Sirtuins and redox signaling interplay in neurogenesis, neurodegenerative diseases, and neural cell reprogramming.

Since the discovery of Neural Stem Cells (NSCs) there are still mechanism to be clarified, such as the role of mitochondrial metabolism in the regulation of endogenous adult neurogenesis and its implication in neurodegeneration. Although stem cells require glycolysis to maintain their stemness, they can perform oxidative phosphorylation and it is becoming more and more evident that mitochondria are central players, not only for ATP production but also for neuronal differentiation's steps regulation, through their ability to handle cellular redox state, intracellular signaling, epigenetic state of the cell, as well as the gut microbiota-brain axis, upon dietary influences. In this scenario, the 8-oxoguanine DNA glycosylase (OGG1) repair system would link mitochondrial DNA integrity to the modulation of neural differentiation. On the other side, there is an increasing interest in NSCs generation, from induced pluripotent stem cells, as a clinical model for neurodegenerative diseases (NDs), although this methodology still presents several drawbacks, mainly related to the reprogramming process. Indeed, high levels of reactive oxygen species (ROS), associated with telomere shortening, genomic instability, and defective mitochondrial dynamics, lead to pluripotency limitation and reprogramming efficiency's reduction. Moreover, while a physiological or moderate ROS increase serves as a signaling mechanism, to activate differentiation and suppress self-renewal, excessive oxidative stress is a common feature of NDs and aging. This ROS-dependent regulatory effect might be modulated by newly identified ROS suppressors, including the NAD+-dependent deacetylase enzymes family called Sirtuins (SIRTs). Recently, the importance of subcellular localization of NAD synthesis has been coupled to different roles for NAD in chromatin stability, DNA repair, circadian rhythms, and longevity. SIRTs have been described as involved in the control of both telomere's chromatin state and expression of nuclear gene involved in the regulation of mitochondrial gene expression, as well as in several NDs and aging. SIRTs are ubiquitously expressed in the mammalian brain, where they play important roles. In this review we summarize the current knowledge on how SIRTs-dependent modulation of mitochondrial metabolism could impact on neurogenesis and neurodegeneration, focusing mainly on ROS function and their role in SIRTs-mediated cell reprogramming and telomere protection.

Revisited role of TRAF2 and TRAF2 C-terminal domain in endoplasmic reticulum stress-induced autophagy in HAP1 leukemia cells.

The scaffold protein Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) has been reported to play a key role in the endoplasmic reticulum (ER) stress-induced activation of c-Jun N-terminal Kinase (JNK) and hence autophagy. Autophagy is a highly conserved catabolic process, whose dysregulation is involved in the pathogenesis of various human diseases, including cancer. We investigated the involvement of TRAF2 in autophagy regulation in the human leukemic HAP1 cell line, under both basal and ER stress conditions. In TRAF2-knockout HAP1 cell line (KO), the basal autophagic flux was higher than in the parental cell line (WT). Moreover, tunicamycin-induced ER stress stimulated JNK activation and autophagy both in WT and KO HAP1. On the other hand, re-expression of a TRAF2 C-terminal fragment (residues ,310-501), in a TRAF2-KO cellular background, rendered HAP1 cells unable to activate both JNK and autophagy upon ER stress induction. Of note, this apparent dominant negative effect of the C-terminal fragment was observed even in the absence of the endogenous, full-length TRAF2 molecule. Furthermore, the expression of the C-terminal fragment resulted in both protein kinase B (AKT) pathway activation and increased resistance to the toxic effects induced by prolonged ER stress conditions. These findings indicate that TRAF2 is dispensable for the activation of both JNK and autophagy in HAP1 cells, while the TRAF2 C-terminal domain may play an autonomous role in regulating the cellular response to ER stress.

Do You Remember Mitochondria?

