Low central venous saturation predicts poor outcome in patients with brain injury after major trauma: a prospective observational study

BACKGROUND: Continuous monitoring of central venous oxygen saturation (ScvO(2)) has been proposed as a prognostic indicator in several pathological conditions, including cardiac diseases, sepsis, trauma. To our knowledge, no studies have evaluated ScvO(2 )in polytraumatized patients with brain injury so far. Thus, the aim of the present study was to assess the prognostic role of ScvO(2 )monitoring during first 24 hours after trauma in this patients' population. METHODS: This prospective, non-controlled study, carried out between April 2006 and March 2008, was performed in a higher level Trauma Center in Florence (Italy). In the study period, 121 patients affected by major brain injury after major trauma were recruited. Inclusion criteria were: 1. Glasgow Coma Scale (GCS) score ≤ 13; 2. an Injury Severity Score (ISS) ≥ 15. Exclusion criteria included: 1. pregnancy; 2. age < 14 years; 3. isolated head trauma; 4. death within the first 24 hours from the event; 5. the lack of ScvO(2 )monitoring within 2 hours from the trauma. Demographic and clinical data were collected, including Abbreviated Injury Scale (AIS), Injury Severity Score (ISS), Simplified Acute Physiologic Score II (SAPS II), Marshall score. The worst values of lactate and ScvO(2 )within the first 24 hours from trauma, ICU length of stay (LOS), and 28-day mortality were recorded. RESULTS: Patients who deceased within 28 days showed higher age (53 ± 16.6 vs 43.8 ± 19.6, P = 0.043), ISS core (39.3 ± 14 vs 30.3 ± 10.1, P < 0.001), AIS score for head/neck (4.5 ± 0.7 vs 3.4 ± 1.2, P = 0.001), SAPS II score (51.3 ± 14.1 vs 42.5 ± 15, P = 0.014), Marshall Score (3.5 ± 0.7 vs 2.3 ± 0.7, P < 0.001) and arterial lactate concentration (3.3 ± 1.8 vs 6.7 ± 4.2, P < 0.001), than survived patients, whereas ScvO(2 )resulted significantly lower (66.7% ± 11.9 vs 70.1% ± 8.9 vs, respectively; P = 0.046). Patients with ScvO(2 )values ≤ 65% also showed higher 28-days mortality rate (31.3% vs 13.5%, P = 0.034), ICU LOS (28.5 ± 15.2 vs 16.6 ± 13.8, P < 0.001), and total hospital LOS (45.1 ± 20.8 vs 33.2 ± 24, P = 0.046) than patients with ScvO(2 )> 65%. CONCLUSION: ScvO(2 )value less than 65%, measured in the first 24 hours after admission in patients with major trauma and head injury, was associated with higher mortality and prolonged hospitalization

Low central venous saturation predicts poor outcome in patients with brain injury after major trauma: a prospective observational study

Abstract Background Continuous monitoring of central venous oxygen saturation (ScvO2) has been proposed as a prognostic indicator in several pathological conditions, including cardiac diseases, sepsis, trauma. To our knowledge, no studies have evaluated ScvO2 in polytraumatized patients with brain injury so far. Thus, the aim of the present study was to assess the prognostic role of ScvO2 monitoring during first 24 hours after trauma in this patients' population. Methods This prospective, non-controlled study, carried out between April 2006 and March 2008, was performed in a higher level Trauma Center in Florence (Italy). In the study period, 121 patients affected by major brain injury after major trauma were recruited. Inclusion criteria were: 1. Glasgow Coma Scale (GCS) score ≤ 13; 2. an Injury Severity Score (ISS) ≥ 15. Exclusion criteria included: 1. pregnancy; 2. age 2 monitoring within 2 hours from the trauma. Demographic and clinical data were collected, including Abbreviated Injury Scale (AIS), Injury Severity Score (ISS), Simplified Acute Physiologic Score II (SAPS II), Marshall score. The worst values of lactate and ScvO2 within the first 24 hours from trauma, ICU length of stay (LOS), and 28-day mortality were recorded. Results Patients who deceased within 28 days showed higher age (53 ± 16.6 vs 43.8 ± 19.6, P = 0.043), ISS core (39.3 ± 14 vs 30.3 ± 10.1, P 2 resulted significantly lower (66.7% ± 11.9 vs 70.1% ± 8.9 vs, respectively; P = 0.046). Patients with ScvO2 values ≤ 65% also showed higher 28-days mortality rate (31.3% vs 13.5%, P = 0.034), ICU LOS (28.5 ± 15.2 vs 16.6 ± 13.8, P 2 > 65%. Conclusion ScvO2 value less than 65%, measured in the first 24 hours after admission in patients with major trauma and head injury, was associated with higher mortality and prolonged hospitalization.</p

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate

Background Lrrk2, a gene linked to Parkinson\u2019s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Results Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Conclusions Given that the most common Parkinson\u2019s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF

GTP binding regulates cellular localization of Parkinso\u144s disease-associated LRRK2

Mutations in LRRK2 comprise the most common cause of familial Parkinso\u144s disease (PD), and sequence variants modify risk for sporadic PD. Previous studies indicate that LRRK2 interacts with microtubules and alters microtubule-mediated vesicular transport processes. However, the molecular determinants within LRRK2 required for such interactions have remained unknown. Here we report that most pathogenic LRRK2 mutants cause relocalization of LRRK2 to filamentous structures which colocalize with a subset of microtubules, and an identical relocalization is seen upon pharmacological LRRK2 kinase inhibition. The pronounced colocalization with microtubules does not correlate with alterations in LRRK2 kinase activity, but rather with increased GTP binding. Synthetic mutations which impair GTP binding, as well as LRRK2 GTP-binding inhibitors profoundly interfere with the abnormal localization of both pathogenic mutant as well as kinase-inhibited LRRK2. Conversely, addition of a non-hydrolyzable GTP analog to permeabilized cells enhances the association of pathogenic or kinase-inhibited LRRK2 with microtubules. Our data elucidate the mechanism underlying the increased microtubule association of select pathogenic LRRK2 mutants or of pharmacologically kinase-inhibited LRRK2, with implications for downstream MT-mediated transport events

