Physiological and pathological roles of LRRK2 in the nuclear envelope integrity.

Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson's disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 -/- mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.

SUMOylation and ubiquitination reciprocally regulate α-synuclein degradation and pathological aggregation.

α-Synuclein accumulation is a pathological hallmark of Parkinson's disease (PD). Ubiquitinated α-synuclein is targeted to proteasomal or lysosomal degradation. Here, we identify SUMOylation as a major mechanism that counteracts ubiquitination by different E3 ubiquitin ligases and regulates α-synuclein degradation. We report that PIAS2 promotes SUMOylation of α-synuclein, leading to a decrease in α-synuclein ubiquitination by SIAH and Nedd4 ubiquitin ligases, and causing its accumulation and aggregation into inclusions. This was associated with an increase in α-synuclein release from the cells. A SUMO E1 inhibitor, ginkgolic acid, decreases α-synuclein levels by relieving the inhibition exerted on α-synuclein proteasomal degradation. α-Synuclein disease mutants are more SUMOylated compared with the wild-type protein, and this is associated with increased aggregation and inclusion formation. We detected a marked increase in PIAS2 expression along with SUMOylated α-synuclein in PD brains, providing a causal mechanism underlying the up-regulation of α-synuclein SUMOylation in the disease. We also found a significant proportion of Lewy bodies in nigral neurons containing SUMO1 and PIAS2. Our observations suggest that SUMOylation of α-synuclein by PIAS2 promotes α-synuclein aggregation by two mutually reinforcing mechanisms. First, it has a direct proaggregatory effect on α-synuclein. Second, SUMOylation facilitates α-synuclein aggregation by blocking its ubiquitin-dependent degradation pathways and promoting its accumulation. Therefore, inhibitors of α-synuclein SUMOylation provide a strategy to reduce α-synuclein levels and possibly aggregation in PD.

The PINK1, synphilin-1 and SIAH-1 complex constitutes a novel mitophagy pathway.

PTEN-induced putative kinase 1 (PINK1) and parkin are mutated in familial forms of Parkinson's disease and are important in promoting the mitophagy of damaged mitochondria. In this study, we showed that synphilin-1 interacted with PINK1 and was recruited to the mitochondria. Once in the mitochondria, it promoted PINK1-dependent mitophagy, as revealed by Atg5 knockdown experiments and the recruitment of LC3 and Lamp1 to the mitochondria. PINK1-synphilin-1 mitophagy did not depend on PINK1-mediated phosphorylation of synphilin-1 and occurred in the absence of parkin. Synphilin-1 itself caused depolarization of the mitochondria and increased the amount of uncleaved PINK1 at the organelle. Furthermore, synphilin-1 recruited seven in absentia homolog (SIAH)-1 to the mitochondria where it promoted mitochondrial protein ubiquitination and subsequent mitophagy. Mitophagy via this pathway was impaired by synphilin-1 knockdown or by the use of a synphilin-1 mutant that is unable to recruit SIAH-1 to the mitochondria. Likewise, knockdown of SIAH-1 or the use of a catalytically inactive SIAH-1 mutant abrogated mitophagy. PINK1 disease mutants failed to recruit synphilin-1 and did not activate mitophagy, indicating that PINK1-synphilin-1-SIAH-1 represents a new parkin-independent mitophagy pathway. Drugs that activate this pathway will provide a novel strategy to promote the clearance of damaged mitochondria in Parkinson's disease.

AF-6 is a positive modulator of the PINK1/parkin pathway and is deficient in Parkinson's disease.

