Elisabetta Ferraro

Endoplasmic Reticulum Stress Induces Apoptosis by an Apoptosome-dependent but Caspase 12-independent Mechanism

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.

Apoptosis is not required for mammalian neural tube closure

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-α to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.

Apoptosome inactivation rescues proneural and neural cells from neurodegeneration

AbstractDeficiency 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-β peptide (typical of Alzheimers 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.

Apoptosome-deficient Cells Lose Cytochrome c through Proteasomal Degradation but Survive by Autophagy-dependent Glycolysis

Cytochrome c release from mitochondria promotes apoptosome formation and caspase activation. The question as to whether mitochondrial permeabilization kills cells via a caspase-independent pathway when caspase activation is prevented is still open. Here we report that proneural cells of embryonic origin, when induced to die but rescued by apoptosome inactivation are deprived of cytosolic cytochrome c through proteasomal degradation. We also show that, in this context, those cells keep generating ATP by glycolysis for a long period of time and that they keep their mitochondria in a depolarized state that can be reverted. Moreover, under these conditions, such apoptosome-deficient cells activate a Beclin 1-dependent autophagy pathway to sustain glycolytic-dependent ATP production. Our findings contribute to elucidating what the point-of-no-return in apoptosis is. They also help in clarifying the issue of survival of apoptosome-deficient proneural cells under stress conditions. Unraveling this issue could be highly relevant for pharmacological intervention and for therapies based on neural stem cell transfer in the treatment of neurological disorders.

Molecular control of neuromuscular junction development

Skeletal muscle innervation is a multi-step process leading to the neuromuscular junction (NMJ) apparatus formation. The transmission of the signal from nerve to muscle occurs at the NMJ level. The molecular mechanism that orchestrates the organization and functioning of synapses is highly complex, and it has not been completely elucidated so far. Neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates (EP). Acetylcholine receptor (AChR) clusterization at the end plates is required for an accurate synaptic transmission. This review will focus on some mechanisms responsible for accomplishing the correct distribution of AChRs at the synapses. Recent evidences support the concept that a dual transcriptional control of AChR genes in subsynaptic and extrasynaptic nuclei is crucial for AChR clusterization. Moreover, new players have been discovered in the agrin–MuSK pathway, the master organizer of postsynaptical differentiation. Mutations in this pathway cause neuromuscular congenital disorders. Alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting. Indeed, recent evidences demonstrate how NMJ misfunctioning has a crucial role at the onset of age-associated sarcopenia.

Foregut separation and tracheo-oesophageal malformations: The role of tracheal outgrowth, dorso-ventral patterning and programmed cell death

Foregut division—the separation of dorsal (oesophageal) from ventral (tracheal) foregut components—is a crucial event in gastro-respiratory development, and frequently disturbed in clinical birth defects. Here, we examined three outstanding questions of foregut morphogenesis. The origin of the trachea is suggested to result either from respiratory outgrowth or progressive septation of the foregut tube. We found normal foregut lengthening despite failure of tracheo-oesophageal separation in Adriamycin-treated embryos, whereas active septation was observed only in normal foregut morphogenesis, indicating a primary role for septation. Dorso-ventral patterning of Nkx2.1 (ventral) and Sox2 (dorsal) expression is proposed to be critical for tracheo-oesophageal separation. However, normal dorso-ventral patterning of Nkx2.1 and Sox2 expression occurred in Adriamycin-treated embryos with defective foregut separation. In contrast, Shh expression shifts dynamically, ventral-to-dorsal, solely during normal morphogenesis, particularly implicating Shh in foregut morphogenesis. Dying cells localise to the fusing foregut epithelial ridges, with disturbance of this apoptotic pattern in Adriamycin, Shh and Nkx2.1 models. Strikingly, however, genetic suppression of apoptosis in the Apaf1 mutant did not prevent foregut separation, indicating that apoptosis is not required for tracheo-oesophageal morphogenesis. Epithelial remodelling during septation may cause loss of cell-cell or cell-matrix interactions, resulting in apoptosis (anoikis) as a secondary consequence.

