ABSTRACT
Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Disease Models, Animal , Indoles , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal , Pyrroles , STAT3 Transcription Factor , Spinal Cord , Sunitinib , Animals , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/pathology , Sunitinib/pharmacology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Indoles/pharmacology , Mice , Spinal Cord/metabolism , Spinal Cord/drug effects , Spinal Cord/pathology , Pyrroles/pharmacology , Superoxide Dismutase/metabolism , Superoxide Dismutase/genetics , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Motor Neurons/drug effects , Motor Neurons/metabolism , Motor Neurons/pathology , Disease ProgressionABSTRACT
BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options. RNS60 is an immunomodulatory and neuroprotective investigational product that has shown efficacy in animal models of ALS and other neurodegenerative diseases. Its administration has been safe and well tolerated in ALS subjects in previous early phase trials. METHODS: This was a phase II, multicentre, randomized, double-blind, placebo-controlled, parallel-group trial. Participants diagnosed with definite, probable or probable laboratory-supported ALS were assigned to receive RNS60 or placebo administered for 24 weeks intravenously (375 ml) once a week and via nebulization (4 ml/day) on non-infusion days, followed by an additional 24 weeks off-treatment. The primary objective was to measure the effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood. Secondary objectives were to measure the effect of RNS60 on functional impairment (ALS Functional Rating Scale-Revised), a measure of self-sufficiency, respiratory function (forced vital capacity, FVC), quality of life (ALS Assessment Questionnaire-40, ALSAQ-40) and survival. Tolerability and safety were assessed. RESULTS: Seventy-four participants were assigned to RNS60 and 73 to placebo. Assessed biomarkers did not differ between arms. The mean rate of decline in FVC and the eating and drinking domain of ALSAQ-40 was slower in the RNS60 arm (FVC, difference 0.41 per week, standard error 0.16, p = 0.0101; ALSAQ-40, difference -0.19 per week, standard error 0.10, p = 0.0319). Adverse events were similar in the two arms. In a post hoc analysis, neurofilament light chain increased over time in bulbar onset placebo participants whilst remaining stable in those treated with RNS60. CONCLUSIONS: The positive effects of RNS60 on selected measures of respiratory and bulbar function warrant further investigation.
Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Humans , Amyotrophic Lateral Sclerosis/diagnosis , Quality of Life , Double-Blind Method , Biomarkers , Treatment OutcomeABSTRACT
BACKGROUND: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis. METHODS: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy. RESULTS: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long-lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26-bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth. CONCLUSIONS: Overall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97-Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.
Subject(s)
Adenosine Triphosphatases , Amyotrophic Lateral Sclerosis , Muscular Atrophy , Neoplasms , Nuclear Proteins , Adenosine Triphosphatases/genetics , Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/pathology , Animals , Cachexia/pathology , Disease Models, Animal , Humans , Membrane Proteins , Mice , Muscular Atrophy/pathology , Neoplasms/complications , Neoplasms/pathology , Nuclear Proteins/genetics , RNA, Messenger/genetics , Superoxide Dismutase-1ABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Hepatocyte Growth Factor/agonists , Immune System , Neurons/cytology , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/immunology , Animals , Astrocytes/cytology , Astrocytes/metabolism , Behavior, Animal , Cell Survival , Coculture Techniques , Disease Models, Animal , Disease Progression , Dogs , Extracellular Signal-Regulated MAP Kinases/metabolism , Gliosis/metabolism , Humans , Interleukin-4/metabolism , Kringles , Ligands , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Motor Neurons/metabolism , Neurons/metabolism , Schwann Cells/metabolism , Spinal Cord/metabolism , T-Lymphocytes/cytologyABSTRACT
The rate of disease progression in amyotrophic lateral sclerosis (ALS) is highly variable, even between patients with the same genetic mutations. Metabolic alterations may affect disease course variability in ALS patients, but challenges in identifying the preclinical and early phases of the disease limit our understanding of molecular mechanisms underlying differences in the rate of disease progression. We examined effects of SOD1G93A on thoracic and lumbar spinal cord metabolites in two mouse ALS models with different rates of disease progression: the transgenic SOD1G93A-C57BL/6JOlaHsd (C57-G93A, slow progression) and transgenic SOD1G93A-129SvHsd (129S-G93A, fast progression) strains. Samples from three timepoints (presymptomatic, disease onset, and late stage disease) were analyzed using Gas Chromatography-Mass Spectrometry metabolomics. Tissue metabolome differences in the lumbar spinal cord were driven primarily by mouse genetic background, although larger responses were observed in metabolic trajectories after the onset of symptoms. The significantly affected lumbar spinal cord metabolites were involved in energy and lipid metabolism. In the thoracic spinal cord, metabolic differences related to genetic background, background-SOD1 genotype interactions, and longitudinal SOD1G93A effects. The largest responses in thoracic spinal cord metabolic trajectories related to SOD1G93A effects before onset of visible symptoms. More metabolites were significantly affected in the thoracic segment, which were involved in energy homeostasis, neurotransmitter synthesis and utilization, and the oxidative stress response. We find evidence that initial metabolic alterations in SOD1G93A mice confer disadvantages for maintaining neuronal viability under ALS-related stressors, with slow-progressing C57-G93A mice potentially having more favorable spinal cord bioenergetic profiles than 129S-G93A. These genetic background-associated metabolic differences together with the different early metabolic responses underscore the need to better characterize the impact of germline genetic variation on cellular responses to ALS gene mutations both before and after the onset of symptoms in order to understand their impact on disease development.
