Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.
amyotrophic lateral sclerosis; interferon-γ; LIGHT; astrocytes; motoneurons
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 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 mSOD1G93A or mSOD1G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1G93A or mSOD1G85R did induce the morphological and functional activation of microglia, increasing their release of pro-inflammatory cytokines and free radicals. Furthermore, only when microglia were co-cultured with motoneurons did extracellular mSOD1G93A injure motoneurons. The microglial activation mediated by mSOD1G93A was attenuated using toll-like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1G93A 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.
mutant SOD1; CD14; Toll-like receptors; microglia; motoneurons
Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study we have examined, using whole-cell patch clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing eGFP driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP+ neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP+ motoneurons prepared from G93A-SOD1 embryos and from their non-transgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell RT-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.
Hb9-eGFP; mutant SOD1; motoneuron culture; GABAA receptor; patch clamp; single-cell RT-PCR
In amyotrophic lateral sclerosis (ALS) the large motoneurons that innervate the fast-contracting muscle fibers (F-type motoneurons) are vulnerable and degenerate in adulthood. In contrast, the small motoneurons that innervate the slow-contracting fibers (S-type motoneurons) are resistant and do not degenerate. Intrinsic hyperexcitability of F-type motoneurons during early postnatal development has long been hypothesized to contribute to neural degeneration in the adult. Here, we performed a critical test of this hypothesis by recording from identified F- and S-type motoneurons in the superoxide dismutase-1 mutant G93A (mSOD1), a mouse model of ALS at a neonatal age when early pathophysiological changes are observed. Contrary to the standard hypothesis, excitability of F-type motoneurons was unchanged in the mutant mice. Surprisingly, the S-type motoneurons of mSDO1 mice did display intrinsic hyperexcitability (lower rheobase, hyperpolarized spiking threshold). As S-type motoneurons are resistant in ALS, we conclude that early intrinsic hyperexcitability does not contribute to motoneuron degeneration.
Amyotrophic lateral sclerosis (ALS), which is also known as Lou Gherig's disease or motoneuron disease, is a neurodegenerative disorder in which muscles throughout the body gradually waste away due to the death of the neurons that control their activity. The disease often begins with weakness of the arms or legs, but progresses to include difficulties with movements such as swallowing and breathing. Around half of those affected die within 3 or 4 years of diagnosis.
Although the causes of the disease are unclear, one leading theory is that the neurons that control muscle activity—motoneurons—are hyperexcitable during early development, and therefore fire too frequently. This causes too much calcium to enter the neurons and, because calcium is toxic to cells in high quantities, leads ultimately to the death of the neurons. But despite the popularity of this idea, and the fact that many therapeutic assays for ALS are based on attempts to reverse this process, there is no direct evidence that early hyperexcitability of motoneurons causes their death in ALS.
Leroy et al. have now tested this theory directly by taking advantage of the fact that not all motoneurons are affected by ALS. The large ‘F-type’ motoneurons that control fast-contracting muscle fibres degenerate in ALS, whereas the small ‘S-type’ motoneurons that control slow-contracting muscle fibres do not. A comparison of F-type and S-type motoneurons in a mouse model of ALS revealed that, surprisingly, S-type motoneurons are hyperexcitable in young ALS mice, whereas F-type motoneurons are not.
Given that S-type motoneurons are resistant to the effects of ALS, this indicates that early hyperexcitability cannot be the cause of motoneuron degeneration. Previous studies have tended to pool different types of motoneurons together, which might explain why this difference has not been seen before. Further experiments are now required to determine whether the hyperexcitability of S-type motoneurons persists into adulthood, and whether it might even contribute to their survival in ALS.
