Oxidative stress and mitochondrial damage in the pathogenesis of ALS: New perspectives.

This review attempts to reconcile the present dual view of the mechanisms operating in Amyotrophic Lateral Sclerosis (ALS). On one side, oxidative stress, mitochondrial damage and protein aggregation are considered as causative of the disease, as strongly supported by evidence obtained in models based on the expression of ALS-typical mutant SOD1. On the other hand, evidence from models expressing ALS-typical mutations in RNA-binding proteins such as FUS and TDP43 indicate that mRNA (dys)metabolism is a major pathway in this disease. A critical analysis of existing literature suggests that there may be more than one point of intersection.

Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells.

TDP-43 is aggregated in patients with ALS and FLTD through mechanisms still incompletely understood. Since aggregation in the cytosol is most probably responsible for the delocalization and loss of proper RNA-binding function of TDP-43 in the nucleus, interception of the formation of aggregates may represent a useful therapeutic option. In this study, we investigated the relative importance of the N-terminal and C-terminal moieties of TDP-43 in the aggregation process and the weight of each of the six cysteine residues in determining unfolding and aggregation of the different domains. We report that cytoplasmic inclusions formed by WT and mutant TDP-43 in motor neuron-like NSC34 cells are redox-sensitive only in part, and contain at least two components, i.e. oligomers and large aggregates, that are made of different molecular species. The two N-terminal cysteine residues contribute to the seeding for the first step in oligomerization, which is then accomplished by mechanisms depending on the four cysteines in the RNA-recognition motifs. Cysteine-independent large aggregates contain unfolded isoforms of the protein, held together by unspecific hydrophobic interactions. Interestingly, truncated isoforms are entrapped exclusively in oligomers. Ab initio modeling of TDP-43 structure, molecular dynamics and molecular docking analysis indicate a differential accessibility of cysteine residues that contributes to aggregation propensity. We propose a model of TDP-43 aggregation involving cysteine-dependent and cysteine-independent stages that may constitute a starting point to devise strategies counteracting the formation of inclusions in TDP-43 proteinopathies.

Pur-alpha functionally interacts with FUS carrying ALS-associated mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. Here we show that Pur-alpha physically interacts with mutated FUS in an RNA-dependent manner. Pur-alpha colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. We observe that both Pur-alpha and mutated FUS upregulate phosphorylation of the translation initiation factor eukaryotic translation initiation factor 2 alpha and consistently inhibit global protein synthesis. In vivo expression of Pur-alpha in different Drosophila tissues significatively exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-alpha in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-alpha, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-alpha produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function.

Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways.

Acetylation homeostasis is thought to play a role in amyotrophic lateral sclerosis, and treatment with inhibitors of histone deacetylases has been considered a potential and attractive therapeutic approach, despite the lack of a thorough study of this class of proteins. In this study, we have considerably extended previous knowledge on the expression of 13 histone deacetylases in tissues (spinal cord and muscle) from mice carrying two different ALS-linked SOD1 mutations (G93A-SOD1 and G86R-SOD1). We have then focused on class III histone deacetylases SIRT1 and SIRT2 that are considered relevant in neurodegenerative diseases. SIRT1 decreases in the spinal cord, but increases in muscle during the progression of the disease, and a similar expression pattern is observed in the corresponding cell models (neuroblastoma and myoblasts). SIRT2 mRNA expression increases in the spinal cord in both G93A-SOD1 and G86R-SOD1 mice but protein expression is substantially unchanged in all the models examined. At variance with other sirtuin modulators (sirtinol, AGK2 and SRT1720), the well-known SIRT1 inhibitor Ex527 has positive effects on survival of neuronal cells expressing mutant SOD1, but this effect is neither mediated by SIRT1 inhibition nor by SIRT2 inhibition. These data call for caution in proposing sirtuin modulation as a target for treatment.

The unfolded protein response in models of human mutant G93A amyotrophic lateral sclerosis.

