completely prevents malonate-induced toxicity, whereas BDNF or NT-3 had no neuroprotective effect. In clinical trials for ALS, predominantly focussed on the survival of spinal motoneurons, BDNF has already been tested with disappointing results; our results suggest that NT-4 may be a better neurotrophin to prevent motoneuron loss.     

Ghrelin protects spinal cord motoneurons against chronic glutamate-induced excitotoxicity via ERK1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways

Excitotoxic degeneration of spinal cord motoneurons has been proposed as a pathogenic mechanism in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R) 1a, functions as a neuroprotective factor in various animal models of neurodegenerative diseases. In this study, the potential neuroprotective effects of ghrelin against chronic glutamate-induced cell death were studied by exposing organotypic spinal cord cultures (OSCC) to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration. Ghrelin receptor was expressed on spinal cord motoneurons. Exposure of OSCC to THA for 3 weeks resulted in a significant loss of motoneurons. However, THA-induced loss of motoneurons was significantly reduced by treatment of ghrelin. Exposure of OSCC to the receptor-specific antagonist D-Lys-3-GHRP-6 abolished the protective effect of ghrelin against THA. Treatment of spinal cord cultures with ghrelin caused rapid phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and glycogen synthase kinase-3β (GSK-3β). The effect of ghrelin on motoneuron survival was blocked by the MEK inhibitor PD98059 and the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. Taken together, these findings indicate that ghrelin has neuroprotective effects against chronic glutamate toxicity by activating the MAPK and PI3K/Akt signaling pathways and suggest that administration of ghrelin may have the potential therapeutic value for the prevention of motoneuron degeneration in human ALS. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β in motoneurons contributes to the protective effect of ghrelin.     

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1 mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1^G93A mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1^G93A mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.     

Muscle-derived but not centrally derived transgene GDNF is neuroprotective in G93A-SOD1 mouse model of ALS

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MNs), and is considered a potential agent for the treatment of amyotrophic lateral sclerosis (ALS) and other MN diseases. The effectiveness of GDNF may depend significantly upon its route of delivery to MNs. In this study we tested the neuroprotective effects of target-derived and centrally derived GDNF in the G93A-SOD1 mouse model of ALS using a transgenic approach. We found that overexpression of GDNF in the skeletal muscle (Myo-GDNF mice) significantly delayed the onset of disease and increased the life span of G93A-SOD1 mice by 17 days. The duration of disease also increased by 8.5 days, indicating that GDNF slowed down the progression of disease. Locomotor performance in Myo-GDNF/G93A-SOD1 mice was also significantly improved. The behavioral improvement correlated well with anatomical and histological data. We demonstrated that muscle-derived GDNF resulted in increased survival of spinal MNs, and twice as many MNs survived in end-stage double transgenic mice compared to end-stage G93A-SOD1 mice. Muscle-derived GDNF also had profound effects on muscle innervation and axonal degeneration. Significantly higher numbers of completely or partially innervated NMJs and large caliber myelinated axons were found in double transgenic mice. In contrast, we demonstrated that overexpression of GDNF in astrocytes in the CNS (GFAP-GDNF mice) failed to demonstrate any neuroprotective effects in G93A-SOD1 mice both on behavioral and histological levels. These data indicate that retrograde transport and signaling of GDNF is more physiological and effective for ALS treatment than anterogradely transported GDNF.