MFN2 mutations in Charcot-Marie-Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT

Concentrations of potentially toxic elements and soil environmental quality evaluation of a typical Prosecco vineyard of the Veneto region (NE Italy)

Purpose The aim of this work was to assess the concentrations of potentially toxic elements and to evaluate the soil quality of a typical Prosecco Denomination of Controlled and Guaranteed Origin vineyard of the Veneto region, NE Italy. Materials and methods Soil samples and leaves of Taraxacum officinale and Vitis vinifera were collected during spring–summer 2014. Element determination (Al, Cd, Cr, Cu, Fe, Mg, Mn, Ni, P, Pb, V, and Zn) were performed with ICP-OES after microwave digestion of samples. Soil quality was assessed via the biological soil quality (BSQ-ar) index. Lipid peroxidation test was performed to evaluate the vegetation oxidative stress, based on malondialdehyde (MDA) content via spectrophotometer. Results and discussion High concentrations of Al,Mg, and P were identified in soil, while high contents of Al, Cu, Fe, and Zn were found in V. vinifera leaves. The high concentrations in soil are probably due to agricultural activities, whereas those in leaves are probably due to atmospheric deposition and repeated use of foliar sprays in viticulture. The bioconcentration factor showed an effective transport of Cu, P, and Zn, from soil to leaf. The BSQ-ar values registered were similar to those obtained in preserved soils; hence, the biological class (VI) of these soils is high. The MDA content in T. officinale and V. vinifera leaves was below the reference value for T. officinale (2.9 ± 0.2 μM), suggesting that the metal content did not stress the vegetation in the investigated site. Conclusions The MDA value for V. vinifera (1.1 ± 0.7 μM) could be adopted as another control value for soil quality, which in our case is of Bgood quality.^ Moreover, our results suggest that high concentrations of elements detected in the analyzed samples do not influence negatively the quality of soil, but a better agronomic management could improve soil quality in the studied area

Novel LRRK2 substrates at the presynaptic site in vesicle recycling pathways in Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) affecting about 1-2% of the population over 65 years of age (Farrer, 2006). The pathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of intracellular proteinaceous inclusions called “Lewy bodies” (LB) in surviving neurons (Damier et al., 1999; Frank et al., 2007). The etiology of PD is unknown, with a complex relationship between environmental and genetic factors, and aging being a required factor. The majority of cases, about 90-95%, is sporadic, while the remaining 5-10% can be explained by mutations in single genes (Van Den Eeden et al., 2003). The discovery of PD-related genes holds the potential to shed light into disease treatments, since the sporadic and familial forms are likely to share common pathogenic mechanisms and compromised pathways (Lesage and Brice, 2012). To date, five genes have been conclusively associated to familial PD, with both autosomal-dominant and autosomal-recessive modes of inheritance (Valente et al., 2004; Lakshminarasimhan et al., 2008; Nemani et al., 2013; Singleton et al., 2013; Kuang et al., 2013). Accordingly, several studies focused on the physiological role of PD-related proteins and on how pathogenic mutations cause the pathology. In 2004, two studies found that mutations in Lrrk2 (PARK8) are responsible for a familial form of PD (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) and, currently, mutations in this gene represent the most common genetic cause of PD (10%). Lrrk2 encodes leucine-rich repeat kinase 2 (LRRK2), a large multi-domain protein with both GTPase and kinase activities (Marín et al., 2008). The majority of LRRK2 pathological mutations are located within the enzymatic core of the protein, and they can affect LRRK2 activity causing impaired cellular functions and cytotoxicity. Among all, the G2019S mutation is the most frequent (Gilks et al. 2005), thus the most studied. This mutation lies in the kinase domain and increases LRRK2 kinase activity (Greggio and Cookson, 2009). Several studies support a role for LRRK2 in synaptic vesicle trafficking, although the exact mechanism is unclear. Thus, understanding which pathways are compromised in pathological conditions is fundamental to develop efficient strategies against synaptic impairment in PD. While LRRK2 pharmacological inhibition may block the PD-related LRRK2 phenotypes, the use of LRRK2 inhibitors has been shown to cause severe side-effects on peripheral organs (Baptista et al., 2013; Luerman et al., 2014). Thus, alternative therapeutic strategies may be directed toward other proteins that take part in LRRK2 pathways. In this scenario, identifying putative LRRK2 substrates at the presynaptic site, among the plethora of possible interactors, is fundamental to understand the downstream effects related to LRRK2 pathological mutations. Previous studies showed that LRRK2 can modulate synaptic vesicle trafficking through phosphorylation of components of the exo- endocytic machinery (Heo et al., 2010; Matta et al., 2012; Yun et al., 2013). For this