Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS\'s pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS\'s pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu+/Zn+ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species.

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS.

The progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS) is caused by a decline in motor neuron function, resulting in worsened motor impairments, malnutrition, respiratory failure and mortality, and there is a lack of effective clinical treatments. The exact mechanism of motor neuronal degeneration remains unclear. Previously, we reported that ferroptosis, which is characterized by the accumulation of lipid peroxide and glutathione depletion in an iron-dependent manner, contributed to motor neuronal death in ALS cell models with the hSOD1G93A (human Cu/Zn-superoxide dismutase) gene mutation. In this study, we further explored the role of ferroptosis in motor neurons and its regulation in mutant hSOD1G93A cell and mouse models. Our results showed that ferroptosis was activated in hSOD1G93A NSC-34 cells and mouse models, which was accompanied by decreased nuclear retention of nuclear factor erythroid 2-related factor 2 (NRF2) and downregulation of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) levels. Moreover, RTA-408, an NRF2 activator, inhibited ferroptosis in hSOD1G93A NSC-34 cells by upregulating the protein expression of SLC7A11 and GPX4. Moreover, hSOD1G93A mice treated with RTA-408 showed obvious improvements in body weight and motor function. Our study demonstrated that ferroptosis contributed to the toxicity of motor neurons and that activating NRF2 could alleviate neuronal degeneration in ALS with the hSOD1G93A mutation.

The mechanisms underlying neurodegeneration in amyotrophic lateral sclerosis (ALS) are multifactorial and include genetic and environmental factors. Nowadays, it is well accepted that neuronal loss is driven by non-cell autonomous toxicity. Non-neuronal cells, such as astrocytes, have been described to significantly contribute to motoneuron cell death and disease progression in cell culture experiments and animal models of ALS. Astrocytes are essential for neuronal survival and function by regulating neurotransmitter and ion homeostasis, immune response, blood flow and glucose uptake, antioxidant defence and growth factor release. Based on their significant functions in \"housekeeping\" the central nervous system (CNS), they are no longer thought to be passive bystanders but rather contributors to ALS pathogenesis. Findings from animal models have broadened our knowledge about different pathomechanisms in ALS, but therapeutic approaches to impede disease progression failed. So far, there is no cure for ALS and effective medication to slow down disease progression is limited. Targeting only a single aspect of this multifactorial disease may exhibit therapeutic limitations. Hence, novel cellular targets must be defined and new pharmaceutical strategies, such as combinatorial drug therapies are urgently needed. The present review discusses the physiological role of astrocytes and current hypotheses of astrocyte pathology in ALS. Furthermore, recent investigation of potential drug candidates in astrocyte cell culture systems and animal models, as well as data obtained from clinical trials, will be addressed. The central role of astrocytes in ALS pathogenesis makes them a promising target for pharmaceutical interventions.

OBJECTIVE: The first part of the experiment identifies and validates MRI biomarkers distinctive of the disease progression in the transgenic superoxide dismutase gene (SOD1(G93A)) animal model. The second part assesses the efficacy of a mesenchymal stem cell-based therapy through the MRI biomarkers previously defined.
METHODS: The first part identifies MRI differences between SOD1(G93A) and healthy mice. The second part of the experiment follows the disease evolution of stem cell-treated and non-stem-cell treated SOD1(G93A) mice. The analysis focused on voxel-based morphometry and T2 mapping on the brain tissues, and T2-weighted imaging and diffusion tensor imaging (DTI) on the hind limbs.
RESULTS: Comparing diseased mice to healthy control revealed gray matter alterations in the brainstem area, accompanied by increased T2 relaxation time. Differences in muscle volume, muscle signal intensity, fractional anisotropy, axial diffusivity, and radial diffusivity were measured in the hind limbs. In the comparison between stem cell-treated mice and nontreated ones, differences in muscle volume, muscle signal intensity, and DTI-derived maps were found.
CONCLUSIONS: MRI-derived biomarkers can be used to identify differences between stem cell-treated and nontreated SOD1(G93A) mice. Magn Reson Med 79:459-469, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Mitochondrial pathology is a seminal pathogenic hallmark of familial amyotrophic lateral sclerosis (FALS) which is extensively manifested by human patients and mutant SOD1(G93A) mammalian models. Rodents expressing human FALS-associated mutations successfully mimic several human disease features; although they are not as amenable to genetic and therapeutic compound screenings as non-mammalian models. In this study, we report a newly generated and characterized Drosophila model that expresses human SOD1(G93A) in muscle fibers. Presence of SOD1(G93A) in thoracic muscles causes mitochondrial pathology and impairs normal motor behavior in these flies. Use of this new FALS-24B-SOD1(G93A) fly model holds promise for better understanding of the mitochondrial affectation process in FALS and for the discovery of novel therapeutic compounds able to reverse mitochondrial dysfunction in this fatal disease.