Parkin E3 ubiquitin-ligase activity and its role in mitochondria homeostasis are thought to play a role in Parkinson's disease (PD). We now report that AF-6 is a novel parkin interacting protein that modulates parkin ubiquitin-ligase activity and mitochondrial roles. Parkin interacts with the AF-6 PDZ region through its C-terminus. This leads to ubiquitination of cytosolic AF-6 and its degradation by the proteasome. On the other hand, endogenous AF-6 robustly increases parkin translocation and ubiquitin-ligase activity at the mitochondria. Mitochondrial AF-6 is not a parkin substrate, but rather co-localizes with parkin and enhances mitochondria degradation through PINK1/parkin-mediated mitophagy. On the other hand, several parkin and PINK1 juvenile disease-mutants are insensitive to AF-6 effects. AF-6 is present in Lewy bodies and its soluble levels are strikingly decreased in the caudate/putamen and substantia nigra of sporadic PD patients, suggesting that decreased AF-6 levels may contribute to the accumulation of dysfunctional mitochondria in the disease. The identification of AF-6 as a positive modulator of parkin translocation to the mitochondria sheds light on the mechanisms involved in PD and underscores AF-6 as a novel target for future therapeutics.

α-Synuclein fate is determined by USP9X-regulated monoubiquitination.

α-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of α-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. α-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in α-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates α-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated α-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated α-synuclein species and enhances the formation of toxic α-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of α-synuclein between different protein degradation systems. Deubiquitinated α-synuclein is mostly degraded by autophagy, while monoubiquitinated α-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of α-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated α-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in α-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated α-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of α-synuclein fate.

SIAH proteins regulate the degradation and intra-mitochondrial aggregation of PINK1: Implications for mitochondrial pathology in Parkinson's disease.

Parkinson's disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α-synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin-ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α-synuclein preformed fibrils (α-SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra-mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin-ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra-mitochondrial aggregation of SIAH3-PINK1 may mediate α-synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.

Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis.

Quantifying the benefit of early antibiotic treatment is crucial for decision making and can be assessed only in observational studies. We performed a systematic review of prospective studies reporting the effect of appropriate empirical antibiotic treatment on all-cause mortality among adult inpatients with sepsis. Two reviewers independently extracted data. Risk of bias was assessed using the Newcastle-Ottawa score. We calculated unadjusted odds ratios (ORs) with 95% confidence intervals for each study and extracted adjusted ORs, with variance, methods, and covariates being used for adjustment. ORs were pooled using random-effects meta-analysis. We examined the effects of methodological and clinical confounders on results through subgroup analysis or mixed-effect meta-regression. Seventy studies were included, of which 48 provided an adjusted OR for inappropriate empirical antibiotic treatment. Inappropriate empirical antibiotic treatment was associated with significantly higher mortality in the unadjusted and adjusted comparisons, with considerable heterogeneity occurring in both analyses (I(2) > 70%). Study design, time of mortality assessment, the reporting methods of the multivariable models, and the covariates used for adjustment were significantly associated with effect size. Septic shock was the only clinical variable significantly affecting results (it was associated with higher ORs). Studies adjusting for background conditions and sepsis severity reported a pooled adjusted OR of 1.60 (95% confidence interval = 1.37 to 1.86; 26 studies; number needed to treat to prevent one fatal outcome, 10 patients [95% confidence interval = 8 to 15]; I(2) = 46.3%) given 34% mortality with inappropriate empirical treatment. Appropriate empirical antibiotic treatment is associated with a significant reduction in all-cause mortality. However, the methods used in the observational studies significantly affect the effect size reported. Methods of observational studies assessing the effects of antibiotic treatment should be improved and standardized.

Involvement of Src tyrosine kinases (SFKs) and of focal adhesion kinase (FAK) in the injurious mechanism in rat primary neuronal cultures exposed to chemical ischemia.