ABSTRACT
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the motor neuromuscular system leading to complete paralysis and premature death. The multifactorial nature of ALS that involves both cell-autonomous and non-cell-autonomous processes contributes to the lack of effective therapies, usually targeted to a single pathogenic mechanism. RNS60, an experimental drug containing oxygenated nanobubbles generated by modified Taylor-Couette-Poiseuille flow with elevated oxygen pressure, has shown anti-inflammatory and neuroprotective properties in different experimental paradigms. Since RNS60 interferes with multiple cellular mechanisms known to be involved in ALS pathology, we evaluated its effect in in vitro and in vivo models of ALS. METHODS: Co-cultures of primary microglia/spinal neurons exposed to LPS and astrocytes/spinal neurons from SOD1G93A mice were used to examine the effect of RNS60 or normal saline (NS) on the selective motor neuron degeneration. Transgenic SOD1G93A mice were treated with RNS60 or NS (300 µl/mouse intraperitoneally every other day) starting at the disease onset and examined for disease progression as well as pathological and biochemical alterations. RESULTS: RNS60 protected motor neurons in in vitro paradigms and slowed the disease progression of C57BL/6-SOD1G93A mice through a significant protection of spinal motor neurons and neuromuscular junctions. This was mediated by the (i) activation of an antioxidant response and generation of an anti-inflammatory environment in the spinal cord; (ii) activation of the PI3K-Akt pro-survival pathway in the spinal cord and sciatic nerves; (iii) reduced demyelination of the sciatic nerves; and (iv) elevation of peripheral CD4+/Foxp3+ T regulatory cell numbers. RNS60 did not show the same effects in 129Sv-SOD1G93A mice, which are unable to activate a protective immune response. CONCLUSION: RNS60 demonstrated significant therapeutic efficacy in C57BL/6-SOD1G93A mice by virtue of its effects on multiple disease mechanisms in motor neurons, glial cells, and peripheral immune cells. These findings, together with the excellent clinical safety profile, make RNS60 a promising candidate for ALS therapy and support further studies to unravel its molecular mechanism of action. In addition, the differences in efficacy of RNS60 in SOD1G93A mice of different strains may be relevant for identifying potential markers to predict efficacy in clinical trials.
Subject(s)
Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/pathology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Neuroglia/drug effects , Peripheral Nervous System Diseases/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Animals , Calcium-Binding Proteins/metabolism , Cells, Cultured , Coculture Techniques , Disease Models, Animal , Embryo, Mammalian , Glial Fibrillary Acidic Protein/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Motor Disorders/drug therapy , Motor Disorders/etiology , Motor Neurons/drug effects , Neuromuscular Junction/drug effects , Neuromuscular Junction/pathology , Neuronal Outgrowth/drug effects , Peripheral Nervous System Diseases/etiology , Signal Transduction/drug effects , Signal Transduction/genetics , Sodium Chloride/therapeutic use , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1ß) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression.