ALS; hyperexcitability; motoneuron; mouse
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motoneurons. Recently we studied glycine receptors (GlyRs) in motoneurons in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). Living motoneurons in dissociated spinal cord cultures were identified by using transgenic mice expressing eGFP driven by the Hb9 promoter. We showed that GlyR-mediated currents were reduced in large-sized (diameter >28 µm) Hb9-eGFP+ motoneurons from G93A-SOD1 embryonic mice. Here we analyze GlyR currents in a morphologically distinct subgroup of medium-sized (diameter 10–28 µm) Hb9-eGFP+ motoneurons, presumably gamma or slow-type alpha motoneurons. We find that glycine-induced current densities were not altered in medium-sized G93A-SOD1 motoneurons. No significant differences in glycinergic mIPSCs were observed between G93A-SOD1 and control medium-sized motoneurons. These results indicate that GlyR deficiency early in the disease process of ALS is specific for large alpha motoneurons.
Hb9-eGFP; mutant SOD1; motoneuron culture; patch clamp; mIPSC; gamma motoneuron; alpha motoneuron
Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1G93A contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Nav) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1G93A and ACM-SOD1G86R) or TDP43 (ACM-TDP43A315T) mutants; we show that such exposure rapidly (within 30–60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Nav channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Nav channel activity.
ALS; non-cell-autonomous; motor neuron; degeneration; ROS/RNS; anti-oxidants
Neuroinflammation is a prominent pathological feature in the spinal cords of patients with amyotrophic lateral sclerosis (ALS), as well as in transgenic mouse models of inherited ALS, and is characterized by activated microglia. Earlier studies showed that activated microglia play important roles in both motoneuron protection and injury. More recent studies investigating the pathoprogression of disease in ALS mice have demonstrated that the in vivo activation states of microglia, including their anti- versus pro-inflammatory responses, are best characterized as a continuum between two extreme activation states which are represented as a neuro-protective M2 (alternatively-activated) phenotypic state or an injurious/toxic M1 (classically-activated) state; a more complete understanding and determination the temporal transformation of microglia activation states in the ALS disease pathoprogression is therefore warranted. In the current study, we demonstrated a phenotypic and functional transformation of adult ALS mice microglia that overexpress mutant superoxide dismutase (mSOD1). mSOD1 microglia isolated from ALS mice at disease onset expressed higher levels of Ym1, CD163 and BDNF (markers of M2) mRNA and lower levels of Nox2 (a marker of M1) mRNA compared with mSOD1 microglia isolated from ALS mice at end-stage disease. More importantly, when co-cultured with motoneurons, these mSOD1 M2 microglia were neuroprotective and enhanced motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to motoneurons. Our study documents that adult microglia isolated from ALS mice at disease onset have an M2 phenotype and protect motoneurons whereas microglia isolated from end-stage disease ALS mice have adopted an M1 phenotype and are neurotoxic supporting the dual pheno-types of microglia and their transformation during disease pathoprogression in these mice. Thus, harnessing the neuroprotective potential of microglia may provide novel avenues for ALS therapies.
Amyotrophic lateral sclerosis; Microglia; Motor neurons; Neuroprotection; Neurotoxicity
Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. It causes the degeneration of motoneurons and is fatal due to paralysis, particularly of respiratory muscles. ALS can be inherited, and specific disease-causing genes have been identified, but the mechanisms causing motoneuron death in ALS are not understood. No effective treatments exist for ALS. One well-studied theory of ALS pathogenesis involves faulty RNA editing and abnormal activation of specific glutamate receptors as well as failure of glutamate transport resulting in glutamate excitotoxicity; however, the excitotoxicity theory is challenged by the inability of anti-glutamate drugs to have major disease-modifying effects clinically. Nevertheless, hyperexcitability of upper and lower motoneurons is a feature of human ALS and transgenic (tg) mouse models of ALS. Motoneuron excitability is strongly modulated by synaptic inhibition mediated by presynaptic glycinergic and GABAergic innervations and postsynaptic glycine receptors (GlyR) and GABAA receptors; yet, the integrity of inhibitory systems regulating motoneurons has been understudied in experimental models, despite findings in human ALS suggesting that they may be affected. We have found in tg mice expressing a mutant form of human superoxide dismutase-1 (hSOD1) with a Gly93 → Ala substitution (G93A-hSOD1), causing familial ALS, that subsets of spinal interneurons degenerate. Inhibitory glycinergic innervation of spinal motoneurons becomes deficient before motoneuron degeneration is evident in G93A-hSOD1 mice. Motoneurons in these ALS mice also have insufficient synaptic inhibition as reflected by smaller GlyR currents, smaller GlyR clusters on their plasma membrane, and lower expression of GlyR1α mRNA compared to wild-type motoneurons. In contrast, GABAergic innervation of ALS mouse motoneurons and GABAA receptor function appear normal. Abnormal synaptic inhibition resulting from dysfunction of interneurons and motoneuron GlyRs is a new direction for unveiling mechanisms of ALS pathogenesis that could be relevant to new therapies for ALS.
Chloride channel; Glutamate receptor; Glycine receptor; Hb9-eGFP; Excitotoxicity; Hyperexcitability; Interneuron; Renshaw cell
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause, characterized by the selective and progressive death of both upper and lower motoneurons, leading to a progressive paralysis. Experimental animal models of the disease may provide knowledge of the pathophysiological mechanisms and allow the design and testing of therapeutic strategies, provided that they mimic as close as possible the symptoms and temporal progression of the human disease. The principal hypotheses proposed to explain the mechanisms of motoneuron degeneration have been studied mostly in models in vitro, such as primary cultures of fetal motoneurons, organotypic cultures of spinal cord sections from postnatal rodents and the motoneuron-like hybridoma cell line NSC-34. However, these models are flawed in the sense that they do not allow a direct correlation between motoneuron death and its physical consequences like paralysis. In vivo, the most widely used model is the transgenic mouse that bears a human mutant superoxide dismutase 1, the only known cause of ALS. The major disadvantage of this model is that it represents about 2%–3% of human ALS. In addition, there is a growing concern on the accuracy of these transgenic models and the extrapolations of the findings made in these animals to the clinics. Models of spontaneous motoneuron disease, like the wobbler and pmn mice, have been used aiming to understand the basic cellular mechanisms of motoneuron diseases, but these abnormalities are probably different from those occurring in ALS. Therefore, the design and testing of in vivo models of sporadic ALS, which accounts for >90% of the disease, is necessary. The main models of this type are based on the excitotoxic death of spinal motoneurons and might be useful even when there is no definitive demonstration that excitotoxicity is a cause of human ALS. Despite their difficulties, these models offer the best possibility to establish valid correlations between cellular alterations and motor behavior, although improvements are still necessary in order to produce a reliable and integrative model that accurately reproduces the cellular mechanisms of motoneuron degeneration in ALS.
During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.
We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.
Repeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.
Here we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.
Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.
motor neuron; SOD1G93A rat; microarray; hierarchical clustering; cranial nerves; cervical spinal cord; IGF-II
Amyotrophic lateral sclerosis (ALS) generally is a late-onset neurodegenerative disease. Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial ALS and 2% of all ALS cases. Although a number of hypotheses have been proposed to explain mutant SOD1 toxicity, the molecular mechanisms of the disease remain unclear. SOD1-linked ALS is thought to function in a non–cell-autonomous manner such that motoneurons are critical for the onset, and glia contribute to progression of the disease. Recently, it has been shown in Drosophila melanogaster that expression of human SOD1 in a subset of neuronal cells causes synaptic transmission defects, modified motor function, and altered sensitivity to compounds that induce oxidative stress. Here we used the Gal4-UAS (Upstream Activation Sequence) system to further characterize flies expressing wild-type Drosophila SOD1 (dSOD1) and the mutant human SOD1G85R (G85R) allele in motoneurons and glia. Cell-specific expression of both dSOD1 and G85R was found to influence lifespan, affect sensitivity to hydrogen peroxide, and alter lipid peroxidation levels. To better understand the genetic consequences of G85R expression in motoneurons and glia, we conducted microarray analysis of both young flies (5 days old) and old flies (45 days old) expressing G85R selectively in motoneurons or glia and concurrently in motoneurons and glia. Results from this microarray experiment identified candidate genes for further investigation and may help elucidate the individual and combined contributions of motoneurons and glia in ALS.
Drosophila; ALS; SOD1; glia; motoneuron
Expression of correct neurotransmitters is crucial for normal nervous system function. How neurotransmitter expression is regulated is not well-understood; however, previous studies provide evidence that both environmental signals and intrinsic differentiation programs are involved. One environmental signal known to regulate neurotransmitter expression in vertebrate motoneurons is Hepatocyte growth factor, which acts through the Met receptor tyrosine kinase and also affects other aspects of motoneuron differentiation, including axonal extension. Here we test the role of Met in development of motoneurons in embryonic zebrafish.
We found that met is expressed in all early developing, individually identified primary motoneurons and in at least some later developing secondary motoneurons. We used morpholino antisense oligonucleotides to knock down Met function and found that Met has distinct roles in primary and secondary motoneurons. Most secondary motoneurons were absent from met morpholino-injected embryos, suggesting that Met is required for their formation. We used chemical inhibitors to test several downstream pathways activated by Met and found that secondary motoneuron development may depend on the p38 and/or Akt pathways. In contrast, primary motoneurons were present in met morpholino-injected embryos. However, a significant fraction of them had truncated axons. Surprisingly, some CaPs in met morpholino antisense oligonucleotide (MO)-injected embryos developed a hybrid morphology in which they had both a peripheral axon innervating muscle and an interneuron-like axon within the spinal cord. In addition, in met MO-injected embryos primary motoneurons co-expressed mRNA encoding Choline acetyltransferase, the synthetic enzyme for their normal neurotransmitter, acetylcholine, and mRNA encoding Glutamate decarboxylase 1, the synthetic enzyme for GABA, a neurotransmitter never normally found in these motoneurons, but found in several types of interneurons. Our inhibitor studies suggest that Met function in primary motoneurons may be mediated through the MEK1/2 pathway.
We provide evidence that Met is necessary for normal development of zebrafish primary and secondary motoneurons. Despite their many similarities, our results show that these two motoneuron subtypes have different requirements for Met function during development, and raise the possibility that Met may act through different intracellular signaling cascades in primary and secondary motoneurons. Surprisingly, although met is not expressed in primary motoneurons until many hours after they have extended axons to and innervated their muscle targets, Met knockdown causes some of these cells to develop a hybrid phenotype in which they co-expressed motoneuron and interneuron neurotransmitters and have both peripheral and central axons.
Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1G93A animals.
Presymptomatic SOD1G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.
These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive loss of motoneurons, motor weakness and death within 1–5 years after disease onset. Therapeutic options remain limited despite a substantial number of approaches that have been tested clinically. In particular, various neurotrophic factors have been investigated. Failure in these trials has been largely ascribed to problems of insufficient dosing or inability to cross the blood–brain barrier (BBB). We have recently uncovered the neurotrophic properties of the haematopoietic protein granulocyte-colony stimulating factor (G-CSF). The protein is clinically well tolerated and crosses the intact BBB. This study examined the potential role of G-CSF in motoneuron diseases. We investigated the expression of the G-CSF receptor in motoneurons and studied effects of G-CSF in a motoneuron cell line and in the SOD1(G93A) transgenic mouse model. The neurotrophic growth factor was applied both by continuous subcutaneous delivery and CNS-targeted transgenic overexpression. This study shows that given at the stage of the disease where muscle denervation is already evident, G-CSF leads to significant improvement in motor performance, delays the onset of severe motor impairment and prolongs overall survival of SOD1(G93A)tg mice. The G-CSF receptor is expressed by motoneurons and G-CSF protects cultured motoneuronal cells from apoptosis. In ALS mice, G-CSF increased survival of motoneurons and decreased muscular denervation atrophy. We conclude that G-CSF is a novel neurotrophic factor for motoneurons that is an attractive and feasible drug candidate for the treatment of ALS.
