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![Lumbar spinal cord TDP-43 Immunohistochemistry. Characterization of TDP-43 staining in a 673 day old VLE mouse. [A] Motor neuron with normal TDP-43 distribution: a darkly stained nucleus and an even, lightly stained cytoplasm graded as a zero. [B] Demonstrates mixed localization with redistribution of TDP-43 into diffuse cytoplasmic staining with reduced label in the nucleus and is graded as a one. [C] Demonstrates motor neuron with redistribution of TDP-43 into punctate staining in the cytoplasm and nucleus and is scored as a two. doi:10.1371/journal.pone.0099879.g006](profile/Guillermo-Alexander/publication/263293067/figure/fig12/AS:669628686671882@1536663328915/Lumbar-spinal-cord-TDP-43-Immunohistochemistry-Characterization-of-TDP-43-staining-in-a.jpg)
Lumbar spinal cord TDP-43 Immunohistochemistry. Characterization of TDP-43 staining in a 673 day old VLE mouse. [A] Motor neuron with normal TDP-43 distribution: a darkly stained nucleus and an even, lightly stained cytoplasm graded as a zero. [B] Demonstrates mixed localization with redistribution of TDP-43 into diffuse cytoplasmic staining with reduced label in the nucleus and is graded as a one. [C] Demonstrates motor neuron with redistribution of TDP-43 into punctate staining in the cytoplasm and nucleus and is scored as a two. doi:10.1371/journal.pone.0099879.g006
Source publication
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS) with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1). There is variability in sporadic ALS as well as FALS where even within the sa...
Contexts in source publication
Context 1
... distribution of TDP-43 immunoreactivity in spinal cord motor neurons is illustrated in Figure 6. In young animals, most TDP-43 is located in the nucleus with light cytoplasmic staining ( Figure 6A). ...
Context 2
... distribution of TDP-43 immunoreactivity in spinal cord motor neurons is illustrated in Figure 6. In young animals, most TDP-43 is located in the nucleus with light cytoplasmic staining ( Figure 6A). As the animals aged, the nuclear staining decreased and there was an increase in cytoplasmic staining ( Figure 6B and Figure 2. Variation in weight with age in male mice. ...
Context 3
... young animals, most TDP-43 is located in the nucleus with light cytoplasmic staining ( Figure 6A). As the animals aged, the nuclear staining decreased and there was an increase in cytoplasmic staining ( Figure 6B and Figure 2. Variation in weight with age in male mice. Weight (mean 6 standard error) of male VLE mice and control litter mates in four age groups. ...
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Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We...
Citations
... Body weight gain was calculated and data were expressed as percentage variation of weight compared to the first measure taken at 6 weeks of age. Monitoring of disease symptoms was achieved by direct observation and annotation of tremors, splay reflex loss, bradykinesia and paralysis (Deitch et al., 2014). Disease onset was set at age of splay reflex loss and peak body weight (Melanie Leitner and Lutz, 2009). ...
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by depletion of motor neurons (MNs), for which effective medical treatments are still required. Previous transcriptomic analysis revealed the up-regulation of C-X-C motif chemokine receptor 2 (CXCR2)-mRNA in a subset of sporadic ALS patients and SOD1G93A mice. Here, we confirmed the increase of CXCR2 in human ALS cortex, and showed that CXCR2 is mainly localized in cell bodies and axons of cortical neurons. We also investigated the effects of reparixin, an allosteric inhibitor of CXCR2, in degenerating human iPSC-derived MNs and SOD1G93A mice. In vitro, reparixin rescued MNs from apoptotic cell death, preserving neuronal morphology, mitochondrial membrane potential and cytoplasmic membrane integrity, whereas in vivo it improved neuromuscular function of SOD1G93A mice. Altogether, these data suggest a role for CXCR2 in ALS pathology and support its pharmacological inhibition as a candidate therapeutic strategy against ALS at least in a specific subgroup of patients.
... Specifically, the enzyme can exist in non-native conformations as a result of inappropriate folding (due to the mutation) or anomalous post-translational modification (e.g., oxidation: [34]). The protein has intrinsic toxic potential depending on the conformation [5,34] and on the specific mutation acquired, and its toxicity may be concentration-dependent [35]. Moreover, the subcellular localization of the mutated protein could influence the severity of the disease [36]. ...