Recent studies indicate that endoplasmic reticulum (ER) stress is involved in the pathogenesis of familial and sporadic amyotrophic lateral sclerosis (ALS). ER stress occurs when the ER-mitochondria calcium cycle (ERMCC) is disturbed and misfolded proteins accumulate in the ER. To cope with ER stress, the cell engages the unfolded protein response (UPR). While activation of the UPR has been shown in some ALS models and tissues, ER stress elements have not been studied directly in motor neurons. Here we investigated the expression of XBP1 and ATF6α and phosphorylation of eIF2α, and their modulation, in mutated SOD1(G93A) NSC34 and animal model of ALS. Expression of XBP1 and ATF6α mRNA and protein was enhanced in SOD1(G93A) NSC34 cells. Activation of ATF6α and XBP1 and phosphorylation of eIF2α were detectable in mutated SOD1(G93A) motor but not in wild-type motor neurons. Treatment with the ER stressor thapsigargin enhanced phosphorylation of eIF2α and activated proteolysis of ATF6α and splicing of XBP1 in NSC34 and motor neurons in a time-dependent manner. The present study thus provides direct evidence of activated UPR in motor neurons which overexpress human pathogenic mutant SOD1(G93A) , providing evidence that ER stress plays a major role in ALS.

Beta-amyloid causes downregulation of calcineurin in neurons through induction of oxidative stress.

Calcineurin is an abundant cytosolic protein that is implicated in the modulation of glutamate release. Here we show that the expression level of this enzyme is reduced in primary neuronal cultures treated with beta-amyloid. Parallel experiments in ETNA cell lines expressing SOD1 suggested that the effect of beta-amyloid on calcineurin expression is mediated by oxidative stress. The relevance of the in vitro experiments was assessed by analysis of tissue from patients with Alzheimer's disease (AD) and tissue from two strains of transgenic mice that mimic aspects of AD. The tissue from the AD brains displayed a pronounced downregulation of calcineurin immunoreactivity in profiles that were negative for glial fibrillary acidic protein (GFAP). In the hippocampus of the transgenic animals (which were analyzed in an early stage of the disease) the downregulation of calcineurin was restricted to mossy fiber terminals. A downregulation of the presynaptic pool of calcineurin may contribute to the dysregulation of glutamate release that is considered a hallmark of AD.

Apoptosome inactivation rescues proneural and neural cells from neurodegeneration.

Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in Apaf1(-/-) embryos escape programmed cell death, proliferate and retain their potential to differentiate. To evaluate the circumstances of Apaf1(-/-) NPC survival and investigate their fate under neurodegenerative conditions, we established cell lines of embryonic origin (ETNA). We found that Apaf1(-/-) NPCs resist common apoptotic stimuli and neurodegenerative inducers such as amyloid-beta peptide (typical of Alzheimer's disease) and mutant G93A superoxide dismutase 1 (typical of familial amyotrophic lateral sclerosis). Similar results were obtained in Apaf1(-/-) primary cells. When death is prevented by Apaf1 deficiency, cytochrome c is released from mitochondria and rapidly degraded by the proteasome, but mitochondria remain intact. Under these conditions, neither activation by cleavage of initiator caspases nor release of alternative apoptotic inducers from mitochondria takes place. In addition, NPCs can still differentiate, as revealed by neurite outgrowth and expression of differentiation markers. Our findings imply that the mitochondrion/apoptosome pathway is the main route of proneural and neural cells to death and that its inhibition prevents them from dismantling in neurodegenerative conditions. Indeed, the ETNA cell model is ideally suited for exploring the potential of novel cell therapies for the treatment of human neurodegenerations.

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis.

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.

Differential role of superoxide and glutathione in S-nitrosoglutathione-mediated apoptosis: a rationale for mild forms of familial amyotrophic lateral sclerosis associated with less active Cu,Zn superoxide dismutase mutants.

SH-SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S-nitrosoglutathione (GSNO)-induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up-regulation, p21 cleavage and Bcl-2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH-SY5Y cells to puromycin-induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide-generating drug paraquat and to cell necrosis provoked by t-butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.

Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase.

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.

A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper,zinc superoxide dismutase.

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.

Cu,Zn-superoxide dismutase-dependent apoptosis induced by nitric oxide in neuronal cells.

Nitric oxide (NO) challenge to human neuroblastoma cells (SH-SY5Y) ultimately results in apoptosis. Tumor suppressor protein p53 and cell cycle inhibitor p21 accumulate as an early sign of S-nitrosoglutathione-mediated toxicity. Cytochrome c release from mitochondria and caspase 3 activation also occurred. Cells transfected with either wild type (WT) or mutant (G93A) Cu, Zn-superoxide dismutase (Cu,Zn-SOD) produced comparable amounts of nitrite/nitrate but showed different degree of apoptosis. G93A cells were the most affected and WT cells the most protected; however, Cu, Zn-SOD content of these two cell lines was 2-fold the SH-SY5Y cells under both resting and treated conditions. We linked decreased susceptibility of the WT cells to higher and more stable Bcl-2 and decreased reactive oxygen species. Conversely, we linked G93A susceptibility to increased reactive oxygen species production since simultaneous administration of S-nitrosoglutathione and copper chelators protects from apoptosis. Furthermore, G93A cells showed a significant decrease of Bcl-2 expression and, as target of NO-derived radicals, showed lower cytochrome c oxidase activity. These results demonstrate that resistance to NO-mediated apoptosis is strictly related to the level and integrity of Cu,Zn-SOD and that the balance between reactive nitrogen and reactive oxygen species regulates neuroblastoma apoptosis.

Copper-dependent oxidative stress and neurodegeneration.

Copper is an essential trace element, but its redox reactivity leads to risks of damage to cell and tissues. These are well exemplified by several forms of neurodegenerative diseases, either arising as inherited disorders of copper metabolism, such as Menkes' and Wilson's disease, or as conformational diseases such as Alzheimer's disease and prion diseases. This review will cover some aspects of the involvement of copper-mediated oxidative stress in degenerative processes in the central nervous system, with special focus on the familial form of amyotrophic lateral sclerosis (FALS). Furthermore, a possible role of copper reactivity in inducing critical steps in the apoptotic pathways leading to neurodegeneration is envisaged.

Aberrant copper chemistry as a major mediator of oxidative stress in a human cellular model of amyotrophic lateral sclerosis.

We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of D-penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild-type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild-type endogenous enzyme compromises the cell's ability to respond to oxidative stress.

Voltage-activated sodium currents in a cell line expressing a Cu,Zn superoxide dismutase typical of familial ALS.

The whole-cell configuration of the patch-clamp recording was used to study the voltage-dependent Na+ currents in a model system for the familial form of amyotrophic lateral sclerosis (ALS) associated with mutations in Cu,Zn superoxide dismutase. Here we report that the amplitude of voltage-gated Na+ currents is significantly reduced in cell lines expressing mutant Cu,Zn superoxide dismutase G93A when compared with the parental, untransfected cell line and to a cell line expressing the wild-type enzyme. This effect is associated with a shift toward positive values of the steady-state inactivation curve of the Na+ currents. These results indicate that expression of a Cu,Zn superoxide dismutase typical of patients affect with familial ALS influence the functionality of the voltage-dependent Na+ channels; this effect may contribute to the pathogenesis of the disease.

Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells.

We have set up a model system for familial amyotrophic lateral sclerosis (FALS) by transfecting human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase (Cu,ZnSOD) or a mutant of this enzyme (G93A) associated with FALS. We have tested mitochondrial function and determined cytosolic Ca2+ concentration in control cells (untransfected) and in cells expressing either wild-type Cu,ZnSOD or G93A. We report that G93A induces a significant loss of mitochondrial membrane potential, an increased sensitivity toward valinomycin and a parallel increase in cytosolic Ca2+ concentration. The above phenomena are not related to total Cu,ZnSOD content and activity in the cell.