ALS; growth factor; drug candidate; functional outcome; motoneuron survival
Granulocyte colony stimulating factor (G-CSF) is a growth factor essential for generation of neutrophilic granulocytes. Apart from this hematopoietic function, we have recently uncovered potent neuroprotective and regenerative properties of G-CSF in the central nervous system (CNS). The G-CSF receptor and G-CSF itself are expressed in α motoneurons, G-CSF protects motoneurons, and improves outcome in the SOD1(G93A) transgenic mouse model for amyotrophic lateral sclerosis (ALS). In vitro, G-CSF acts anti-apoptotically on motoneuronal cells. Due to the pleiotrophic effects of G-CSF and the complexity of the SOD1 transgenic ALS models it was however not possible to clearly distinguish between directly mediated anti-apoptotic and indirectly protective effects on motoneurons. Here we studied whether G-CSF is able to protect motoneurons from purely apoptotic cell death induced by a monocausal paradigm, neonatal sciatic nerve axotomy.
We performed sciatic nerve axotomy in neonatal mice overexpressing G-CSF in the CNS and found that G-CSF transgenic mice displayed significantly higher numbers of surviving lumbar motoneurons 4 days following axotomy than their littermate controls. Also, surviving motoneurons in G-CSF overexpressing animals were larger, suggesting additional trophic effects of this growth factor.
In this model of pure apoptotic cell death the protective effects of G-CSF indicate direct actions of G-CSF on motoneurons in vivo. This shows that G-CSF exerts potent anti-apoptotic activities towards motoneurons in vivo and suggests that the protection offered by G-CSF in ALS mouse models is due to its direct neuroprotective activity.
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
Tumor necrosis factor-alpha (TNF-α) may cause apoptosis and inflammation in amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI). Recent studies suggest that estrogen (EST) provides neuroprotection against SCI. We tested whether 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (EST receptor alpha (ERα) agonist), 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) (EST receptor beta (ERβ) agonist), or EST itself would prevent apoptosis in VSC4.1 motoneurons following exposure to TNF-α. Cells were exposed to TNF-α and 15 min later treated with PPT, DPN, or EST. Posttreatment with 50 nM PPT, 50 nM DPN, or 150 nM EST prevented cell death in VSC4.1 motoneurons. Treatment of VSC4.1 motoneurons with PPT, DPN, or EST induced overexpression of ERα, ERβ, or both, which contributed to neuroprotection by upregulating expression of anti-apoptotic proteins (p-AKT, p-CREB, Bcl-2, and p-Src). Our analyses also revealed that EST agonists and EST increased phosphorylation of extracellular signal-regulated kinase (ERK). The L-type Ca2+ channel inhibitor, nifedipine (10 μM), partially inhibited EST agonist and EST-induced increase in phosphorylated ERK expression. The mitogen-activated protein kinase inhibitor, PD98059 (5 μM), partially prevented ER agonists and EST from providing neuroprotection to TNF-α toxicity. Presence of the nuclear ER antagonist, ICI 182 780 (10 μM), blocked the neuroprotection provided by all three ER agonists tested. Taken together, our data indicate that both ERα and ERβ contribute to PPT, DPN, or EST-mediated neuroprotection with similar signaling profiles. Our data strongly imply that PPT, DPN, or EST can be used as effective neuroprotective agents to attenuate motoneuron death in ALS and SCI.
Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.
amyotrophic lateral sclerosis; autophagy; motoneuron diseases; muscle cells; proteasome; SOD1
Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.