In recent years, the “non-autonomous motor neuron death” hypothesis has become more consolidated behind amyotrophic lateral sclerosis (ALS). It postulates that cells other than motor neurons participate in the pathology. In fact, the involvement of the autonomic nervous system is fundamental since patients die of sudden death when they become unable to compensate for cardiorespiratory arrest. Mitochondria are thought to play a fundamental role in the physiopathology of ALS, as they are compromised in multiple ALS models in different cell types, and it also occurs in other neurodegenerative diseases. Our study aimed to uncover mitochondrial alterations in the sympathoadrenal system of a mouse model of ALS, from a structural, bioenergetic and functional perspective during disease instauration. We studied the adrenal chromaffin cell from mutant SOD1G93A mouse at pre-symptomatic and symptomatic stages. The mitochondrial accumulation of the mutated SOD1G93A protein and the down-regulation of optic atrophy protein-1 (OPA1) provoke mitochondrial ultrastructure alterations prior to the onset of clinical symptoms. These changes affect mitochondrial fusion dynamics, triggering mitochondrial maturation impairment and cristae swelling, with increased size of cristae junctions. The functional consequences are a loss of mitochondrial membrane potential and changes in the bioenergetics profile, with reduced maximal respiration and spare respiratory capacity of mitochondria, as well as enhanced production of reactive oxygen species. This study identifies mitochondrial dynamics regulator OPA1 as an interesting therapeutic target in ALS. Additionally, our findings in the adrenal medulla gland from presymptomatic stages highlight the relevance of sympathetic impairment in this disease. Specifically, we show new SOD1G93A toxicity pathways affecting cellular energy metabolism in non-motor neurons, which offer a possible link between cell specific metabolic phenotype and the progression of ALS.
... 008230, Jackson Laboratories) [27], and VLE G93A mice (B6SJL-Tg(SOD1*G93A)1Gur/ThpaJ; stock no. 032166, Jackson Laboratories [28]. The G85R-SOD1:YFP, WT-SOD1:YFP, and Thy1-G93A mice were maintained on the FVB/ NJ background. ...
... The GurG93A-SOD1 mice have been reported to have inherently high levels of misfolded SOD1 from very young ages [41], and thus it could be argued that these mice are not responsive to seeding because the pathogenic misfolding of SOD1 is already at saturation. Thus, we turned to lines of mice that express G93A-SOD1 at much lower levels; Thy1-G93A mice (line T3) [27] and a substrain of the G93A mice termed the very low expressing (VLE) G93A mice [28]. Mice from the T3 line that are heterozygous for the transgene do not develop paralysis by 20 months of age; whereas, homozygous mice become paralyzed between 15 and 24 months of age [27]. ...
... Heterozygous Thy-1 G93A mice injected with spinal homogenates from paralyzed GurG93A or G37R-SOD1 mice did not develop paralysis or pathology (Table 2; Additional file 2: Fig. S8). The VLE-G93A mice do not develop paralysis until > 22 months of age [28]. For these experiments, the source of the seed was spinal homogenates from paralyzed GurG93A-SOD1 (FVB/NJ strain). ...
Misfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.
... Hind limbs splay reflex was scored weekly in transgenic mice from the age of 8 weeks. Each mouse was lifted by the tail for 2 s and a score from 0 (full hind limbs extension) to 4 (absence of righting reflex, humane endpoint) were assigned based on the degree of legs extension from the body's lateral midline (Leitner et al., 2009;Deitch et al., 2014). Symptoms like tremors, loss of splay reflex, BW decline, delayed righting reflex were daily monitored at the late stage of the disease and used to determine the humane endpoint (BW loss > 20%, complete loss of righting reflex). ...
... We considered previously developed metric such as DVC R activity (Iannello, 2019;Pernold et al., 2019) and developed a new metric, referred to as Regularity Disruption Index (RDI), which is designed to capture irregularities in the activity pattern of mice. We also collected measurements on the grid hanging test, grip strength test, hind limbs splay reflex and body weight, which are classically used to monitor the progression of the disease (Knippenberg et al., 2010;Deitch et al., 2014;Olivan et al., 2015). ...