Subject(s)
Amyotrophic Lateral Sclerosis/therapy , Mesenchymal Stem Cell Transplantation , Motor Neurons/cytology , Superoxide Dismutase-1/genetics , Umbilical Cord/transplantation , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Animals , Female , Humans , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/metabolism , Point Mutation , Superoxide Dismutase-1/metabolism , Umbilical Cord/cytology , Umbilical Cord/metabolism , Umbilical Cord/ultrastructureABSTRACT
Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNFα is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNFα levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNFα toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNFα is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNFα in ALS in the light of its multisystem nature.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , T-Lymphocytes, Regulatory/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/pathology , Animals , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , T-Lymphocytes, Regulatory/immunologyABSTRACT
Increased intracellular calcium (Ca), which might be the consequence of an excess influx through Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, plays a crucial role in degeneration of motor neurons. Previously we demonstrated that the presymptomatic application of AMPA receptor antagonist, talampanel, could reduce Ca elevation in spinal motor neurons of mice carrying the G93A mutation of superoxide dismutase 1 (SOD1), modeling amyotrophic lateral sclerosis (ALS). It remained to be examined whether the remote, functionally semi-autonomous motor axon terminals could be rescued from the Ca overload, or if the terminals, where the degeneration possibly starts, already experience intractable changes at early time points. Thus using electron microscopic techniques, we measured the Ca level of motor axon terminals in the interosseus muscle of the SOD1 mutant animals, which are prototypes of vulnerable nerve endings in ALS. In line with the results obtained in the perikarya, talampanel treatment could reduce Ca increase evoked by the presence of mutant SOD1 in the axon terminals if the treatment was started presymptomatically but not at an early symptomatic stage. We also tested the Ca level in the cell bodies and axon terminals of the oculomotor neurons, which are resistant to the disease. Neither Ca increase, nor talampanel effect could be demonstrated at either time point. This is consistent with the observations that oculomotor neurons contain increased level of Ca buffer, which could reduce excess Ca load, and they also express glutamate receptor subunit type 2, which renders AMPA receptors impermeable to Ca.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Benzodiazepines/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Presynaptic Terminals/metabolism , Receptors, AMPA/antagonists & inhibitors , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Presynaptic Terminals/pathology , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
Non-cell autonomous processes involving astrocytes have been shown to contribute to motor neuron degeneration in amyotrophic lateral sclerosis. Mutant superoxide dismutase 1 (SOD1G93A) expression in astrocytes is selectively toxic to motor neurons in co-culture, even when mutant protein is expressed only in astrocytes and not in neurons. To examine metabolic changes in astrocyte-spinal neuron co-cultures, we carried out metabolomic analysis by 1H NMR spectroscopy of media from astrocyte-spinal neuron co-cultures and astrocyte-only cultures. We observed increased glucose uptake with SOD1G93A expression in all co-cultures, but while co-cultures with only SOD1G93A neurons had lower extracellular lactate, those with only SOD1G93A astrocytes exhibited the reverse. Reduced branched-chain amino acid uptake and increased accumulation of 3-methyl-2-oxovalerate were observed in co-culture with only SOD1G93A neurons while glutamate was reduced in all co-cultures expressing SOD1G93A. The shifts in these coupled processes suggest a potential block in glutamate processing that may impact motor neuron survival. We also observed metabolic alterations which may relate to oxidative stress responses. Overall, the different metabolite changes observed with the two SOD1G93A cell types highlight the role of the astrocyte-motor neuron interaction in the resulting metabolic phenotype, requiring further examination of altered met abolic pathways and their impact on motor neuron survival.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Astrocytes/metabolism , Motor Neurons/metabolism , Superoxide Dismutase-1/metabolism , Animals , Coculture Techniques , Disease Models, Animal , Glucose/metabolism , Glutamic Acid/metabolism , Metabolome , Mice , Oxidative StressABSTRACT
Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Cyclophilin A/metabolism , Disease Models, Animal , Extracellular Fluid/metabolism , Inflammation Mediators/metabolism , Adult , Aged , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/mortality , Animals , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Coculture Techniques , Cyclophilin A/antagonists & inhibitors , Drug Delivery Systems/methods , Enzyme Inhibitors/administration & dosage , Extracellular Fluid/drug effects , Female , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation Mediators/antagonists & inhibitors , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Neurons/drug effects , Neurons/metabolism , Survival Rate/trendsABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous disease in terms of progression rate and survival. This is probably one of the reasons for the failure of many clinical trials and the lack of effective therapies. Similar variability is also seen in SOD1(G93A) mouse models based on their genetic background. For example, when the SOD1(G93A) transgene is expressed in C57BL6 background the phenotype is mild with slower disease progression than in the 129Sv mice expressing the same amount of transgene but showing faster progression and shorter lifespan. This review summarizes and discusses data obtained from the analysis of these two mouse models under different aspects such as the motor phenotype, neuropathological alterations in the central nervous system (CNS) and peripheral nervous system (PNS) and the motor neuron autonomous and non-cell autonomous mechanisms with the aim of finding elements to explain the different rates of disease progression. We also discuss the identification of promising prognostic biomarkers by comparative analysis of the two ALS mouse models. This analysis might possibly suggest new strategies for effective therapeutic intervention in ALS to slow significantly or even block the course of the disease.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/physiopathology , Disease Models, Animal , Superoxide Dismutase-1/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Animals , Disease Progression , Humans , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Superoxide Dismutase-1/geneticsABSTRACT
The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.
