INTERAZIONE TRA LE PROTEINE VSP ED ARMS

Le interazioni proteina-proteina e la formazione di complessi multiproteici sono meccanismi fondamentali in molti processi biologici, quali la trasmissione dei segnali provenienti dall’esterno, l’adesione cellulare, lo sviluppo e la comunicazione tra cellule. Specifiche proteine svolgono un ruolo cruciale per mediare queste interazioni e reclutare recettori, enzimi, adattatori in determinati compartimenti cellulari. Questa tesi si propone di caratterizzare la proteina VSP (Ventral Eye Staining Protein), il cui gene, identificato in Danio rerio, è prevalentemente espresso nella retina neurale ventrale, nel tratto iniziale del peduncolo ottico e nei gangli dorsali durante gli stadi precoci dello sviluppo; successivamente la sua espressione è estesa anche ad altre regioni del sistema nervoso. Un potenziale interattore di VSP è la proteina ARMS (Ankiryn Rich-repeat Membrane Spanning), precedentemente isolata nel nostro laboratorio mediante esperimenti di phage display. Anche questa proteina è espressa specificatamente nei gangli dorsali e in varie regioni del sistema nervoso; inoltre è stato dimostrato che si concentra alla punta dei neuriti in cellule neuroendocrine PC12 indotte con NGF (Bracale et al., 2006). Durante il mio progetto di tesi ho analizzato l’espressione di queste due proteine, prodotte in forma ricombinante come prodotti di fusione ad epitopi facilmente riconoscibili da anticorpi di uso commerciale (HA e FLAG), utilizzando come sistemi modello per studiarne le interazioni cellule neuroendocrine di ratto, PC12 e cellule umane embrionali di rene, Hek293. Mediante trasfezione, analisi elettroforetica e Western blotting ho potuto verificare la corretta produzione delle proteine ricombinanti. Il passo successivo è stato effettuare esperimenti di co-immunoprecipitazione e co-localizzazione, che hanno permesso di dimostrare l’interazione diretta tra le due proteine. In particolare, la proteina VSP lega l’estremità carbossi-terminale di ARMS, tramite un dominio strutturale PDZ, specializzato per riconoscere specificamente gli ultimi quattro residui del bersaglio. Ulteriori esperimenti di co-immunoprecipitazione e co-localizzazione per confermare l’interazione tra le corrispondenti proteine endogene sono attualmente in fase di esecuzione

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (A\u3b2) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4R\u394C20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10\u202fnm) and the long- (40-50\u202fnm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4R\u394C20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

UNLABELLED Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but thatbokwas not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found thatbok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injuryin vitroand seizure-induced neuronal injuryin vivo Deletion ofbokalso increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death inbax-deficient neurons. Single-cell imaging demonstrated thatbok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bokdeficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death inbok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injuryin vitroandin vivo SIGNIFICANCE STATEMENT Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activitiesin vitroandin vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok

Neuronal cell death is often triggered by events that involve intracellular increases in Ca(2+). Under resting conditions, the intracellular Ca(2+) concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca(2+) overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca(2+)-dependent cell death. Excessive Ca(2+) entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer\u27s disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca(2+) homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca(2+)-dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca(2+) homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the \u27pro-apoptotic\u27 Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such \u27non-apoptotic/daytime\u27 functions

Characterisation of calpain activation in response to excitotoxic events in primary neurons

Excitotoxicity resulting from excessive Ca²+ influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca²+ levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that underwent either: (i) excitotoxic necrosis, characterized by immediate deregulation of Ca²+ homeostasis, a persistent depolarization of mitochondrial membrane potential (Ay/m), and insensitivity to bax-gene deletion; (ii) excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca²+ increases, sensitivity to bax gene deletion, and delayed Ai//m depolarization and Ca²+ deregulation; or (iii) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis, but did not influence excitotoxic nccrosis. Calpeptin failed to exert a protective effect in oax-deficient neurons, but protected bid- and e/'m-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy, using a calpain-sensitive Forster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injuiy. Oxygen/glucose deprivationinduced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during .ax-dependent apoptosis and in this setting function as downstream cell death executioners. Lastly, box gene deletion prevented NMDA-induced alterations to Ay/m, hyperpolarisation and depolarisation were notably diminished, and affected neuronal calcium handling, resulting in increased neuronal survival.</p

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions.

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult

AMPK‐regulated miRNA‐210‐3p is activated during ischaemic neuronal injury and modulates PI3K‐p70S6K signalling

AbstractProgressive neuronal injury following ischaemic stroke is associated with glutamate‐induced depolarization, energetic stress and activation of AMP‐activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR‐210‐3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR‐210‐3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3‐phosphoinositide‐dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR‐210‐3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over‐activation in primary neurons, we demonstrated that induction of miR‐210‐3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR‐210‐3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR‐210‐3p to control p70S6K activity in ischaemic stroke and excitotoxic injury. imag

AMPK-regulated miRNA-210-3p is activated during ischaemic neuronal injury and modulates P13K-p70S6K signalling

Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.<br