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease that affects both central and peripheral nervous system, leading to the degeneration of motor neurons, which eventually results in muscle atrophy, paralysis, and death. Sleep disturbances are common in patients with ALS, leading to even further deteriorated quality of life. Investigating methods to potentially assess sleep and rest disturbances in animal models of ALS is thus of crucial interest. We used an automated home cage monitoring system (DVC®) to capture irregular activity patterns that can potentially be associated with sleep and rest disturbances and thus to the progression of ALS in the SOD1G93A mouse model. DVC® enables non-intrusive 24/7 long term animal activity monitoring, which we assessed together with body weight decline and neuromuscular function deterioration measured by grid hanging and grip strength tests in male and female mice from 7 until 24 weeks of age. We show that as the ALS progresses over time in SOD1G93A mice, activity patterns start becoming irregular, especially during day time, with frequent activity bouts that are neither observed in control mice nor in SOD1G93A at a younger age. The increasing irregularities of activity pattern are quantitatively captured by designing a novel digital biomarker, referred to as Regularity Disruption Index (RDI). We show that RDI is a robust measure capable of detecting home cage activity patterns that could be related to rest/sleep-related disturbances during the disease progression. Moreover, the RDI rise during the early symptomatic stage parallels grid hanging and body weight decline. The non-intrusive long-term continuous monitoring of animal activity enabled by DVC® has been instrumental in discovering novel activity patterns potentially correlated, once validated, with sleep and rest disturbances in the SOD1G93A mouse model of the ALS disease.
... However, disease severity in GEMMs harboring GBA1 mutations is markedly variable and is affected by modifier genes that vary according to the genetic background of the mouse strain (Xu et al. 2003;Goker-Alpan et al. 2005;Klein et al. 2016). Similarly, mutations in SOD1 are implicated in familial ALS (FALS); however, the complete disease phenotype cannot be replicated in Sod1 knockout mice or in transgenic lines bearing the human SOD1 mutations, suggesting modifying genes or environmental factors play a role in the disease (Gurney et al. 1994;Alexander et al. 2004;Deitch et al. 2014). Yet, the SOD1 and GBA1 GEMMs are useful for evaluating compounds that target these pathways or downstream consequences of pathway activation or inhibition. ...
Considering high drug attrition rates in clinical studies and the overall complexity and challenging environment of drug development, it is increasingly important to understand the therapeutic molecule and target and how they intersect with disease biology as fully as possible. This requires one to use numerous tools and investigative approaches in combination. Genetically engineered mouse models are a critical component to the drug development toolbox as they can provide key insights across multiple steps of the drug development process. While knock-out and knock-in mice can inform questions of basic biology, genetically engineered mice can also be applied to model diseases for efficacy studies, to discriminate on-target and off-target effects of novel therapeutics, and to inform an array of biologic and pharmacologic questions, including pharmacodynamics, pharmacokinetics, and biomarker discovery. However, use of these models requires not only an understanding of their strengths and limitations but also a careful consideration of the context in which they are being used and the hypotheses being addressed by them. Additionally, they should not be used in isolation, but instead in combination with other biochemical, in vitro, and clinical data to create a broad understanding of the drug, target, and disease biology.
... Overexpression mutants with human transgenes are the still the most widely used models of neurodegeneration but they may have a severe disease, sometimes with a relatively short lifespan that can make study of ageing-related processes difficult. As discussed above, 'low-copy' transgenics can exhibit a slower disease course, but gene dosage is often still artificially high; for example, 'low-copy' SOD1-G93A mice have between 4 and 10 copies of human SOD1 (Acevedo-Arozena et al. 2011;Deitch et al. 2014). However, the use of knock-in models that have a slower rate of progression, with physiological expression of the mutation, is more informative for both early-stage disease processes and for following the effects of ageing; such models include genetically humanised FUS-ALS (Devoy et al. 2017) and HD (Heng et al. 2007) knock-in mice that exhibit age-dependent, progressive neurodegeneration. ...
Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer’s Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.
... The survival time of commonly used mouse ALS model (B6SJL-Tg(SOD1*G93A)1Gur/J) would likely be too short to realize full benefits of stem cell therapy. There are reports indicating that the severity of the disease highly depends on the copy number of mutated SOD1 gene, as well as genetic background [11][12][13] . In brief, the lower the copy number the less severe the phenotype, and finally the longer the lifespan. ...