Subject(s)
Amyotrophic Lateral Sclerosis/therapy , Cell Tracking , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Staining and Labeling , Umbilical Cord/cytology , Amyotrophic Lateral Sclerosis/pathology , Animals , Cell Size , Disease Models, Animal , Humans , Injections, Intravenous , Injections, Intraventricular , Mice, Inbred C57BL , Mice, Transgenic , Organ Specificity , Tissue DistributionABSTRACT
Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , Receptors, Tumor Necrosis Factor, Type II/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , Astrocytes/metabolism , Axons/metabolism , Cell Death/physiology , Cells, Cultured , Coculture Techniques , DNA-Binding Proteins/metabolism , Disease Models, Animal , Disease Progression , Mice , Neuroglia/metabolism , Receptors, Tumor Necrosis Factor, Type II/deficiencyABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease caused by the degeneration of motor neurons. The transgenic mouse model carrying the human SOD1G93A mutant gene (hSOD1G93A mouse) represents one of the most reliable and widely used model of this pathology. In the present work, the innovative technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was applied in the study of pathological alterations at the level of small brain regions such as facial and trigeminal nuclei, which in rodents are extremely small and would be difficult to analyze with classical proteomics approaches. Comparing slices from three mice groups (transgenic hSOD1G93A, transgenic hSOD1WT, and nontransgenic, Ntg), this technique allowed us to evidence the accumulation of hSOD1G93A in the facial and trigeminal nuclei, where it generates aggregates. This phenomenon is likely to be correlated to the degeneration observed in these regions. Moreover, a statistical analysis allowed us to highlight other proteins as differentially expressed among the three mice groups analyzed. Some of them were identified by reverse-phase HPLC fractionation of extracted proteins and mass spectrometric analysis before and after trypsin digestion. In particular, the 40S ribosomal protein S19 (RPS19) was upregulated in the parenkyma and reactive glial cells in facial nuclei of hSOD1G93A mice when compared to transgenic hSOD1WT and nontransgenic ones.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Brain Chemistry/genetics , Molecular Imaging/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Superoxide Dismutase/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Tissue DistributionABSTRACT
Evidence is accumulating that an imbalance between pathways for degeneration or survival in motor neurons may play a central role in mechanisms that lead to neurodegeneration in amyotrophic lateral sclerosis (ALS). We and other groups have observed that downregulation, or lack of induction, of the PI3K/Akt prosurvival pathway may be responsible for defective response of motor neurons to injury and their consequent cellular demise. Some of the neuroprotective effects mediated by growth factors may involve activation of Akt, but a proof of concept of Akt as a target for therapy is lacking. We demonstrate that specific expression of constitutively activated Akt3 in motor neurons through the use of the promoter of homeobox gene Hb9 prevents neuronal loss induced by SOD1.G93A both in vitro (in mixed neuron/astrocyte cocultures) and in vivo (in a mouse model of ALS). Inhibition of ASK1 and GSK3beta was involved in the neuroprotective effects of activated Akt3, further supporting the hypothesis that induction of Akt3 may be a key step in activation of pathways for survival in the attempt to counteract motor neuronal degeneration in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Disease Models, Animal , Motor Neurons/enzymology , Proto-Oncogene Proteins c-akt/biosynthesis , Spinal Cord/enzymology , Amyotrophic Lateral Sclerosis/pathology , Animals , Cell Survival/genetics , Coculture Techniques , Enzyme Induction/genetics , Female , HEK293 Cells , Humans , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/pathology , Neuroprotective Agents/metabolism , Proto-Oncogene Proteins c-akt/genetics , Spinal Cord/pathologyABSTRACT