Src family of kinases (SFKs) and focal adhesion kinase (FAK) are two important cellular signaling components known to act cooperatively in the transduction of death and survival signals. We investigated the involvement of these proteins in the mechanism of the injurious response in rat primary neuronal cultures exposed to an insult composed of chemical ischemia (poisoning with iodoacetic acid; 100 muM, for 150 min) followed by 1 h of incubation in the regular medium, an insult shown before to be associated with generation of reactive oxygen species and with the depletion of adenosine triphisphate. The exposure of the neuronal cultures to the insult resulted in cell injury, assessed by the increased release of cytoplasmic lactate dehydrogenase (LDH) into the culture media, which could be attenuated markedly by the presence of the antioxidant LY 231617. The insult resulted in the decreased level of phosphorylation of the SFKs members Src, Fyn, and Yes at the Src Y416-equivalent activation sites and of the FAK Y397 activation site, degradation of FAK to a p85 fragment, and disassembling of the FAK-SFKs complexes. The inhibition of SFKs was found to be responsible for part of the insult-induced cell damage manifested in increased LDH release. Pervanadate, an inhibitor of the phosphotyrosine phosphatases (PTPs), abrogated the inactivation of SFKs and attenuated cell injury, indicating that insult-induced activation of PTPs is involved in SFKs inhibition and the ensued damage. The inhibition of SFKs and FAK is probably the cause of the disassembling of SFKs-FAK complexes, a process known to be associated with apoptosis.

Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1.

A common genetic form of Parkinson's disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

α-Synuclein ubiquitination and novel therapeutic targets for Parkinson's disease.

Accumulation of α-synuclein is key to the pathogenesis of Parkinson's disease (PD), though the exact mechanisms involved in its toxicity are still subject to debate. Increased α-synuclein expression or reduced degradation may play a role in the proteotoxicity observed in PD. Here we review the mechanisms of α-synuclein ubiquitination by different E3 ubiquitin-ligases, and its degradation via the proteasome, autophagy and lysosomes. Activators of α- synuclein ubiquitination and degradation pathways represent a plausible strategy to decrease α-synuclein burden in the disease. Nevertheless, since proteasomes and autophagy might be impaired in the disease, and because proteolytic impairment causes the accumulation of monoubiquitinated α-synuclein and the formation of toxic inclusions, compounds that promote α-synuclein monoubiquitination should be used in concert with compounds that boost these proteolytic pathways. This combined approach may therefore ease the accumulation of α-synuclein in PD and may represent a promising new avenue for the development of novel treatments for the disease.

A model for diagnosis of pulmonary infections in solid-organ transplant recipients.

BACKGROUND: Opportunistic pulmonary infections are a major cause of morbidity and mortality among solid organ transplant recipients. The diagnosis of these infections is challenging because of the broad spectrum of bacteria, fungi and viruses affecting these patients and the lack of specific signs and symptoms. Treatment directed at the offending organism started as soon as possible improves survival. OBJECTIVE: To develop a decision support system for the diagnosis of pulmonary infections in solid-organ transplant recipients. The model's goal is to improve the accuracy of the diagnosis and thus the appropriateness of empirical treatment. DESIGN: The model is built using a Bayesian network (also known as causal probabilistic network). The network is based on pathogen segments which are the main building blocks of the model. Segments share common risk factors, such as time after transplantation, latent infections of donor/recipient and organ transplanted. The segments are linked at symptoms, signs and diagnostic tests common to all pathogens. The outputs of the model are predicted probabilities of infectious pathogens. To populate the model with data we have mainly abstracted data from the literature, using a systematic approach. The structure of the model and its adaptation for decision support will be presented. EVALUATION: The first evaluation phase assessed the model's diagnosis in a series of 20 representative cases of opportunistic infections. A match between the case's diagnosis and the model's prediction was achieved in 17/20 of cases. The next evaluation phase will consist of a prospective observational study comparing the accuracy of the model's diagnosis vs. that of the physician within 24h of episode onset, as compared with a gold-standard diagnosis ascribed to the patients at the end of the infectious episode by two independent experts. Data for this phase are currently collected prospectively.

Synphilin-1A inhibits seven in absentia homolog (SIAH) and modulates alpha-synuclein monoubiquitylation and inclusion formation.

Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates alpha-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward alpha-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as alpha-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of alpha-synuclein by SIAH and the formation of alpha-synuclein inclusions, supporting a role for monoubiquitylation in alpha-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.

Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells.

alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.