Dementia is one among the consequences of aging, and amnesia is often one of the most common symptoms. The lack of memory, as a consequence of both "healthy" aging or neurodegenerative conditions, such as in Alzheimer's disease, has a dramatic impact on the patient's lifestyle. In fact, the inability to recall information made by a previous experience could not only alter the interaction with the environment, but also lead to a loss of identity. Mitochondria are key regulators of brain's activity; thanks to their "dynamic organelles" nature they constantly rearrange in the cell body and move along axons and dendrites, changing in dimension, shape, and location, accordingly to the cell's energy requirements. Indeed, the energy they can provide is essential to maintain synaptic plasticity and to ensure transmission through presynaptic terminals and postsynaptic spines. Stressful conditions, like the ones found in neurodegenerative diseases, seriously impair mitochondria bioenergetic, leading to both loss of proper neuronal interaction and of neuron themselves. Here, we highlighted the current knowledge about the role of mitochondria and mitochondrial dynamics in relation to neurodegenerative disorders linked to aging. Furthermore, we discuss the obstacles as well as the future perspectives aimed to enlarge our knowledge about mitochondria as target for new therapeutic strategies to slow down aging and neurodegenerative disease's symptoms.

A structure-based mechanism of cisplatin resistance mediated by glutathione transferase P1-1.

Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.

Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control.

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson's disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein-protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis.

Mitophagy in neurodegenerative diseases.

Neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and Amyotrophic Lateral Sclerosis (ALS), are a complex "family" of pathologies, characterised by the progressive loss of neurons and/or neuronal functions, leading to severe physical and cognitive inabilities in affected patients. These syndromes, despite differences in the causative events, the onset, and the progression of the disease, share as common features the presence of aggregate-prone neuro-toxic proteins, in the form of aggresomes and/or inclusion bodies, perturbing cellular homeostasis and neuronal function (Popovic et al., 2014), and the presence of dysfunctional mitochondria. The removal of protein aggregates and of damaged organelles, through the ubiquitin-proteasome system (UPS) and/or the autophagy/lysosome machinery, is a crucial step for the maintenance of neuronal homeostasis. Indeed, their impairment has been reported as associated with the development of these diseases. In this review, we focus on the role played by mitophagy, a specialised form of autophagy, in the onset and progression of major neurodegenerative diseases, as well as on possible therapeutic approaches involving mitophagy modulation.

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation.

Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold.

Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells.

Ophiobolin A, a fungal toxin from Bipolaris species known to affect different cellular processes in plants, has recently been shown to have anti-cancer activity in mammalian cells. In the present study, we investigated the anti-proliferative effect of Ophiobolin A on human melanoma A375 and CHL-1 cell lines. This cellular model was chosen because of the incidence of melanoma malignant tumor on human population and its resistance to chemical treatments. Ophyobolin A strongly reduced cell viability of melanoma cells by affecting mitochondrial functionality. The toxin induced depolarization of mitochondrial membrane potential, reactive oxygen species production and mitochondrial network fragmentation, leading to autophagy induction and ultimately resulting in cell death by activation of the mitochondrial pathway of apoptosis. Finally, a comparative proteomic investigation on A375 cells allowed to identify several Ophiobolin A down-regulated proteins, which are involved in fundamental processes for cell homeostasis and viability.

Autophagy in stem and progenitor cells.

Autophagy is a highly conserved cellular process, responsible for the degradation and recycling of damaged and/or outlived proteins and organelles. This is the major cellular pathway, acting throughout the formation of cytosolic vesicles, called autophagosomes, for the delivering to lysosome. Recycling of cellular components through autophagy is a crucial step for cell homeostasis as well as for tissue remodelling during development. Impairment of this process has been related to the pathogenesis of various diseases, such as cancer and neurodegeneration, to the response to bacterial and viral infections, and to ageing. The ability of stem cells to self-renew and differentiate into the mature cells of the body renders this unique type of cell highly crucial to development and tissue renewal, not least in various diseases. During the last two decades, extensive knowledge about autophagy roles and regulation in somatic cells has been acquired; however, the picture about the role and the regulation of autophagy in the different types of stem cells is still largely unknown. Autophagy is a major player in the quality control and maintenance of cellular homeostasis, both crucial factors for stem cells during an organism's life. In this review, we have highlighted the most significant advances in the comprehension of autophagy regulation in embryonic and tissue stem cells, as well as in cancer stem cells and induced pluripotent cells.

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states.

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293(TG)) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

BACKGROUND: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. METHODS: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. RESULTS: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. CONCLUSIONS: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

Proteomic analysis of mitochondrial dysfunction in neurodegenerative diseases.