... The number of generated 8-copy mice was significantly lower than 4-copy mice (79% of 4-copy comparing to 21% of 8-copy). Decrease in the copy number of SOD1 gene was reported previously during mating process and might be caused by spontaneous intra-locus recombination during meiosis 11,12 . Some of the researchers managed to obtain SOD1 G93A mice with low copy number of the transgene and prolonged survival, however only 80% of mice have shown symptoms of the disease and mice that presented the symptoms had prolonged asymptomatic period and consequently longer mean lifespan in comparison to mice in our colony 12,17 . ...
... Decrease in the copy number of SOD1 gene was reported previously during mating process and might be caused by spontaneous intra-locus recombination during meiosis 11,12 . Some of the researchers managed to obtain SOD1 G93A mice with low copy number of the transgene and prolonged survival, however only 80% of mice have shown symptoms of the disease and mice that presented the symptoms had prolonged asymptomatic period and consequently longer mean lifespan in comparison to mice in our colony 12,17 . This fact might be related to different background of our mice, as our colony has mixed background of SOD (B6SJL) and rag2 (129S6) mice. ...
The most recent research concerning amyotrophic lateral sclerosis (ALS) emphasizes the role of glia in disease development. Thus, one can suspect that the effective therapeutic strategy in treatment of ALS would be replacement of defective glia. One of the basic problems with human glial progenitors (hGRPs) replacement strategies is the time needed for the cells to become fully functional in vivo. The lifespan of most popular high copy number SOD1 mutant mice might be too short to acknowledge benefits of transplanted cells. We focused on developing immunodeficient rag2−/− model of ALS with lower number of transgene copies and longer lifespan. The obtained hSOD1/rag2 double mutant mice have been characterized. QPCR analysis revealed that copy number of hSOD1 transgene varied in our colony (4–8 copies). The difference in transgene copy number may be translated to significant impact on the lifespan. The death of long- and short-living hSOD1/rag2 mice is preceded by muscular weakness as early as one month before death. Importantly, based on magnetic resonance imaging we identified that mutant mice demonstrated abnormalities within the medullar motor nuclei. To conclude, we developed long-living double mutant hSOD1/rag2 mice, which could be a promising model for testing therapeutic utility of human stem cells.
... During the past decade, significant advances have been made in understanding this disease, from linkage analysis to isolation of defective genes and identification of their protein product (3). The development of animal models for the study of this disease, in particular, mouse models for ALS (1), has now made it possible to understand the molecular basis of this disease and has demonstrated the feasibility of using the whole affected gene, which is found in all patients with ALS, as a means of treating this disorder. ...
... Hemizygotes. 2 This includes the promoter sequence of a human gene. 3 This depends on the mouse line used. 4 GOF at disease-onset and LOF during disease progression. ...
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There are several genetic mutations that lead to ALS development, such as chromosome 9 hexanucleotide repeat 72 (C9ORF72), transactive response DNA-binding protein (TARDBP), superoxide dismutase 1 (SOD1) and fused in sarcoma (FUS). ALS is associated with disrupted gene homeostasis causing aberrant RNA processing or toxic pathology. Several animal models of ALS disease have been developed to understand whether TARDBP-mediated neurodegeneration results from a gain or a loss of function of the protein, however, none exactly mimic the pathophysiology and the phenotype of human ALS. Here, the pathophysiology of specific ALS-linked gene mutations is discussed. Furthermore, some of the generated mouse models, as well as the similarities and differences between these models, are comprehensively reviewed. Further refinement of mouse models will likely aid the development of a better form of model that mimics human ALS. However, disrupted gene homeostasis that causes mutation can result in an ALS-like syndrome, increasing concerns about whether neurodegeneration and other effects in these models are due to the mutation or to gene overexpression. Research on the pleiotropic role of different proteins present in motor neurons is also summarized. The development of better mouse models that closely mimic human ALS will help identify potential therapeutic targets for this disease.
