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.

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.

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.

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.

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.

Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis.

Several point mutations in the gene coding for human Cu,Zn superoxide dismutase have been reported as being responsible for familial amyotrophic lateral sclerosis (FALS). However, no direct demonstration has been provided for a correlation between total superoxide dismutase activity and severity of the FALS pathology. In order to get a better insight into the mechanism(s) underlying the FALS phenotype, we have investigated the activity and the copper binding properties of the single mutant H46R, which is associated with a Japanese form of FALS. We have shown that this mutant is structurally stable but lacks significant enzyme activity and has impaired capability of binding catalytic copper. The mutant protein can be fully reconstituted with copper in vitro but its ESR spectrum displays an axial shape quite different from that of the wild-type.

Chaperonins dependent increase of Cu,Zn superoxide dismutase production in Escherichia coli.

Over-expression of the chaperonins GroEL and GroES significantly suppressed the temperature-dependent pattern of expression of Cu,Zn superoxide dismutases in Escherichia coli and increased the yield of active enzyme. The results obtained indicate that chaperonins prevent degradation of metal-deficient enzyme molecules. GroEL was shown to form a complex with unfolded Cu,Zn superoxide dismutase in vitro, confirming that GroEL can interact with beta-stranded proteins.

Evidence for co-regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor.

Saccharomyces cerevisiae mutant strain DTY26, lacking ACE1, the protein mediator for the induction of metallothionein gene expression, is unable to increase Cu,Zn superoxide dismutase mRNA in response to copper. In the wild-type strain DTY22 transcription of both Cu,Zn superoxide dismutase and metallothionein genes is induced by copper and silver, as expected on the basis of previous results indicating that ACE1 binds only Ag(I) besides Cu(I). We conclude that at the transcriptional level Cu,ZnSOD is co-regulated with metallothionein. Furthermore, structural similarities between the two promoters were found, which could explain the co-regulation effect and the quantitative differences in the response of the two genes to copper.

Mutation of Lys-120 and Lys-134 drastically reduces the catalytic rate of Cu,Zn superoxide dismutase.

Lys-120 and Lys-134, located at the edge of the active site channel in most Cu,Zn superoxide dismutases, have been suggested to play a major role in steering the anionic substrate towards the catalytic copper ion. In this study, mutants of Xenopus laevis Cu,Zn superoxide dismutase have been engineered, with Lys-120 and Lys-134 changed into leucine and threonine, respectively, and their catalytic properties have been investigated by pulse radiolysis. Results obtained demonstrate that both residues decrease the catalytic rate by about 40%, in partial disagreement with previous brownian dynamics calculations, carried out on bovine Cu,Zn superoxide dismutase.

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.

Crystal structure of the cyanide-inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K.

The crystal structure of cyanide-inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R-factor for the 27,299 reflections in the 10.0-1.7 A resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 A in subunit A, and 0.27 A in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

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.

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.

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.

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.

Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1.

We show that thiol-groups confer redox-susceptibility to the zinc-finger transcription factor Sp1 and that this redox-susceptibility is prevented by DNA-binding and depends on zinc-coordination of the protein. Apo-Sp1 contained in metal depleted nuclear extracts of human K562 cells exhibited a markedly increased susceptibility towards oxidizing and alkylating agents, as compared to holo-Sp 1. Moreover, DNA binding of apo-Sp1, but not of the holo-protein, was dramatically decreased in the presence of GSH/GSSG ratios within the physiological range. We compared these results with the redox behaviour of two other transcription factors, OTF-1 and NF1, which was found to be different in several aspects from that of Sp1.