UsnRNP trafficking is regulated by stress granules and compromised by mutant ALS proteins.

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-ß1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-ß1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-ß1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.

Differential toxicity of TAR DNA-binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.

Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS.

A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.

Pathways to mitochondrial dysfunction in ALS pathogenesis.

Alterations in the structure and functions of mitochondria are a typical trait of Amyotrophic Lateral Sclerosis, a neurodegenerative disease characterized by a prominent degeneration of upper and lower motor neurons. The known gene mutations that are responsible for a small fraction of ALS cases point to a complex interplay between different mechanisms in the disease pathogenesis. Here we will briefly overview the genetic and mechanistic evidence that make dysfunction of mitochondria a candidate major player in this process.

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells.

BACKGROUND: Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated. METHODS: In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters. RESULTS: SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO4 treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1). CONCLUSION: Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.

The NADPH oxidase pathway is dysregulated by the P2X7 receptor in the SOD1-G93A microglia model of amyotrophic lateral sclerosis.

Inflammation and oxidative stress are thought to play determinant roles in the pathogenesis of amyotrophic lateral sclerosis (ALS). Degenerating motor neurons produce signals that activate microglia to release reactive oxygen species (ROS) and proinflammatory cytokines, resulting in a vicious cycle of neurodegeneration. The ALS-causing mutant protein Cu(+)/Zn(+) superoxide dismutase SOD1-G93A directly enhances the activity of the main ROS-producing enzyme in microglia, NADPH oxidase 2 (NOX2), a well-known player in the pathogenesis of ALS. Considering that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS pathology, we used primary microglial cells derived from transgenic SOD1-G93A mice and SOD1-G93A mice lacking the P2X7 receptor to investigate the effects of both pharmacological induction and genetic ablation of receptor activity on the NOX2 pathway. We observed that, in SOD1-G93A microglia, the stimulation of P2X7 receptor by 2'-3'-O-(benzoyl-benzoyl) ATP enhanced NOX2 activity in terms of translocation of p67(phox) to the membrane and ROS production; this effect was totally dependent on Rac1. We also found that, following P2X7 receptor stimulation, the phosphorylation of ERK1/2 was augmented in ALS microglia, and there was a mutual dependency between the NOX2 and ERK1/2 pathways. All of these microglia-mediated damaging mechanisms were prevented by knocking out P2X7 receptor and by the use of specific antagonists. These findings suggest a noxious mechanism by which P2X7 receptor leads to enhanced oxidative stress in ALS microglia and identify the P2X7 receptor as a promising target for the development of therapeutic strategies to slow down the progression of ALS.

S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis.

Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based in vitro models of neurodegeneration, namely, Parkinson's disease (PD) and familial amyotrophic lateral sclerosis (fALS). In particular, we provide evidence that GSNOR-knocking down protects SH-SY5Y against PD toxins, while, by contrast, its upregulation is required for G93A-SOD1 expressing cells resistance to NO-releasing drugs. Although completely opposite, both conditions are characterized by Nrf2 localization in the nuclear compartment: in the first case induced by GSNOR silencing, while in the second one underlying the antinitrosative response. Overall, our results demonstrate that GSNOR expression has different effect on neuronal viability in dependence on the stimulus applied and suggest that GSNOR could be a responsive gene downstream of Nrf2 activation.

Cystatin B and SOD1: protein­protein interaction and possible relation to neurodegeneration.

Cystatin B (CSTB), an inhibitor of the cysteine proteases, belongs to the cathepsin family and it is known to interact with a number of proteins involved in cytoskeletal organization. CSTB has an intrinsic tendency to form aggregates depending on the redox environment. The gene encoding for CSTB is frequently mutated in association with the rare neurodegenerative condition progressive myoclonus epilepsy. Increased levels of CSTB have been observed in the spinal cord of transgenic mice modeling SOD1-linked familial amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motoneurons. In the present study, we have investigated the relationship occurring between the expression of SOD1 and CSTB either wild-type or double-cysteine substitution mutant (Cys 3 and Cys 64). Whether or not there is a physical interaction between the two proteins was also investigated in overexpression experiments using a human neuroblastoma cell line and mouse-immortalized motoneurons. Here we report evidences for a reciprocal influence of CSTB and SOD1 at the gene expression level and for a direct interaction of the two proteins.