reason, an outstanding question is to identify physiologically relevant, substrates of LRRK2 kinase activity and the effects of LRRK2 PD-related mutations on substrate phosphorylation. In this thesis, we focused on N-ethylmaleimide Sensitive Fusion protein (NSF) and Rab7L1. These two proteins have been previously indicated as LRRK2 interactors in neurons (Piccoli et al., 2011; MacLeod et al., 2013, Beilina et al., 2014). Here, our aim was to test whether they are also substrates of LRRK2 kinase activity. NSF is an AAA+ (ATPases Associated with various cellular Activities) and its function is pivotal at the presynaptic site for proper synaptic vesicle recycling. More in detail, NSF uses the energy produced from ATP hydrolysis to disassemble SNARE proteins (Soluble N-ethylmaleimide Attachment protein REceptors), together with its adaptor protein alpha-SNAP (Soluble NSF Associated Protein), allowing them to another fusion cycle (Zhao and Brunger, 2015). NSF displays a homo-hexameric structure, where each monomer is composed by three different domains: the N-terminal domain (N-ter) required for alpha-SNAP:SNARE complex interaction, the D1 domain with an ATP-binding site necessary for the ATPase activity and a D2 domain with another ATP-binding site required for oligomerization (Zhao et al., 2015). Since NSF has been largely studied using non-human orthologous proteins (Chang et al., 2012; Cipriano et al., 2013; Vivona et al., 2013; Zhao et al., 2015), we set up a protocol to purify human Flag-tagged NSF from HEK293T mammalian cells. Firstly, we investigated the biochemical properties of NSF and subsequently its interaction with LRRK2. We demonstrated that the purified protein is an active ATPase and is able to interact with LRRK2. In particular, data obtained from pull-down assays revealed an interaction between NSF D2 domain and LRRK2. Moreover, we demonstrated that NSF is also a substrate of LRRK2 kinase activity and this phosphorylation preferentially occurs at Thr-645 in the D2 domain. We confirmed this result by measuring the 33P incorporation with kinase assays incubating NSF non-phosphorylatable mutants (NSF-T645A, T646A and S647A) together with LRRK2 G2019S. Kinetic studies of NSF ATPase activity revealed that NSF is 2-fold more active upon LRRK2 G2019S phosphorylation. Noteworthy, NSF-T645A, which resulted in a 50% reduction of 33P incorporation compared to wild-type, abolishes LRRK2-mediated increased ATPase activity. In addition, we demonstrated that NSF disassembles the SNARE complex at a higher rate after phosphorylation by LRRK2 G2019S. Taken together, these results highlight a possible regulatory mechanism in which LRRK2 is involved in synaptic vesicle recycling through phosphorylation of NSF. Importantly, NSF ATPase activity could be compromised by excessive phosphorylation due to LRRK2 G2019S pathological mutation. As mentioned, an important aspect in the development of PD may be linked to impaired synaptic vesicle trafficking, which may trigger neurodegeneration at early stages. A major class of proteins orchestrating vesicle sorting inside the cell are Rab proteins. Rabs constitute a large family of monomeric small GTPases associated with all cellular compartments (Grosshans et al., 2006). There are more than 60 different Rab proteins in humans (Schwartz et al., 2007; Pereira-Leal et al., 2001) and a switch between the GTP- (active) and GDP- (inactive) bound form regulates the interactions with their effectors. To date, several studies highlight a possible interaction between LRRK2 and a number of Rabs (MacLeod et al., 2013; Gomez-Suaga et al., 2014; Dodson et al., 2014; Waschbusch et al., 2014). Here, we tested whether different Rab proteins are also substrates of LRRK2 kinase activity. We performed kinase experiments on Rab7, Rab7L1, Rab9, Rab11 and Rab32. Kinase assays revealed that, among them, only Rab7L1 is a substrate of both LRRK2 wild-type and G2019S kinase activity. Rab7L1 is located within the PARK16 locus, a non-familial PD risk-associated locus, and Rab7L1 has been found to associate with LRRK2 to regulate the degradation of trans-Golgi derived vesicles (Beilina 2014). Mass spectrometry analysis revealed that phosphorylation by LRRK2 G2019S preferentially occurs on Rab7L1-T21, a residue localized in the highly conserved region responsible for GTP binding (P-loop region). In addition, we detected Rab7L1-S22 as a less probable phosphorylation site. To test which residue was phosphorylated, we generated Rab7L1-T21A and S22A mutants. Kinase assays indicate that Rab7L1 likely possesses additional sites, other than T21 or S22, able to be phosphorylated by LRRK2 G2019S. In summary, this work identified two novel substrates of LRRK2 kinase activity in vitro with potential relevance for disease. Our studies revealed an increased NSF ATPase activity upon LRRK2 G2019S phosphorylation and highlighted a novel regulatory mechanism that might be compromised in PD. In addition, we found that Rab7L1 is another substrate of LRRK2 kinase activity. Future studies should uncover whether NSF and Rab7L1 are substrates of LRRK2 also in the cellular context and whether pathological phosphorylation is relevant for PD.La malattia di Parkinson (PD) è la seconda malattia neurodegenerative più comune dopo la malattia di Alzheimer (AD) e colpisce circa l’1-2% della popolazione oltre I 65 anni (Farrer, 2006). Gli elementi caratteristici del morbo di Parkinson sono la perdita dei neuroni nella sustantia nigra pars compacta (SNpc), e la presenza di aggregati proteici intracellulari denominati corpi di Lewy (LB) nei neuroni che sopravvivono (Damier et al., 1999; Frank et al., 2007). L’eziologia della malattia di Parkinson è sconosciuta, con una complessa