Apaf1 plays a pro-survival role by regulating centrosome morphology and function

The apoptotic protease activating factor 1 (Apaf1) is the main component of the apoptosome, and a crucial factor in the mitochondria-dependent death pathway. Here we show that Apaf1 plays a role in regulating centrosome maturation. By analyzing Apaf1-depleted cells, we have found that Apaf1 loss induces centrosome defects that impair centrosomal microtubule nucleation and cytoskeleton organization. This, in turn, affects several cellular processes such as mitotic spindle formation, cell migration and mitochondrial network regulation. As a consequence, Apaf1-depleted cells are more fragile and have a lower threshold to stress than wild-type cells. In fact, we found that they exhibit low Bcl-2 and Bcl-XL expression and, under apoptotic treatment, rapidly release cytochrome c. We also show that Apaf1 acts by regulating the recruitment of HCA66, with which it interacts, to the centrosome. This function of Apaf1 is carried out during the cell life and is not related to its apoptotic role. Therefore, Apaf1 might also be considered a pro-survival molecule, whose absence impairs cell performance and causes a higher responsiveness to stressful conditions.

Apaf1 mediates apoptosis and mitochondrial damage induced by mutant human SOD1s typical of familial amyotrophic lateral sclerosis

Several studies have indicated that apoptotic pathways are responsible for the loss of motor neurons that constitute the hallmark of amyotrophic lateral sclerosis (ALS). In this study, we demonstrate that apoptosis induced by the expression of several mutant Cu,Zn superoxide dismutases (SOD1) typical of familial ALS is mediated by Apaf1, a scaffold protein involved in neural development. Using different cell lines of neuronal origin and modulating the expression of both mutant SOD1s and Apaf1, we show that the removal of Apaf1 prevents cells death. Interestingly, intercepting activation of the caspases cascade is also effective in preventing both the mitochondrial damage and the increase in the production of reactive oxygen species induced by fALS-SOD1, even in the presence of cytochrome c release. This death pathway may be crucial also for the pathogenesis of the sporadic form of the disease, where markers of increased oxidative stress and mitochondria damage have been found.

S-Nitrosoglutathione Reductase Deficiency-Induced S-Nitrosylation Results in Neuromuscular Dysfunction

AIMS Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. RESULTS We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. INNOVATION Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. CONCLUSION These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

The metabolic modulator trimetazidine triggers autophagy and counteracts stress-induced atrophy in skeletal muscle myotubes

It has recently been demonstrated that trimetazidine (TMZ), an anti‐ischemic antianginal agent, is also able to improve exercise performance in patients with peripheral arterial disease. TMZ is a metabolic modulator, and the mechanisms underlying its cytoprotective anti‐ischemic activity could be ascribed, at least in cardiomyocytes, to optimization of metabolism. However, regarding the cytoprotection exerted by TMZ on skeletal muscle and allowing the improvement of exercise performance, no information is yet available. In the present study, we investigated in detail the protective effects of this drug on in vitro skeletal muscle models of atrophy. Experiments carried out with murine C2C12 myotubes treated with TMZ revealed that this drug could efficiently counteract the cytopathic effects induced by the proinflammatory cytokine tumor necrosis factor‐α and by the withdrawal of growth factors. Indeed, TMZ significantly counteracted the reduction in myotube size induced by these treatments. TMZ also increased myosin heavy chain expression and induced hypertrophy in C2C12 myotubes, both effects strongly suggesting a role of TMZ in counteracting atrophy in vitro. In particular, we found that TMZ was able to activate the phosphoinositide 3‐kinase–Akt–mammalian target of rapamycin 2 pathway and to reduce the stress‐induced transcriptional upregulation of atrogin‐1, muscle ring finger protein 1, and myostatin, all of which are key molecules involved in muscle wasting. Moreover, this is the first demonstration that TMZ induces autophagy, a key mechanism involved in muscle mass regulation. On the basis of these results, it can be hypothesized that the improvement in exercise performance previously observed in patients could be ascribed to a cytoprotective mechanism exerted by TMZ on skeletal muscle integrity.