Amino Acids as biomarkers in the SOD1G93A mouse model of ALS

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1^G93A transgenic mouse model for ALS. Presymptomatic SOD1^G93A mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70 days of age in both the SOD1^G93A and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1^G93A-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.     

Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional gel electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress.     

Altered macroautophagy in the spinal cord of SOD1 mutant mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by selective loss of motor neurons (MNs). About 20% familial cases of ALS (fALS) carried the Cu, Zn-superoxide dismutase (SOD1) gene mutation, which plays a crucial role in the pathogenesis of fALS. There is evidence suggesting that macroautophagy can degrade mutated SOD1 in vitro. To investigate whether the mutant SOD1 can induce macroautophagy in vivo, we examined the LC3 processing in spinal cord and the activation status of macroautophagy in MNs of SOD1(G93A) transgenic mice at different stages. Our data demonstrated that autophagy was activated in spinal cord of SOD1(G93A) mice indicating a possible role of macroautophagy in the pathogenesis of ALS.     

Human Glial-Restricted Progenitor Transplantation into Cervical Spinal Cord of the SOD1G93A Mouse Model of ALS

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1^G93A rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1^G93A rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1^G93A mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1^G93A mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.     

Transplantation of human glial-restricted neural precursors into injured spinal cord promotes functional and sensory recovery without causing allodynia

Background aimsTraumatic injuries of the central nervous system cause damage and degeneration of specific cell populations with subsequent functional loss. Cell transplantation is a strategy to treat such injuries by replacing lost or damaged cell populations. Many kinds of cells are considered candidates for intraspinal transplantation. Human neural precursor cells (hNPC) derived from post-mortem fetal tissue are easy to isolate and expand, and are capable of producing large numbers of neuronal and glial cells. After transplantation into the nervous system, hNPC produce mature neural phenotypes and permit functional improvement in some models of neurodegenerative disease. In this study, we aimed to elucidate the therapeutic effect of different neuronal and glial progenitor populations of hNPC on locomotor and sensory functions of spinal cord-injured (SCI) ratsMethodsDifferent populations of progenitor cells were obtained from hNPC by cell sorting and neural induction, resulting in cell cultures that were NCAM⁺ A2B5⁺, NCAM⁺ A2B5^? or A2B5⁺ NG2⁺. These different cell populations were then tested for efficacy in repair of the injured spinal cord by transplantation into rats with SCIResultsThe A2B5⁺ NG2⁺ population of hNPC significantly improved locomotor and sensory (hindlimb) functional recovery of SCI rats. Importantly, no abnormal pain responses were observed in the forelimbs following transplantationConclusionsThis treatment approach can improve functional recovery after SCI without causing allodynia. Further studies will be conducted to investigate the ability of A2B5⁺ NG2⁺ cells to survive, differentiate and integrate in the injured spinal cord.     

Late stage treatment with arimoclomol delays disease progression and prevents protein aggregation in the SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motoneuron degeneration, resulting in muscle paralysis and death, typically within 1-5 years of diagnosis. Although the pathogenesis of ALS remains unclear, there is evidence for the involvement of proteasome dysfunction and heat shock proteins in the disease. We have previously shown that treatment with a co-inducer of the heat shock response called arimoclomol is effective in the SOD(G93A) mouse model of ALS, delaying disease progression and extending the lifespan of SOD(G93A) mice (Kieran et al. 2004). However, this previous study only examined the effects arimoclomol when treatment was initiated in pre- or early symptomatic stages of the disease. Clearly, to be of benefit to the majority of ALS patients, any therapy must be effective after symptom onset. In order to establish whether post-symptomatic treatment with arimoclomol is effective, in this study we carried out a systematic assessment of different treatment regimes in SOD(G93A) mice. Treatment with arimoclomol from early (75 days) or late (90 days) symptomatic stages significantly improved muscle function. Treatment from 75 days also significantly increased the lifespan of SOD(G93A) mice, although treatment from 90 days has no significant effect on lifespan. The mechanism of action of arimoclomol involves potentiation of the heat shock response, and treatment with arimoclomol increased Hsp70 expression. Interestingly, this up-regulation in Hsp70 was accompanied by a decrease in the number of ubiquitin-positive aggregates in the spinal cord of treated SOD(G93A) mice, suggesting that arimoclomol directly effects protein aggregation and degradation.     

Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS

Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1(G93A) transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.     