correlazione tra fattori ambientali e genetici, e fattori legati all’invecchiamento. La maggior parte dei casi, circa 95%, è di origina sporadica, mentre il rimanente 5-10% può essere collegato a mutazioni in singoli geni (Van Den Eeden et al., 2003). La scoperta di geni collegati al morbo di Parkinson ha messo in luce possibili trattamenti terapeutici, dato che la forma sporadica e quella familiare hanno in comune molti meccanismi patologici e pathway compromessi (Lesage and Brice, 2012). Ad oggi, cinque geni sono stati associati alla forma familiare del morbo di Parkinson, sia ad una forma genetica autosomica dominante che recessiva (Valente et al., 2004; Lakshminarasimhan et al., 2008; Nemani et al., 2013; Singleton et al., 2013; Kuang et al., 2013). Per questo motivo, numerosi studi si sono focalizzati sul ruolo fisiologico delle proteine collegate alla malattia di Parkinson e come le mutazioni patologiche causino la patologia. Nel 2004, due studi hanno identificato come mutazioni nel gene Lrrk2 (PARK8) siano causa della forma familiare di morbo di Parkinson (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) e, ad oggi, mutazioni all’interno di questo gene rappresentano la più comune causa genetica di malattia di Parkinson (10%). Lrrk2 Questo gene codifica per la proteina leucine-rich repeat kinase 2 (LRRK2), una grande proteina composta da vari domini con attività GTPasica e chinasica (Marín et al., 2008). La maggior parte delle mutazioni sono localizzate all’interno del core enzimatico della proteina, e possono colpire l’attività di LRRK2 causando un danno alle funzioni cellulari e citotossicità. Tra tutte, la mutazione G2019S è la più frequente (Gilks et al. 2005), quindi la più studiata. Questa mutazione avviene all’interno del dominio chinasico e aumenta l’attività chinasica di LRRK2 (Greggio and Cookson, 2009). Numerosi studi sostengono un ruolo di LRRK2 a livello del traffico delle vescicole sinaptiche, anche se il meccanismo esatto non è ancora chiaro. Perciò, capire quali pathway sono compromessi in condizioni patologiche è fondamentale per sviluppare strategie efficienti contro i danni provocati dalla malattia di Parkinson. Mentre l’inibizione farmacologica di LRRK2 potrebbe bloccare il fenotipo patologico, l’uso di inibitori di LRRK2 ha mostrato effetti secondari gravi a livello degli organi periferici (Baptista et al., 2013; Luerman et al., 2014). Per questo motivo, strategie terapeutiche alternative potrebbero essere dirette verso altre proteine che sono coinvolte all’interno dei pathway di LRRK2. In questo scenario, l’identificazione di substrati di LRRK2 a livello presinaptico, tra tutti i possibili interattori, è fondamentale per capire gli effetti a valle relativi a mutazioni patologiche di LRRK2. Studi precedenti hanno dimostrato che LRRK2 può modulare il traffico delle vescicole sinaptiche attraverso la fosforilazione di componenti facenti parte dei processi di eso- ed endocitosi (Heo et al., 2010; Matta et al., 2012; Yun et al., 2013). Per questa ragione, una domanda ancora senza risposta è quella di identificare possibili substrati dell’attività chinasica di LRRK2, rilevanti a livello fisiologico, e gli effetti che le mutazioni di LRRK2 associate al morbo di Parkinson hanno sulla fosforilazione. In questa tesi, ci siamo focalizzati sulla proteina N-ethylmaleimide Sensitive Fusion (NSF) protein e Rab7L1. Queste due proteine sono state precedentemente indicate come interattori di LRRK2 nei neuroni (Piccoli et al., 2011; MacLeod et al., 2013, Beilina et al., 2014). In questo lavoro, il nostro scopo era quello di testare se queste due proteine siano anche substrati dell’attività chinasica di LRRK2. NSF è classificata come proteina AAA+ (ATPases Associated with various cellular Activities) e la sua funzione è fondamentale a libello presinaptico per un corretto riciclo delle vescicole sinaptiche. In dettaglio, NSF usa l’energia prodotta dall’idrolisi dell’ATP per disassemblare le proteine del complesso SNARE (Soluble N-ethylmaleimide Attachment protein REceptors), insieme alla sua proteina adattatrice alpha-SNAP (Soluble NSF Associated Protein), consentendo un nuovo ciclo di fusione (Zhao and Brunger, 2015). NSF mostra una struttura omo-esamerica, dove ogni monomero è composto da tre differenti domini: l’N-terminale (N-ter) necessario all’interazione con alpha-SNAP ed il complesso SNARE, il dominio D1 con un sito di legame dell’ATP necessaria per l’attività ATPasica, ed un dominio D2 con un altro dito di legame per l’ATP richiesta all’oligomerizzazione (Zhao et al., 2015). Dato che NSF è stato largamente studiato usando una proteina ortologa non umana, abbiamo messo a punto un protocollo per purificare NSF umano con un Flag-tag da cellule di mammifero HEK293T. Per prima cosa, abbiamo deciso di studiare la biochimica di NSF e successivamente la sua interazione con LRRK2. Abbiamo dimostrato che la proteina purificata è attiva ed è in grado di interagire con LRRK2. In particolare, i risultati ottenuti attraverso esperimenti di pull-down rivelano una interazione tra il dominio D2 di NSF e LRRK2. Inoltre, abbiamo dimostrato che NSF è anche un substrato dell’attività chinsica di LRRK2 e questa fosforilazione avviene preferenzialmente nella treonina-645 all’interno del dominio D2. Abbiamo confermato questo risultato misurando l’incorporazione di 33P con saggi chinasici incubando dei mutanti non-fosforilabili di NSF (NSF-T645A, T646A and S647A) insieme a LRRK2 G2019S, Studi cinetici sull’attività ATPasica di NSF hanno rivelato che NSF è due volte più attivo dopo la fosforilazione da parte di LRRK2 G2019S. E’ importante notare che il mutante NSF-T645A, dove l’incorporazione di 33P risulta essere il 50% in meno rispetto al wild-type, non