... From this aspect of view, using soluble SOD1 in the body fluids can moderate such reactions and LDL oxidation, which renders the body resistant to cardiovascular problems. The important aspect of the present study was using human wild-type of SOD1 gene, in contrast to other studies that used the mutant form of the SOD1 enzyme, which could trigger the characteristics of ALS in the corresponding cells in order to investigate the adverse effects of the mutant form of this enzyme (29)(30)(31). However, a similar study demonstrated the efficacy of SOD1 in the treatment of cataracts, as the overexpression of SOD1 in the whole lens prevents H 2 O 2induced oxidative damage to the lens (32). ...
Objective(s)
Human superoxide dismutase 1 (SOD1) is the cytosolic form of this enzyme it detoxifies superoxide anions and attenuates their toxicities and concomitant detrimental effects on the cells. It is believed that the amount of these enzymes present in the oxidative stress-induced diseases is crucial for preventing disease progression. Transfection of rat bone marrow stromal cells (BMSCs) by a constructed vector carrying the human wild-type SOD1 gene, a non-viral gene transfer method, was the main aim of this study.
Materials and Methods
For this purpose, the rat BMSCs were transfected with the vector using Turbofect reagent and then stabilized. Western-blot and real-time PCR were also used for evaluation of SOD1 expression.
Results
Data analysis from RT-PCR and Western-blot techniques revealed that the stable transfected cells could secrete human wild-type SOD1 in the supernatant. Also, the total activity of SOD1 was about 0.5±0.09 U/ml and 0.005±0.002 U/ml in the supernatants of the transfected and not-transfected of rat BMSCs, respectively.
Conclusion
This study showed that expansion of the stable transfected rat BMSCs by a constructed vector carrying the human wild-type SOD1 gene is capable of secreting the active SOD1 enzyme under ex-vivo conditions. The recommendation of this study is that the same experiment would be applicable for expression of the other form of this enzyme, SOD3, as well. More valuable information could probably be provided about the variety of the diseases caused by superoxide anions toxicities by intervention and application of the non-viral method for expressions of SOD1 and SOD3 enzymes.
... In fact, patients who harbor a recognized pathogenic mutation in the SOD1 gene represent a very limited subgroup of all ALS patients. Moreover, the ALS phenotype showed by these mice is greatly dependent on four factors: gender, genetic background, SOD1 mutation, and expression levels of the human SOD1 transgene [210]. The standard SOD1 G93A mouse model carries 25 copies of the mSOD1 transgene under the control of the human SOD1 promoter [211], but it is known that loss of several transgene copies can occur spontaneously during meiosis [210]. ...
... Moreover, the ALS phenotype showed by these mice is greatly dependent on four factors: gender, genetic background, SOD1 mutation, and expression levels of the human SOD1 transgene [210]. The standard SOD1 G93A mouse model carries 25 copies of the mSOD1 transgene under the control of the human SOD1 promoter [211], but it is known that loss of several transgene copies can occur spontaneously during meiosis [210]. This phenomenon can lead to a variable phenotype expression and cause a variable degree of severity and response to treatment [212]. ...
Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by the progressive loss of cortical, bulbar, and spinal motor neurons (MNs). The cardinal manifestation of ALS is a progressive paralysis which leads to death within a time span of 3 to 5 years after disease onset. Despite similar final output of neuronal death, the underlying pathogenic causes are various and no common cause of neuronal damage has been identified to date. Inflammation-mediated neuronal injury is increasingly recognized as a major factor that promotes disease progression and amplifies the MN death-inducing processes. The neuroimmune activation is not only a physiological reaction to cell-autonomous death but is an active component of nonautonomous cell death. Such injury-perpetuating phenomenon is now proved to be a common mechanism in many human disorders characterized by progressive neurodegeneration. Therefore, it represents an interesting therapeutic target. To date, no single cell population has been proved to play a major role. The existing evidence points to a complex cross talk between resident immune cells and nonresident cells, like monocytes and T lymphocytes, and to a dysregulation in cytokine profile and in phenotype commitment. After a summary of the most important mechanisms involved in the inflammatory reaction in ALS, this review will focus on novel therapeutic tools that rely on tackling inflammation to improve motor function and survival. Herein, completed, ongoing, or planned clinical trials, which aim to modify the rapidly fatal course of this disease, are discussed. Anti-inflammatory compounds that are currently undergoing preclinical study and novel suitable molecular targets are also mentioned.