Abnormal medial prefrontal cortex connectivity and defective fear extinction in the presymptomatic G93A SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neuropathy associated with the degeneration of spinal and brainstem motor neurons. Although ALS is essentially considered as a lower motor neuron disease, prefrontal cortex atrophy underlying executive function deficits have been extensively reported in ALS patients. Here, we examine whether prefrontal cortex neuronal abnormalities and related cognitive impairments are present in presymptomatic G93A Cu/Zn superoxide dismutase mice, a mouse model for familial ALS. Structural characteristics of prelimbic/infralimbic (PL/IL) medial prefrontal cortex (mPFC) neurons were studied in 3-month-old G93A and wild-type mice with the Golgi–Cox method, while mPFC-related cognitive operations were assessed using the conditioned fear extinction paradigm. Sholl analysis performed on the dendritic material showed a reduction in dendrite length and branch nodes on basal dendrites of PL/IL neurons in G93A mice. Spine density was also decreased on basal dendrite segments of branch order five. Consistent with the altered morphology of PL/IL cortical regions, G93A mice showed impaired extinction of conditioned fear. Our findings indicate that abnormal prefrontal cortex connectivity and function are appreciable before the onset of motor disturbances in this model.     

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Background

The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.

Methodology/Principal Findings

Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.

Conclusions/Significance

Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.     

Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1

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Structures of mouse SOD1 and human/mouse SOD1 chimeras

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS). Inclusions enriched in pathogenic SOD1 accumulate in the spinal cords of transgenic mice expressing these proteins, but endogenous mouse SOD1 is not found as a component of these aggregates. In the accompanying paper, Karch and colleagues analyze aggregation propensities of human/mouse SOD1 chimeras in cell culture and identify two sequence elements in the human enzyme that seem to enhance its aggregation relative to the mouse enzyme. Here, we report the first structure of mouse SOD1 along with those of SOD1 chimeras in which residues 1-80 come from human SOD1 and residues 81-153 come from mouse SOD1 and vice versa. Taken together, the structural and cell-based data suggest a model in which residues Q42 and Q123 in mouse SOD1 modulate non-native SOD1-SOD1 intermolecular interactions at edge strands in the SOD1 Greek key β-barrel.     

Lessons from models of SOD1-linked familial ALS

Ten years ago, the linkage between mutations in the gene coding for the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) and the neurodegenerative disease known as familial amyotrophic lateral sclerosis (FALS) was established. This finding has prompted a myriad of new studies in experimental models aimed at investigating the toxic function of the mutant enzymes. The cellular functions that are impaired in motoneurons as a consequence of molecular alterations induced by the expression of FALS SOD1 converge on pathways that might be activated in sporadic ALS by other toxic factors. Recent data demonstrate that, although motoneurons are lost in patients, other cell types are also affected and actively contribute to the pathogenesis of the disease.     

Endocannabinoids accumulate in spinal cord of SOD1 G93A transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1(G93A) transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.     

小鼠脊髓损伤模型的建立及其评价

通过对模型的制备模拟脊髓损伤,研究其病理和影像的变化及脊髓组织的病理分析,为后期的唔疗提供了实验信息。使用7～8周龄小鼠,咬除T9～T10棘突及相应椎板,用重物压迫脊髓,缝合皮肤,制成脊髓损伤模型。分不同的时间进行行为学评分及病理和影像学的检测。结果显示对照组在不同时间行为学评分较高,而实验组评分较低。脊髓损伤区出现明显的病理改变和影像学的改变。可见在实验组中小鼠脊髓损伤区无脊髓组织残留,且出现明显的组织和影像改变,在行为学上两组相比具有显著差异,适用于脊髓再生的研究,从而为进一步研究脊髓损伤提供了较为可靠的模型。     

Direct interstitial infusion of NK1-targeted neurotoxin into the spinal cord: a computational model

Convection-enhanced delivery of substance P (SP) nocitoxins to the spinal cord interstitium is under consideration for the treatment of chronic pain. To characterize treatment protocols, a three-dimensional finite-element model of infusion into the human dorsal column was developed to predict the distribution of SP-diphtheria toxin fusion protein (SP-DT') within normal and target tissue. The model incorporated anisotropic convective and diffusive transport through the interstitial space, hydrolysis by peptidases, and intracellular trafficking. For constant SP-DT' infusion (0.1 microl/min), the distribution of cytotoxicity in NK1 receptor-expressing neurons was predicted to reach an asymptotic limit at 6-8 h in the transverse direction at the level of the infusion cannula tip ( approximately 60% ablation of target neurons in lamina I/II). Computations revealed that SP-DT' treatment was favored by a stable SP analog (half-life approximately 60 min), high infusate concentration (385 nM), and careful catheter placement (adjacent to target lamina I/II). Sensitivity of cytotoxic regions to NK1 receptor density and white matter protease activity was also established. These data suggest that intraparenchymal infusions can be useful for treatment of localized chronic pain.