presenta un aumento dell’attività ATPasoca dovuto alla fosforilazione ad opera di LRRK2 G2019S. Inoltre, abbiamo dimostrato che NSF dopo essere stato fosforilato da LRRK2 G2019S disassembla il complesso SNARE più velocemente. Tutti questi risultati evidenziano un possibile meccanismo regolatorio in cui LRRK2 è implicato all’interno del riciclo delle vescicole sinaptiche attraverso la fosforilazione di NSF. L’attività ATPasica di NSF potrebbe quindi essere compromessa da una sua aumentata fosforilazione ad opera del mutante patologico G2019S. Come illustrato precedentemente, un aspetto importante nello sviluppo della malattia di Parkinson potrebbere essere collegato ad un danno nel traffico delle vescicole, che potrebbe far scaturire la neurodegenerazione a stadi precoci (Fernandez-Chacon et al., 2004; Burre et al., 2010). La principale classe di proteine che governa l’organizzazione delle vescicole all’interno della cellula, è composta dalle proteine Rab. Esse costituiscono una vasta famiglia di piccole GTPasi monomeriche associate a tutti i compartimenti cellulari (Grosshans et al., 2006). Nell’uomo ci sono più di 60 tipi di Rab ((Schwartz et al., 2007; Pereira-Leal et al., 2001)) che passando da una forma attiva, legata al GTP, ad una forma inattiva, legata al GDP, regolando la loro interazione con proteine effettrici. Ad oggi, numerosi studi hanno evidenziato una possibile interazione tra LRRK2 e varie proteine Rab (MacLeod et al., 2013; Gomez-Suaga et al., 2014; Dodson et al., 2014; Waschbusch et al., 2014). Nel nostro lavoro, abbiamo testato se alcune proteine Rab siano anche substrati dell’attività chinasica di LRRK2. Abbiamo eseguito saggi chinasici con varie Rab, su Rab7, Rab7L1, Rab9, Rab11 e Rab32. I saggi chinasici hanno rivelato che, tra tutte, solamente Rab7L1 è un substrato di LRRK2, sia wild-type che G2019S. Il gene Rab7L1 è localizzato all’interno del locus PARK16, un locus associato al richio di insorgenza della forma di malattia di Parkinson non-familiare, Rab7L1 è stato trovato associato a LRRK2 nella regolazione della degradazione delle vescicole a livello dell’apparato del Golgi (Beilina 2014). Analisi di spettrometria di massa hano rivelato comee la fosforilazione ad opera di LRRK2 G2019S avvenga preferenzialmente sulla treonina-21 di Rab7L1, un residuo localizzato nella regione altamente conservata responsabile del legame col GTP (P-loop). In aggiunta, abbiamo individuato come meno probabile sito di fosforilazione la serina-22. Per testare quale residuo fosse quello fosforilato, abbiamo generato i mutati non fosforilabili Rab7L1-T21A ed S22A. I saggi chinasici ci hanno indicato che Rab7L1 possiede verosimilmente siti di fosforilazione aggiuntivi, oltre la T21 e la S22, che possono essere fosforilati da LRRK2 G2019S. Riassumendo, questo lavoro ha identificato due nuovi substrati dell’attività chinasica di LRRK2 in vitro con una potenziale rilevanza a livello della malattia. I nostri studi hanno rivelato un aumento dell’attività ATPasica di NSF successivamente alla fosforilazione da parte di LRRK2 G2019S ed hanno evidenziato un nuovo meccanismo regolatorio che potrebbe essere compromesso nella malattia di Parkinson. In aggiunta, abbiamo trovato che Rab7L1 è un altro substrato dell’attività chinasica di LRRK2. Studi futuri sono necessari per scoprire se NSF e Rab7L1 siano substrati di LRRK2 anche in un contesto cellulare, e se la fosforilazione in condizioni patologiche è rilevante nel morbo di Parkinson

Novel LRRK2 substrates at the presynaptic site in vesicle recycling pathways in Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) affecting about 1-2% of the population over 65 years of age (Farrer, 2006). The pathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of intracellular proteinaceous inclusions called “Lewy bodies” (LB) in surviving neurons (Damier et al., 1999; Frank et al., 2007). The etiology of PD is unknown, with a complex relationship between environmental and genetic factors, and aging being a required factor. The majority of cases, about 90-95%, is sporadic, while the remaining 5-10% can be explained by mutations in single genes (Van Den Eeden et al., 2003). The discovery of PD-related genes holds the potential to shed light into disease treatments, since the sporadic and familial forms are likely to share common pathogenic mechanisms and compromised pathways (Lesage and Brice, 2012). To date, five genes have been conclusively associated to familial PD, with both autosomal-dominant and autosomal-recessive modes of inheritance (Valente et al., 2004; Lakshminarasimhan et al., 2008; Nemani et al., 2013; Singleton et al., 2013; Kuang et al., 2013). Accordingly, several studies focused on the physiological role of PD-related proteins and on how pathogenic mutations cause the pathology. In 2004, two studies found that mutations in Lrrk2 (PARK8) are responsible for a familial form of PD (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) and, currently, mutations in this gene represent the most common genetic cause of PD (10%). Lrrk2 encodes leucine-rich repeat kinase 2 (LRRK2), a large multi-domain protein with both GTPase and kinase activities (Marín et al., 2008). The majority of LRRK2 pathological mutations are located within the enzymatic core of the protein, and they can affect LRRK2 activity causing impaired cellular functions and cytotoxicity. Among all, the G2019S mutation is the most frequent (Gilks et al. 2005), thus the most studied. This mutation lies in the kinase domain and increases LRRK2 kinase activity (Greggio and Cookson, 2009). Several studies support a role for LRRK2 in synaptic vesicle trafficking, although the exact mechanism is unclear. Thus, understanding which pathways are