Mitochondria and ALS: implications from novel genes and pathways.

Evidence from patients with sporadic and familiar amyotrophic lateral sclerosis (ALS) and from models based on the overexpression of mutant SOD1 found in a small subset of patients, clearly point to mitochondrial damage as a relevant facet of this neurodegenerative condition. In this mini-review we provide a brief update on the subject in the light of newly discovered genes (such as TDP-43 and FUS/TLS) associated to familial ALS and of a deeper knowledge of the mechanisms of derangement of mitochondria. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

Mislocalised FUS mutants stall spliceosomal snRNPs in the cytoplasm.

Genes encoding RNA-binding proteins have frequently been implicated in various motor neuron diseases, but the particular step in RNA metabolism that is vulnerable in motor neurons remains unknown. FUS, a nuclear protein, forms cytoplasmic aggregates in cells affected by amyotrophic lateral sclerosis (ALS), and mutations disturbing the nuclear import of FUS cause the disease. It is extremely likely that the cytoplasmic aggregates are cytotoxic because they trap important factors; the nature of these factors, however, remains to be elucidated. Here we show that FUS associates in a neuronal cell line with SMN, the causative factor in spinal muscular atrophy (SMA). The two genes work on the same pathway, as FUS binds to spliceosomal snRNPs downstream of the SMN function. Pathogenic FUS mutations do not disturb snRNP binding. Instead, cytoplasmic mislocalisation of FUS causes partial mis-localisation of snRNAs to the cytoplasm, which in turn causes a change in the behaviour of the alternative splicing machinery. FUS, and especially its mutations, thus have a similar effect as SMN1 deletion in SMA, suggesting that motor neurons could indeed be particularly sensitive to changes in alternative splicing.

Mitochondrial redox signalling by p66Shc mediates ALS-like disease through Rac1 inactivation.

Increased oxidative stress and mitochondrial damage are among the mechanisms whereby mutant SOD1 (mutSOD1) associated with familial forms of amyotrophic lateral sclerosis (ALS) induces motoneuronal death. The 66 kDa isoform of the growth factor adapter Shc (p66Shc) is known to be central in the control of mitochondria-dependent oxidative balance. Here we report that expression of mutSOD1s induces the activation of p66Shc in neuronal cells and that the overexpression of inactive p66Shc mutants protects cells from mutSOD1-induced mitochondrial damage. Most importantly, deletion of p66Shc ameliorates mitochondrial function, delays onset, improves motor performance and prolongs survival in transgenic mice modelling ALS. We also show that p66Shc activation by mutSOD1 causes a strong decrease in the activity of the small GTPase Rac1 through a redox-sensitive regulation. Our results provide new insight into the potential mechanisms of mutSOD1-mediated mitochondrial dysfunction.

The intriguing case of motor neuron disease: ALS and SMA come closer.

MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND. In the present paper we review recent developments showing a functional link between SMN (survival of motor neuron), the causative factor of SMA (spinal muscular atrophy), and FUS (fused in sarcoma), a genetic factor in ALS (amyotrophic lateral sclerosis). SMN is long known to have a crucial role in the biogenesis and localization of the spliceosomal snRNPs (small nuclear ribonucleoproteins), which are essential assembly modules of the splicing machinery. Now we know that FUS interacts with SMN and pathogenic FUS mutations have a significant effect on snRNP localization. Together with other recently published evidence, this finding potentially links ALS pathogenesis to disturbances in the splicing machinery, and implies that pre-mRNA splicing may be the common weak point in MND, although other steps in mRNA metabolism could also play a role. Certainly, further comparison of the RNA metabolism in different MND will greatly help our understanding of the molecular causes of these devastating diseases.