Alzheimer's, Parkinson's and Huntington's disease, and amyotrophic lateral sclerosis are the most relevant neurodegenerative syndromes worldwide. The identification of the etiology and additional factors contributing to the onset and progression of these diseases is of great importance in order to develop both preventive and therapeutic intervention. A common feature of these pathologies is the formation of aggregates, containing mutated and/or misfolded proteins, in specific subsets of neurons, which progressively undergo functional impairment and die. The relationship between protein aggregation and the molecular events leading to neurodegeneration has not yet been clarified. In the last decade, several lines of evidence pointed to a major role for mitochondrial dysfunction in the onset of these pathologies. Here, we review how proteomics has been applied to neurodegenerative diseases in order to characterize the relationship existing between protein aggregation and mitochondrial alterations. Moreover, we highlight recent advances in the use of proteomics to identify protein modifications caused by oxidative stress. Future developments in this field are expected to significantly contribute to the full comprehension of the molecular mechanisms at the heart of neurodegeneration.

More than two sides of a coin? How to detect the multiple activities of type 2 transglutaminase.

Programmed cell death (PCD) by apoptosis has been widely characterized as a process in which the expression and protein activation of a gene must be regulated in a very precise way in order to achieve the elimination of the dying cell without disturbing the neighborhoods. One of the first genes observed to be induced during the onset of PCD is the one coding for type 2 transglutaminase (TG2). Since the late 1990s, the unveiling of different new properties and enzymatic activities suggested the involvement of TG2 in a variety of cellular processes other than PCD and rendered the study of this protein more and more complicated.

Type 2 transglutaminase in neurodegenerative diseases: the mitochondrial connection.

"Tissue" or type 2 Transglutaminase (TG2) is a peculiar multifunctional enzyme able to catalyse Ca(2+)-dependent post-translational modification of proteins, by establishing covalent bonds between peptide-bound glutamine residues and either lysine residues or mono- and poly-amines. In addition, it may act also as a G protein in transmembrane signalling, as a kinase, as a protein disulphide isomerase and as a cell surface adhesion mediator. The vast array of biochemical functions exerted by TG2 characterises and distinguishes it from all the other members of the transglutaminase family. Multiple lines of evidence suggest an involvement of the enzyme in neurodegenerative diseases, such as Huntington's (HD) and Parkinson (PD), and that its inhibition, either via drug treatments or genetic approaches, might be beneficial for the treatment of these syndromes. This review will exploit the recent developments in the comprehension of the role played by type 2 transglutaminase in eukaryotic cells, focusing on the role exerted by TG2 on mitochondrial physiology and on the regulation of cell death pathways at the basis of neurodegenerative diseases.

"Tissue" transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes.

In this study we provide the first in vivo evidences showing that, under physiological conditions, "tissue" transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype, resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function.

Tissue transglutaminase is a multifunctional BH3-only protein.

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.

Bak: a downstream mediator of fenretinide-induced apoptosis of SH-SY5Y neuroblastoma cells.

Unlike 13-cis-retinoic acid, the synthetic retinoid fenretinide [N-(4-hydroxyphenyl)retinamide] induces apoptosis of neuroblastoma cells by mechanisms involving retinoic acid receptors and oxidative stress. After screening a cDNA array for apoptosis-related genes, the Bcl2-related protein Bak was identified as a fenretinide-inducible gene in SH-SY5Y neuroblastoma cells, and this was confirmed by Western blotting and flow cytometry. Although fenretinide acts synergistically in vitro with chemotherapeutic drugs, these drugs did not induce Bak expression. Retinoic acid receptor antagonists did not block the induction of Bak by fenretinide. Conversely, Bak induction was blocked by the antioxidant vitamin C. Overexpression of Bak increased apoptosis in both the presence and absence of fenretinide, whereas expression of antisense Bak inhibited fenretinide-induced apoptosis. Bak expression was also induced in cells overexpressing the stress-induced transcription factor GADD153, but Bak expression was inhibited in cells expressing an antisense GADD153 construct. These results suggest that Bak is a downstream mediator of an oxidative stress pathway leading to apoptosis of SH-SY5Y neuroblastoma cells in response to fenretinide.