compromised in pathological conditions is fundamental to develop efficient strategies against synaptic impairment in PD. While LRRK2 pharmacological inhibition may block the PD-related LRRK2 phenotypes, the use of LRRK2 inhibitors has been shown to cause severe side-effects on peripheral organs (Baptista et al., 2013; Luerman et al., 2014). Thus, alternative therapeutic strategies may be directed toward other proteins that take part in LRRK2 pathways. In this scenario, identifying putative LRRK2 substrates at the presynaptic site, among the plethora of possible interactors, is fundamental to understand the downstream effects related to LRRK2 pathological mutations. Previous studies showed that LRRK2 can modulate synaptic vesicle trafficking through phosphorylation of components of the exo- endocytic machinery (Heo et al., 2010; Matta et al., 2012; Yun et al., 2013). For this reason, an outstanding question is to identify physiologically relevant, substrates of LRRK2 kinase activity and the effects of LRRK2 PD-related mutations on substrate phosphorylation. In this thesis, we focused on N-ethylmaleimide Sensitive Fusion protein (NSF) and Rab7L1. These two proteins have been previously indicated as LRRK2 interactors in neurons (Piccoli et al., 2011; MacLeod et al., 2013, Beilina et al., 2014). Here, our aim was to test whether they are also substrates of LRRK2 kinase activity. NSF is an AAA+ (ATPases Associated with various cellular Activities) and its function is pivotal at the presynaptic site for proper synaptic vesicle recycling. More in detail, NSF uses the energy produced from ATP hydrolysis to disassemble SNARE proteins (Soluble N-ethylmaleimide Attachment protein REceptors), together with its adaptor protein alpha-SNAP (Soluble NSF Associated Protein), allowing them to another fusion cycle (Zhao and Brunger, 2015). NSF displays a homo-hexameric structure, where each monomer is composed by three different domains: the N-terminal domain (N-ter) required for alpha-SNAP:SNARE complex interaction, the D1 domain with an ATP-binding site necessary for the ATPase activity and a D2 domain with another ATP-binding site required for oligomerization (Zhao et al., 2015). Since NSF has been largely studied using non-human orthologous proteins (Chang et al., 2012; Cipriano et al., 2013; Vivona et al., 2013; Zhao et al., 2015), we set up a protocol to purify human Flag-tagged NSF from HEK293T mammalian cells. Firstly, we investigated the biochemical properties of NSF and subsequently its interaction with LRRK2. We demonstrated that the purified protein is an active ATPase and is able to interact with LRRK2. In particular, data obtained from pull-down assays revealed an interaction between NSF D2 domain and LRRK2. Moreover, we demonstrated that NSF is also a substrate of LRRK2 kinase activity and this phosphorylation preferentially occurs at Thr-645 in the D2 domain. We confirmed this result by measuring the 33P incorporation with kinase assays incubating NSF non-phosphorylatable mutants (NSF-T645A, T646A and S647A) together with LRRK2 G2019S. Kinetic studies of NSF ATPase activity revealed that NSF is 2-fold more active upon LRRK2 G2019S phosphorylation. Noteworthy, NSF-T645A, which resulted in a 50% reduction of 33P incorporation compared to wild-type, abolishes LRRK2-mediated increased ATPase activity. In addition, we demonstrated that NSF disassembles the SNARE complex at a higher rate after phosphorylation by LRRK2 G2019S. Taken together, these results highlight a possible regulatory mechanism in which LRRK2 is involved in synaptic vesicle recycling through phosphorylation of NSF. Importantly, NSF ATPase activity could be compromised by excessive phosphorylation due to LRRK2 G2019S pathological mutation. As mentioned, an important aspect in the development of PD may be linked to impaired synaptic vesicle trafficking, which may trigger neurodegeneration at early stages. A major class of proteins orchestrating vesicle sorting inside the cell are Rab proteins. Rabs constitute a large family of monomeric small GTPases associated with all cellular compartments (Grosshans et al., 2006). There are more than 60 different Rab proteins in humans (Schwartz et al., 2007; Pereira-Leal et al., 2001) and a switch between the GTP- (active) and GDP- (inactive) bound form regulates the interactions with their effectors. To date, several studies highlight a possible interaction between LRRK2 and a number of Rabs (MacLeod et al., 2013; Gomez-Suaga et al., 2014; Dodson et al., 2014; Waschbusch et al., 2014). Here, we tested whether different Rab proteins are also substrates of LRRK2 kinase activity. We performed kinase experiments on Rab7, Rab7L1, Rab9, Rab11 and Rab32. Kinase assays revealed that, among them, only Rab7L1 is a substrate of both LRRK2 wild-type and G2019S kinase activity. Rab7L1 is located within the PARK16 locus, a non-familial PD risk-associated locus, and Rab7L1 has been found to associate with LRRK2 to regulate the degradation of trans-Golgi derived vesicles (Beilina 2014). Mass spectrometry analysis revealed that phosphorylation by LRRK2 G2019S preferentially occurs on Rab7L1-T21, a residue localized in the highly conserved region responsible for GTP binding (P-loop region). In addition, we detected Rab7L1-S22 as a less probable phosphorylation site. To test which residue was phosphorylated, we generated Rab7L1-T21A and S22A mutants. Kinase assays indicate that Rab7L1 likely possesses additional sites, other than T21 or S22, able to be phosphorylated by LRRK2 G2019S. In summary, this work identified two novel substrates of LRRK2 kinase activity in vitro with potential relevance for disease. Our studies revealed an increased NSF ATPase activity upon LRRK2 