Interaction of cisplatin with human superoxide dismutase.

cis-Diamminedichloroplatinum(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 ± 3 µM through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu(2)-Zn(2) and Zn(2)-Zn(2) forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers in vitro and in cell, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis.

Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity.

Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration.

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant G93A-SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre-mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A-SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS.

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Bcl2-A1 interacts with pro-caspase-3: implications for amyotrophic lateral sclerosis.

Expression of mutant SOD1 typical of familial amyotrophic lateral sclerosis (ALS) induces the expression of Bcl2-A1, a member of the Bcl2 family of proteins, specifically in motor neurons of transgenic mice. In this work, we have used immortalized motor neurons (NSC-34) and transgenic mice expressing mutant SOD1 to unravel the molecular mechanisms and the biological meaning of this up-regulation. We report that up-regulation of Bcl2-A1 by mutant SOD1 is mediated by activation of the redox sensitive transcription factor AP1 and that Bcl2-A1 interacts with pro-caspase-3 via its C-terminal helix α9. Furthermore, Bcl2-A1 inhibits pro-caspase-3 activation in immortalized motor neurons expressing mutant SOD1 and thus induction of Bcl2-A1 in ALS mice represents a pro-survival strategy aimed at counteracting the toxic effects of mutant SOD1. These data provide significant new insights on how molecular signaling, driven by expression of the ALS-causative gene SOD1, affects regulation of apoptosis in motor neurons and thus may have implications for ALS therapy, where prevention of motor neuronal cell death is one of the major aims.

SOD1 and mitochondria in ALS: a dangerous liaison.

Mutant Cu,Zn superoxide dismutase (mutSOD1) is found in a subset of patients with familial amyotrophic lateral sclerosis (ALS), a fatal progressive paralysis due to loss of motor neurons. In the present article, we review existing evidence linking the expression of mutSOD1 to the many facets of mitochondrial dysfunction in ALS, with a focus on recent studies suggesting that the association and misfolding of the mutant protein (and possibly of the wild type protein as well) within these organelles is causally linked to their functional and structural alterations. Energy deficit, calcium mishandling and oxidative stress are paralleled by alteration in mitochondrial motility, dynamics and turnover and most probably lead to mitochondria-dependent cell death. Thus, the development of new, selective mitochondria-targeted therapies may constitute a promising approach in the treatment of SOD1-linked ALS.

The proinflammatory action of microglial P2 receptors is enhanced in SOD1 models for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of lower and upper motoneurons. The pathology is imputable in approximately 2% of cases to mutations in the ubiquitous enzyme Cu, Zn superoxide dismutase (SOD1). Common theories to explain the pathogenic mechanisms of ALS include activation of microglia, responsible for the release of proinflammatory factors. However, how mutant SOD1 affects microglial activation and subsequently injures neurons is still unclear. Considering that extracellular ATP, through purinergic P2 receptors, constitutes a well recognized neuron-to-microglia alarm signal, the aim of this study was to investigate how the expression of mutant SOD1 affects P2 receptor-mediated proinflammatory microglial properties. We used primary and immortalized microglial cells from mutant SOD1 mice to explore several aspects of activation by purinergic ligands and to analyze the overall effect of such stimulation on the viability of NSC-34 and SH-SY5Y neuronal cell lines. We observed up-regulation of P2X(4), P2X(7), and P2Y(6) receptors and down-regulation of ATP-hydrolyzing activities in mutant SOD1 microglia. This potentiation of the purinergic machinery reflected into enhanced sensitivity mainly to 2'-3'-O-(benzoyl-benzoyl) ATP, a P2X(7) receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-alpha and cyclooxygenase-2 content, and finally into toxic effects exerted on neuronal cell lines by microglia expressing mutant SOD1. All these parameters were prevented by the antagonist Brilliant Blue G. The purinergic activation of microglia may thus constitute a new route involved in the progression of ALS to be exploited to potentially halt the disease.