Extensive clinical investigations, in hand with biochemical and biophysical research, have associated brain iron accumulation with the pathogenesis of the amyotrophic lateral sclerosis (ALS) disease. The origin of iron is still not identified, but it is proposed that it forms redox active complexes that can participate in the Fenton reaction generating the toxic hydroxyl radical. In this paper, the state of iron in the neural tissues isolated from SOD1(G93A) transgenic rats was investigated using low temperature EPR spectroscopy and is compared with that of nontransgenic (NTg) littermates. The results showed that iron in neural tissues is present as high- and low-spin, heme and non-heme iron. It appears that the SOD1(G93A) rat neural tissues were most likely exposed in vivo to higher amounts of reactive oxygen species when compared to the corresponding NTg tissues, as they showed increased oxidized [3Fe-4S](1+) cluster content relative to [4Fe-4S](1+). Also, the activity of cytochrome c oxidase (CcO) was found to be reduced in these tissues, which may be associated with the observed uncoupling of heme a3 Fe and CuB in the O2-reduction site of the enzyme. Furthermore, the SOD1(G93A) rat spinal cords and brainstems contained more manganese, presumably from MnSOD, than those of NTg rats. The addition of potassium superoxide to all neural tissues ex vivo, led to the [4Fe-4S]→[3Fe-4S] cluster conversion and concurrent release of Fe. These results suggest that the superoxide anion may be the cause of the observed oxidative damage to SOD1(G93A) rat neural tissues and that the iron-sulfur clusters may be the source of poorly liganded redox active iron implicated in ALS pathogenesis. Low temperature EPR spectroscopy appears to be a valuable tool in assessing the role of metals in neurodegenerative diseases.

In amyotrophic lateral sclerosis (ALS), it has been suggested that the process of neurodegeneration starts at the neuromuscular junction and is propagated back along axons towards motor neurons. Caspase-dependent pathways are well established as a cause of motor neuron death, and recent work in other disease models indicated a role for caspase 6 in axonal degeneration. Therefore we hypothesised that caspase 6 may be involved in motor neuron death in ALS. To investigate the role of caspase 6 in ALS we profiled protein levels of caspase-6 throughout disease progression in the ALS mouse model SOD1(G93A); this did not reveal differences in caspase 6 levels during disease. To investigate the role of caspase 6 further we generated a colony with SOD1(G93A) transgenic mice lacking caspase 6. Analysis of the transgenic SOD1(G93A); Casp6(-/-) revealed an exacerbated phenotype with motor dysfunction occurring earlier and a significantly shortened lifespan when compared to transgenic SOD1(G93A); Casp6(+/+) mice. Immunofluorescence analysis of the neuromuscular junction revealed no obvious difference between caspase 6(+/+) and caspase 6(-/-) in non-transgenic mice, while the SOD1(G93A) transgenic mice showed severe degeneration compared to non-transgenic mice in both genotypes. Our data indicate that caspase-6 does not exacerbate ALS pathogenesis, but may have a protective role.

[This corrects the article on p. 204 in vol. 7, PMID: 26539112.].

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease causing death of motor neurons. This study investigated the roles of energy metabolism in the pathogenesis of ALS in the SOD1(G93A) transgenic mouse model. Control and SOD1(G93A) mice were administered with shcontrol or shPGC-1α in combination with PBS or thiazolidinedione (TZD) for 8 weeks. Gene expression was analyzed by quantitative real-time PCR and Western blot. ROS and fibrosis were assessed with a colorimetric kit and Sirius staining, respectively. Inflammatory cytokines were measured using ELISA kits. The levels of tissue ROS and serum inflammatory cytokines were significantly higher in SOD1(G93A) mice compared to control mice, and knocking down peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) drastically increased cytokine levels in both control and SOD1(G93A) mice. Muscle fibrosis was much severer in SOD1(G93A) mice, and worsened by silencing PGC-1α and attenuated by TZD. The expression levels of PGC-1α, SOD1, UCP2, and cytochrome C were substantially reduced by shPGC-1α and increased by TZD in muscle of both control and SOD1(G93A) mice, whereas the level of NF-κB was significantly elevated in SOD1(G93A) mice, which was further increased by PGC-1α silencing. These data indicated that disruption of energy homeostasis would exacerbate the pathological changes caused by SOD1 mutations to promote the pathogenesis of ALS.