G2019S phosphorylation and highlighted a novel regulatory mechanism that might be compromised in PD. In addition, we found that Rab7L1 is another substrate of LRRK2 kinase activity. Future studies should uncover whether NSF and Rab7L1 are substrates of LRRK2 also in the cellular context and whether pathological phosphorylation is relevant for PD.La malattia di Parkinson (PD) è la seconda malattia neurodegenerative più comune dopo la malattia di Alzheimer (AD) e colpisce circa l’1-2% della popolazione oltre I 65 anni (Farrer, 2006). Gli elementi caratteristici del morbo di Parkinson sono la perdita dei neuroni nella sustantia nigra pars compacta (SNpc), e la presenza di aggregati proteici intracellulari denominati corpi di Lewy (LB) nei neuroni che sopravvivono (Damier et al., 1999; Frank et al., 2007). L’eziologia della malattia di Parkinson è sconosciuta, con una complessa correlazione tra fattori ambientali e genetici, e fattori legati all’invecchiamento. La maggior parte dei casi, circa 95%, è di origina sporadica, mentre il rimanente 5-10% può essere collegato a mutazioni in singoli geni (Van Den Eeden et al., 2003). La scoperta di geni collegati al morbo di Parkinson ha messo in luce possibili trattamenti terapeutici, dato che la forma sporadica e quella familiare hanno in comune molti meccanismi patologici e pathway compromessi (Lesage and Brice, 2012). Ad oggi, cinque geni sono stati associati alla forma familiare del morbo di Parkinson, sia ad una forma genetica autosomica dominante che recessiva (Valente et al., 2004; Lakshminarasimhan et al., 2008; Nemani et al., 2013; Singleton et al., 2013; Kuang et al., 2013). Per questo motivo, numerosi studi si sono focalizzati sul ruolo fisiologico delle proteine collegate alla malattia di Parkinson e come le mutazioni patologiche causino la patologia. Nel 2004, due studi hanno identificato come mutazioni nel gene Lrrk2 (PARK8) siano causa della forma familiare di morbo di Parkinson (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) e, ad oggi, mutazioni all’interno di questo gene rappresentano la più comune causa genetica di malattia di Parkinson (10%). Lrrk2 Questo gene codifica per la proteina leucine-rich repeat kinase 2 (LRRK2), una grande proteina composta da vari domini con attività GTPasica e chinasica (Marín et al., 2008). La maggior parte delle mutazioni sono localizzate all’interno del core enzimatico della proteina, e possono colpire l’attività di LRRK2 causando un danno alle funzioni cellulari e citotossicità. Tra tutte, la mutazione G2019S è la più frequente (Gilks et al. 2005), quindi la più studiata. Questa mutazione avviene all’interno del dominio chinasico e aumenta l’attività chinasica di LRRK2 (Greggio and Cookson, 2009). Numerosi studi sostengono un ruolo di LRRK2 a livello del traffico delle vescicole sinaptiche, anche se il meccanismo esatto non è ancora chiaro. Perciò, capire quali pathway sono compromessi in condizioni patologiche è fondamentale per sviluppare strategie efficienti contro i danni provocati dalla malattia di Parkinson. Mentre l’inibizione farmacologica di LRRK2 potrebbe bloccare il fenotipo patologico, l’uso di inibitori di LRRK2 ha mostrato effetti secondari gravi a livello degli organi periferici (Baptista et al., 2013; Luerman et al., 2014). Per questo motivo, strategie terapeutiche alternative potrebbero essere dirette verso altre proteine che sono coinvolte all’interno dei pathway di LRRK2. In questo scenario, l’identificazione di substrati di LRRK2 a livello presinaptico, tra tutti i possibili interattori, è fondamentale per capire gli effetti a valle relativi a mutazioni patologiche di LRRK2. Studi precedenti hanno dimostrato che LRRK2 può modulare il traffico delle vescicole sinaptiche attraverso la fosforilazione di componenti facenti parte dei processi di eso- ed endocitosi (Heo et al., 2010; Matta et al., 2012; Yun et al., 2013). Per questa ragione, una domanda ancora senza risposta è quella di identificare possibili substrati dell’attività chinasica di LRRK2, rilevanti a livello fisiologico, e gli effetti che le mutazioni di LRRK2 associate al morbo di Parkinson hanno sulla fosforilazione. In questa tesi, ci siamo focalizzati sulla proteina N-ethylmaleimide Sensitive Fusion (NSF) protein e Rab7L1. Queste due proteine sono state precedentemente indicate come interattori di LRRK2 nei neuroni (Piccoli et al., 2011; MacLeod et al., 2013, Beilina et al., 2014). In questo lavoro, il nostro scopo era quello di testare se queste due proteine siano anche substrati dell’attività chinasica di LRRK2. NSF è classificata come proteina AAA+ (ATPases Associated with various cellular Activities) e la sua funzione è fondamentale a libello presinaptico per un corretto riciclo delle vescicole sinaptiche. In dettaglio, NSF usa l’energia prodotta dall’idrolisi dell’ATP per disassemblare le proteine del complesso SNARE (Soluble N-ethylmaleimide Attachment protein REceptors), insieme alla sua proteina adattatrice alpha-SNAP (Soluble NSF Associated Protein), consentendo un nuovo ciclo di fusione (Zhao and Brunger, 2015). NSF mostra una struttura omo-esamerica, dove ogni monomero è composto da tre differenti domini: l’N-terminale (N-ter) necessario all’interazione con alpha-SNAP ed il complesso SNARE, il dominio D1 con un sito di legame dell’ATP necessaria per l’attività ATPasica, ed un dominio D2 con un altro dito di legame per l’ATP richiesta all’oligomerizzazione (Zhao et al., 2015). Dato che NSF è stato largamente studiato usando una proteina ortologa non umana, abbiamo messo a punto un protocollo per purificare NSF umano con un Flag-tag da cellule di mammifero HEK293T. Per prima cosa, abbiamo deciso di studiare la biochimica di NSF e successivamente la sua interazione