Nuclear localization of human SOD1 and mutant SOD1-specific disruption of survival motor neuron protein complex in transgenic amyotrophic lateral sclerosis mice.
ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that causes degeneration of motor neurons and paralysis. Approximately 20% of familial ALS cases have been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene, but it is unclear how mutations in the protein result in motor neuron degeneration. Transgenic (tg) mice expressing mutated forms of human SOD1 (hSOD1) develop clinical and pathological features similar to those of ALS. We used tg mice expressing hSOD1-G93A, hSOD1-G37R, and hSOD1-wild-type to investigate a new subcellular pathology involving mutant hSOD1 protein prominently localizing to the nuclear compartment and disruption of the architecture of nuclear gems. We developed methods for extracting relatively pure cell nucleus fractions from mouse CNS tissues and demonstrate a low nuclear presence of endogenous SOD1 in mouse brain and spinal cord, but prominent nuclear accumulation of hSOD1-G93A, -G37R, and -wild-type in tg mice. The hSOD1 concentrated in the nuclei of spinal cord cells, particularly motor neurons, at a young age. The survival motor neuron protein (SMN) complex is disrupted in motor neuron nuclei before disease onset in hSOD1-G93A and -G37R mice; age-matched hSOD1-wild-type mice did not show SMN disruption despite a nuclear presence. Our data suggest new mechanisms involving hSOD1 accumulation in the cell nucleus and mutant hSOD1-specific perturbations in SMN localization with disruption of the nuclear SMN complex in ALS mice and suggest an overlap of pathogenic mechanisms with spinal muscular atrophy.
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes skeletal muscle paralysis. Familial forms of ALS are linked to mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human SOD1 (hSOD1) toxicity to MNs are unknown. We hypothesized that skeletal muscle is a primary site of pathogenesis in ALS that triggers MN degeneration. We created transgenic (tg) mice expressing wild-type-, G37R- and G93A-hSOD1 gene variants only in skeletal muscle. These tg mice developed age-related neurologic and pathologic phenotypes consistent with ALS. Affected mice showed limb weakness and paresis with motor deficits. Skeletal muscles developed severe pathology involving oxidative damage, protein nitration, myofiber cell death and marked neuromuscular junction (NMJ) abnormalities. Spinal MNs developed distal axonopathy and formed ubiquitinated inclusions and degenerated through an apoptotic-like pathway involving capsase-3. Mice expressing wild-type and mutant forms of hSOD1 developed MN pathology. These results demonstrate that human SOD1 in skeletal muscle has a causal role in ALS and identify a new non-autonomous mechanism for MN degeneration explaining their selective vulnerability. The discovery of instigating molecular toxicities or disease progression determinants within skeletal muscle could be very valuable for the development of new effective therapies for the treatment and cure of ALS.
Project description:Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.
Project description:Approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1(G93A) and SOD1(G37R). Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1(G93A) mice, and from 9 months until end stage in SOD1(G37R) mice. The beneficial effects of WA in the SOD1(G93A) mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1(G93A) mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1(G93A) mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.
Project description:Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.
Project description:Amyotrophic lateral sclerosis (ALS) is characterized by predominant vulnerability and central degeneration of both corticospinal/corticobulbar motor neurons (CSMN; "upper motor neurons") in cerebral cortex, and spinal/bulbar motor neurons (SMN; "lower motor neurons") in spinal cord and brainstem. Increasing evidence indicates broader cerebral cortex pathology in cognitive, sensory, and association systems in select cases. It remains unclear whether widely accepted transgenic ALS models, in particular hSOD1(G93A) mice, undergo degeneration of CSMN and molecularly/developmentally closely related populations of nonmotor projection neurons [e.g., other subcerebral projection neurons (SCPN)], and whether potential CSMN/SCPN degeneration is specific and early. This relative lack of knowledge regarding upper motor neuron pathology in these ALS model mice has hindered both molecular-pathophysiologic understanding of ALS and their use toward potential CSMN therapeutic approaches. Here, using a combination of anatomic, cellular, transgenic labeling, and newly available neuronal subtype-specific molecular analyses, we identify that CSMN and related nonmotor SCPN specifically and progressively degenerate in hSOD1(G93A) mice. Degeneration starts quite early and presymptomatically, by postnatal day 30. Other neocortical layers, cortical interneurons, and other projection neuron populations, even within layer V, are not similarly affected. Nonneuronal pathology in neocortex (activated astroglia and microglia) is consistent with findings in human ALS cortex and in affected mouse and human spinal cord. These results indicate previously unknown neuron type-specific vulnerability of CSMN/sensory and association SCPN, and identify that characteristic dual CSMN and SMN degeneration is conserved in hSOD1(G93A) mice. These results provide a foundation for detailed investigation of CSMN/SCPN vulnerability and toward potential CSMN therapeutics in ALS.
Project description:Co-expression of wild-type human superoxide dismutase 1 (WT-hSOD1) with ALS mutant hSOD1 accelerates disease onset relative to mice expressing only mutant protein. Here, we analyzed the effect of co-expressed WT-hSOD1 in two established mutant mouse models (L126Z and G37R), and a new model that expresses the first 102 amino acids of SOD1 with mutations at histidines 46, 48 and 63 to eliminate Cu binding (Cu-V103Z). A subset of Cu-V103Z mice developed paralysis between 500 and 730 days. Similar to mice expressing L126Z-SOD1, the spinal cords of this new model showed SOD1 immunoreactive fibrillar inclusions. Co-expression of WT-hSOD1 with Cu-V103Z SOD1 moderately accelerated the age to paralysis, similar in magnitude to WT/L126Z mice. In either combination of these bigenic mice, the severity of fibrillar inclusion pathology was diminished and unreactive to antibodies specific for the C terminus of WT protein. Co-expression of WT-hSOD1 fused to yellow fluorescent protein (WT-hSOD1:YFP) with G37R-hSOD1 produced earlier disease, and spinal cords of paralyzed bigenic mice showed YFP fluorescent inclusion-like structures. In bigenic L126Z/WT-hSOD1:YFP mice, disease was not accelerated and WT-hSOD1:YFP remained diffusely distributed. A combination of split luciferase complementation assays and affinity capture-binding assays demonstrated that soluble G37R-hSOD1 efficiently and tightly bound soluble WT-hSOD1, whereas soluble forms of the Cu-V103Z and L126Z variants demonstrated low affinity. These data indicate that WT-hSOD1 may indirectly augment the toxicity of mutant protein by competing for protective factors, but disease onset seems to be most accelerated when WT-hSOD1 interacts with mutant SOD1 and becomes misfolded.
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood-spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood-spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood-spinal cord barrier endothelial cells of presymptomatic hSOD1(G93A) mice and bEnd.3 cells stably expressing hSOD1(G93A). In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the ?-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1(G93A) cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood-spinal cord barrier.
Project description:There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with late-onset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations.
Project description:Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.
Project description:Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A.Our results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS.