Alberto Ferri

Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease

Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimers disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimers disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3–dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimers disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages.

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.

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

Neurodegeneration in amyotrophic lateral sclerosis: the role of oxidative stress and altered homeostasis of metals

Amyotrophic lateral sclerosis is one of the most common neurodegenerative disorders, with an incidence of about 1/100,000. One of the typical features of this progressive, lethal disease, occurring both sporadically and as a familial disorder, is degeneration of cortical and spinal motor neurones. Present evidence indicates that loss of neurones in patients results from a complex interplay among oxidative injury, excitotoxic stimulation, dysfunction of critical proteins and genetic factors. This review focuses on existing evidence that oxidative stress is a major culprit in the pathogenesis of amyotrophic lateral sclerosis. An increase in reactive oxygen species and in products of oxidation has been observed both in post-mortem samples and in experimental models for ALS. This increase may be consequent to altered metabolism of copper and iron ions, that share the property to undergo redox cycling and generate reactive oxygen species. Metal-mediated oxidative stress would lead to several intracellular alterations and contribute to the induction of cell death pathways.

Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive degeneration of motor neurons occurring both as a sporadic and a familial disease. The etiology of ALS remains unknown, but one fifth of instances are due to specific gene defects, the best characterized of which is point mutations in the gene coding for Cu/Zn superoxide dismutase (SOD1). Because sporadic and familial ALS affect the same neurons with similar pathology, it is hoped that understanding these gene defects will help in devising therapies effective in both forms. A wealth of evidence has been collected in rodents made transgenic for mutant SOD1, which represent the best available models for familial ALS. Mutant SOD1 likely induces selective vulnerability of motor neurons through a combination of several mechanisms, including protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities and defective axonal transport, excitotoxicity, inadequate growth factor signaling, and inflammation. Damage within motor neurons is enhanced by noxious signals originating from nonneuronal neighboring cells, where mutant SOD1 induces an inflammatory response that accelerates disease progression. The clinical implication of these findings is that promising therapeutic approaches can be derived from multidrug treatments aimed at the simultaneous interception of damage in both motor neurons and nonmotor neuronal cells.

Cysteine 111 affects aggregation and cytotoxicity of mutant CU/ZN superoxide dismutase associated with familial amyotrophic lateral sclerosis

Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.ResultsTo gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.ConclusionIn this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Phosphatidylinositol 3‐kinase is recruited to a specific site in the activated IL‐1 receptor I

Interleukin 1 (IL‐1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF‐κB. The IL‐1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C‐terminal portion of the type I IL‐1 receptor (IL‐1RI) is essential for IL‐1 signalling and for IL‐1 dependent activation of NF‐κB. This portion contains a putative phosphatidylinositol 3‐kinase (PI 3‐kinase) binding domain (Tyr‐E‐X‐Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3‐kinase in IL‐1 signalling. Here we report evidence that PI 3‐kinase is recruited by the activated IL‐1RI, causing rapid and transient activation of PI 3‐kinase. We also show that the receptor is tyrosine phosphorylated in response to IL‐1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3‐kinase activation by IL‐1. Our results indicate that PI 3‐kinase is likely to be an important mediator of some IL‐1 effects, providing docking sites for additional signalling molecules.

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 ∼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 P2X4, P2X7, and P2Y6 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 P2X7 receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-α 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.

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.