Oxidative stress and mitochondrial impairment are the main pathogenic mechanisms of Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease still lacking of effective therapy. Recently, the coenzyme-Q (CoQ) complex, a key component of mitochondrial function and redox-state modulator, has raised interest for ALS treatment. However, while the oxidized form ubiquinone10 was ineffective in ALS patients and modestly effective in mouse models of ALS, no evidence was reported on the effect of the reduced form ubiquinol10, which has better bioavailability and antioxidant properties. In this study we compared the effects of ubiquinone10 and a new stabilized formulation of ubiquinol10 on the disease course of SOD1(G93A) transgenic mice, an experimental model of fALS. Chronic treatments (800 mg/kg/day orally) started from the onset of disease until death, to mimic the clinical trials that only include patients with definite ALS symptoms. Although the plasma levels of CoQ10 were significantly increased by both treatments (from <0.20 to 3.0-3.4 µg/mL), no effect was found on the disease progression and survival of SOD1(G93A) mice. The levels of CoQ10 in the brain and spinal cord of ubiquinone10- or ubiquinol10-treated mice were only slightly higher (≤10%) than the endogenous levels in vehicle-treated mice, indicating poor CNS availability after oral dosing and possibly explaining the lack of pharmacological effects. To further examine this issue, we measured the oxidized and reduced forms of CoQ9/10 in the plasma, brain and spinal cord of symptomatic SOD1(G93A) mice, in comparison with age-matched SOD1(WT). Levels of ubiquinol9/10, but not ubiquinone9/10, were significantly higher in the CNS, but not in plasma, of SOD1(G93A) mice, suggesting that CoQ redox system might participate in the mechanisms trying to counteract the pathology progression. Therefore, the very low increases of CoQ10 induced by oral treatments in CNS might be not sufficient to provide significant neuroprotection in SOD1(G93A) mice.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Central Nervous System/metabolism , Ubiquinone/analogs & derivatives , Amyotrophic Lateral Sclerosis/blood , Amyotrophic Lateral Sclerosis/pathology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Central Nervous System/drug effects , Central Nervous System/pathology , Disease Models, Animal , Disease Progression , Humans , Mice , Mice, Transgenic , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase/metabolism , Ubiquinone/administration & dosage , Ubiquinone/blood , Ubiquinone/metabolism , Ubiquinone/pharmacologyABSTRACT
Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.
Subject(s)
Biocompatible Materials/pharmacology , Cell Tracking/methods , Nanoparticles/chemistry , Stem Cells/cytology , Animals , Biomarkers/metabolism , Endocytosis/drug effects , Flow Cytometry , Fluorescence , Fluorescent Dyes/chemistry , Humans , Implants, Experimental , Magnetic Resonance Imaging , Mice , Microscopy, Confocal , Nanoparticles/ultrastructure , Staining and Labeling , Stem Cell Transplantation , Stem Cells/drug effects , Stem Cells/metabolism , Time-Lapse ImagingABSTRACT
Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.
Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Astrocytes/enzymology , Exosomes/enzymology , Motor Neurons/enzymology , Nerve Tissue Proteins/metabolism , Superoxide Dismutase/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Death/genetics , Exosomes/genetics , Exosomes/pathology , Humans , Mice , Mice, Transgenic , Motor Neurons/pathology , Mutation , Nerve Tissue Proteins/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Valosin Containing ProteinABSTRACT
Recombinant lentiviral vectors (rLVs) have emerged as versatile tools for gene delivery applications due to a number of favorable features, such as the possibility to maintain long-term transgene expression, the flexibility in the design of the expression cassettes and recent improvements in their biosafety profile. Since rLVs are able to infect multiple cell types including post-mitotic cells such as neurons and skeletal muscle cells, several studies have been exploring their application for the study and cure of neurodegenerative diseases. In particular, the introduction of rLVs carrying cell-type specific promoters could restrict the transgene expression either to neuronal or glial cells, thus helping to better dissect in vivo the role played by these cell populations in several neurodegenerative processes. In this study we developed rLVs carrying motor neuron specific regulatory sequences derived from the promoter of homeobox gene Hb9, and demonstrated that these constructs can represent a suitable platform for selective gene-targeting of murine spinal cord motor neurons, in vivo. This tool could be instrumental in the dissection of the molecular mechanisms involved in the selective degeneration of motor neurons occurring in Motor Neuron Diseases.