Ghrelin; Neuroinflammation; Microglial activation; Excitotoxicity; Motoneuron
Vascular endothelial growth factor (VEGF), originally described as a factor with a regulatory role in vascular growth and development, it is also known for its direct effects on neuronal cells. The discovery in the past decade that transgenic mice expressing reduced levels of VEGF developed late-onset motoneuron pathology, reminiscent of amyotrophic lateral sclerosis (ALS), opened a new field of research on this disease. VEGF has been shown to protect motoneurons from excitotoxic death, which is a relevant mechanism involved in motoneuron degeneration in ALS. Thus, VEGF delays motoneuron degeneration and increases survival in animal models of ALS. VEGF exerts its anti-excitotoxic effects on motoneurons through molecular mechanisms involving the VEGF receptor-2 resulting in the activation of the PI3-K/Akt signaling pathway, upregulation of GluR2 subunit of AMPA receptors, inhibition of p38MAPK, and induction of the anti-apoptotic molecule Bcl-2. In addition, VEGF acts on astrocytes to reduce astroglial activation and to induce the release of growth factors. The potential use of VEGF as a therapeutic tool in ALS is counteracted by its vascular effects and by its short effective time frame. More studies are needed to assess the optimal isoform, route of administration, and time frame for using VEGF in the treatment of ALS.
VEGF; motoneuron; ALS; AMPA receptors; excitotoxicity; Akt
Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1G93A ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders.
Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motoneuron death. Mutations in the gene for superoxide dismutase 1 (SOD1) cause a familial form of ALS and have been used to develop transgenic mice which overexpress human mutant SOD1 (mSOD) and these mice exhibit a motoneuron disease which is pathologically and phenotypically similar to ALS. Neuroinflammation is a pathological hallmark of many neurodegenerative diseases including ALS and is typified by the activation and proliferation of microglia and the infiltration of T cells into the brain and spinal cord. Although the neuroinflammatory response has been considered a consequence of neuronal dysfunction and death, evidence indicates that manipulation of this response can alter disease progression. Previously viewed as deleterious to neuronal survival, recent reports suggest a trophic role for activated microglia in the mSOD mouse during the early stages of disease that is dependent on instructive signals from infiltrating T cells. However, at advanced stages of disease, activated microglia acquire increased neurotoxic potential, warranting further investigation into factors capable of skewing microglial activation towards a neurotrophic phenotype as a means of therapeutic intervention in ALS.
The authors have previously demonstrated a neuroprotective mechanism of facial motoneuron (FMN) survival after facial nerve transection that is dependent on CD4+T helper 2 (Th2) cell interactions with peripheral antigen presenting cells, as well as central nervous system (CNS) resident microglia. Pituitary adenylyl cyclase activating polypeptide is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these data suggest a model involving CD4+ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. In this study, the authors tested the hypothesis that Th2-associated chemokine expression occurs in the facial motor nucleus after facial nerve axotomy at the stylomastoid foramen. Initial microarray analysis of Th2-associated and Th1-associated chemokine mRNA levels was accomplished after facial nerve axotomy in wild type (WT) and presymptomatic mutant superoxide dismutase 1 (mSOD1) [model of familial amyotrophic lateral sclerosis (ALS)] mice. Based on that initial microarray analysis, the Th2-associated chemokine, CCL11, and Th1-associated chemokine, CXCL11, were further analyzed by RT-PCR. The results indicate that facial nerve injury predominantly increases Th2-associated chemokine, but not Th1-associated chemokine mRNA levels in the mouse facial motor nucleus. Interestingly, no differences were detected between WT and mSOD1 mice for CCL11 and CXCL11 after injury. These data provide a basis for further investigation into Th2-associated chemokine expression in the facial motor nucleus after FMN injury, which may lead to more specifically targeted therapeutics in motoneuron diseases, such as ALS.
CCL11; CXCL11; Neuroprotection; Chemokine