con LRRK2. Abbiamo dimostrato che la proteina purificata è attiva ed è in grado di interagire con LRRK2. In particolare, i risultati ottenuti attraverso esperimenti di pull-down rivelano una interazione tra il dominio D2 di NSF e LRRK2. Inoltre, abbiamo dimostrato che NSF è anche un substrato dell’attività chinsica di LRRK2 e questa fosforilazione avviene preferenzialmente nella treonina-645 all’interno del dominio D2. Abbiamo confermato questo risultato misurando l’incorporazione di 33P con saggi chinasici incubando dei mutanti non-fosforilabili di NSF (NSF-T645A, T646A and S647A) insieme a LRRK2 G2019S, Studi cinetici sull’attività ATPasica di NSF hanno rivelato che NSF è due volte più attivo dopo la fosforilazione da parte di LRRK2 G2019S. E’ importante notare che il mutante NSF-T645A, dove l’incorporazione di 33P risulta essere il 50% in meno rispetto al wild-type, non presenta un aumento dell’attività ATPasoca dovuto alla fosforilazione ad opera di LRRK2 G2019S. Inoltre, abbiamo dimostrato che NSF dopo essere stato fosforilato da LRRK2 G2019S disassembla il complesso SNARE più velocemente. Tutti questi risultati evidenziano un possibile meccanismo regolatorio in cui LRRK2 è implicato all’interno del riciclo delle vescicole sinaptiche attraverso la fosforilazione di NSF. L’attività ATPasica di NSF potrebbe quindi essere compromessa da una sua aumentata fosforilazione ad opera del mutante patologico G2019S. Come illustrato precedentemente, un aspetto importante nello sviluppo della malattia di Parkinson potrebbere essere collegato ad un danno nel traffico delle vescicole, che potrebbe far scaturire la neurodegenerazione a stadi precoci (Fernandez-Chacon et al., 2004; Burre et al., 2010). La principale classe di proteine che governa l’organizzazione delle vescicole all’interno della cellula, è composta dalle proteine Rab. Esse costituiscono una vasta famiglia di piccole GTPasi monomeriche associate a tutti i compartimenti cellulari (Grosshans et al., 2006). Nell’uomo ci sono più di 60 tipi di Rab ((Schwartz et al., 2007; Pereira-Leal et al., 2001)) che passando da una forma attiva, legata al GTP, ad una forma inattiva, legata al GDP, regolando la loro interazione con proteine effettrici. Ad oggi, numerosi studi hanno evidenziato una possibile interazione tra LRRK2 e varie proteine Rab (MacLeod et al., 2013; Gomez-Suaga et al., 2014; Dodson et al., 2014; Waschbusch et al., 2014). Nel nostro lavoro, abbiamo testato se alcune proteine Rab siano anche substrati dell’attività chinasica di LRRK2. Abbiamo eseguito saggi chinasici con varie Rab, su Rab7, Rab7L1, Rab9, Rab11 e Rab32. I saggi chinasici hanno rivelato che, tra tutte, solamente Rab7L1 è un substrato di LRRK2, sia wild-type che G2019S. Il gene Rab7L1 è localizzato all’interno del locus PARK16, un locus associato al richio di insorgenza della forma di malattia di Parkinson non-familiare, Rab7L1 è stato trovato associato a LRRK2 nella regolazione della degradazione delle vescicole a livello dell’apparato del Golgi (Beilina 2014). Analisi di spettrometria di massa hano rivelato comee la fosforilazione ad opera di LRRK2 G2019S avvenga preferenzialmente sulla treonina-21 di Rab7L1, un residuo localizzato nella regione altamente conservata responsabile del legame col GTP (P-loop). In aggiunta, abbiamo individuato come meno probabile sito di fosforilazione la serina-22. Per testare quale residuo fosse quello fosforilato, abbiamo generato i mutati non fosforilabili Rab7L1-T21A ed S22A. I saggi chinasici ci hanno indicato che Rab7L1 possiede verosimilmente siti di fosforilazione aggiuntivi, oltre la T21 e la S22, che possono essere fosforilati da LRRK2 G2019S. Riassumendo, questo lavoro ha identificato due nuovi substrati dell’attività chinasica di LRRK2 in vitro con una potenziale rilevanza a livello della malattia. I nostri studi hanno rivelato un aumento dell’attività ATPasica di NSF successivamente alla fosforilazione da parte di LRRK2 G2019S ed hanno evidenziato un nuovo meccanismo regolatorio che potrebbe essere compromesso nella malattia di Parkinson. In aggiunta, abbiamo trovato che Rab7L1 è un altro substrato dell’attività chinasica di LRRK2. Studi futuri sono necessari per scoprire se NSF e Rab7L1 siano substrati di LRRK2 anche in un contesto cellulare, e se la fosforilazione in condizioni patologiche è rilevante nel morbo di Parkinson

Metallo-responsive self-assembly of lipophilic guanines in hydrocarbon solvents: a systematic SAXS structural characterization

none7siLipophilic guanines (LipoGs) in aprotic solvents undergo different self-assembly processes based on different H-bonded motifs. Cylindrical nanotubes made by π–π stacked guanine tetramers (G-quadruplexes) and flat, tape-like aggregates (G-ribbons) have been observed depending on the presence of alkali metal ions. To obtain information on the structural properties and stability of these LipoG aggregates, Small-Angle X-ray Scattering (SAXS) experiments have been performed in dodecane, both in the presence and in the absence of potassium ions. As a result, the occurrence of the two different metallo-responsive architectures (nanoribbons or columnar nanotubes) was confirmed and we reported here for the first time a systematic study on the dependence of the aggregate properties on composition, temperature and molecular unit structure. Even if dodecane was selected to favour LipoG solubility, a strong tendency to self-organize into ordered lyotropic phases was indeed detected.mixedAdriano Gonnelli, Silvia Pieraccini, Enrico J. Baldassarri, Sergio Funari, Stefano Masiero, Maria Grazia Ortore, Paolo MarianiAdriano Gonnelli, Silvia Pieraccini, Enrico J. Baldassarri, Sergio Funari, Stefano Masiero, Maria Grazia Ortore, Paolo Marian