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The Cosmic Gems arc is among the brightest and highly magnified galaxies observed at redshift z ≈ 10.2 (ref.  1 ). However, it is an intrinsically ultraviolet faint galaxy, in the range of those now thought to drive the reionization of the Universe 2-4 . Hitherto the smallest features resolved in a galaxy at a comparable redshift are between a few hundreds and a few tens of parsecs (pc) 5,6 . Here we report JWST observations of the Cosmic Gems. The light of the galaxy is resolved into five star clusters located in a region smaller than 70 pc. They exhibit minimal dust attenuation and low metallicity, ages younger than 50 Myr and intrinsic masses of about 10 6 M ⊙ . Their lensing-corrected sizes are approximately 1 pc, resulting in stellar surface densities near 10 5 M ⊙  pc -2 , three orders of magnitude higher than typical young star clusters in the local Universe 7 . Despite the uncertainties inherent to the lensing model, they are consistent with being gravitationally bound stellar systems, that is, proto-globular clusters. We conclude that star cluster formation and feedback likely contributed to shaping the properties of galaxies during the epoch of reionization., (© 2024. The Author(s).)

The placenta plays a key role in several adverse obstetrical outcomes, such as preeclampsia, intrauterine growth restriction and gestational diabetes mellitus. The early identification of at-risk pregnancies could significantly improve the management, therapy and prognosis of these pregnancies, especially if these at-risk pregnancies are identified in the first trimester. The aim of this review was to summarize the possible biomarkers that can be used to diagnose early placental dysfunction and, consequently, at-risk pregnancies. We divided the biomarkers into proteins and non-proteins. Among the protein biomarkers, some are already used in clinical practice, such as the sFLT1/PLGF ratio or PAPP-A; others are not yet validated, such as HTRA1, Gal-3 and CD93. In the literature, many studies analyzed the role of several protein biomarkers, but their results are contrasting. On the other hand, some non-protein biomarkers, such as miR-125b, miR-518b and miR-628-3p, seem to be linked to an increased risk of complicated pregnancy. Thus, a first trimester heterogeneous biomarkers panel containing protein and non-protein biomarkers may be more appropriate to identify and discriminate several complications that can affect pregnancies.

We are witnessing a revolution in the treatment of metastatic renal cell carcinoma (mRCC). Indeed, several immune-based combinations (ICI [immune checkpoint inhibitor] + ICI, or ICI + antiangiogenic agents) have been approved as first-line therapy for mRCC after demonstrating superior efficacy over the previous standard. Despite all the improvements made, safety remains a critical issue, adverse events (AEs) being the main reason for drug discontinuations or dose reductions, ultimately resulting in an increased risk of losing efficacy. Thus, a good understanding of the AEs associated with the use of immune-based combinations, their prevention, and management, are key in order to maximize therapeutic effectiveness. Among these AEs, renal ones are relatively frequent, but always difficult to be diagnosed, not to take into account that it is often difficult to determine which drug is to blame for such toxicities. Chronic kidney disease (CKD) is a common finding in patients with RCC, either as a pre-existing condition and/or as a consequence of cancer and its treatment; furthermore, CKD, especially in advanced stages and in patients undergoing dialysis, may influence the pharmacokinetics and pharmacodynamics properties of anticancer agents. Finally, managing cancer therapy in kidney transplanted patients is another challenge. In this review, we discuss the therapy management of immune-based combinations in patients with CKD, on dialysis, or transplanted, as well as their renal toxicities, with a focus on their prevention, detection and practical management, taking into account the crucial role of the consulting nephrologist within the multidisciplinary care of these patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Short- and medium-term cardio-pulmonary sequelae after COVID-19 have been extensively studied. However, studies with longer follow-ups are required. This study aims to identify and characterise cardio-pulmonary sequelae, in patients hospitalised for SARS-CoV-2 pneumonia, at 24 months follow-up. This is a prospective, observational cohort study conducted on consecutive patients hospitalised for COVID-19 and acute respiratory failure. Patients were followed up at 24 months with complete pulmonary function tests (PFTs), 6-min walking test and a dyspnoea score (Modified Medical Research Council scale). A subgroup of patients with at least one clinical or functional sign suggestive of increased pulmonary pressures also underwent transthoracic echocardiography (TTE) to evaluate the presence of direct or indirect signs of pulmonary hypertension (PH). Ninety consecutive patients (74% men, median age 59.1 years) were enrolled in the study. In regard to PFTs, carbon monoxide diffusion capacity (DLCO) impairment was observed in 23 cases (26%), in all cases of mild entity. When considering the dyspnoea, 30 (34%) patients showed some degree of breathlessness. Forty patients underwent TTE. No patients had overt PH or chronic thromboembolic PH. However, all patients showed a hyperdynamic state of the right ventricle, and 8 (20%) patients had a decreased acceleration time on pulmonary valve, signs of increased pulmonary vasculature resistances and afterload elevation. At 24-month follow-up after severe COVID-19, DLCO and TTE prove to be the most sensitive tool to detect cardio-pulmonary sequelae. Dyspnoea is still present in about one-third of patients and requires a multidisciplinary approach., (© 2023. The Author(s).)

Background and Objectives: This study aimed to identify CSF proteomic signatures characteristic of Parkinson disease (PD) and evaluate their clinical utility., Methods: This observational study used data from the Parkinson's Progression Markers Initiative (PPMI), which enrolled patients with PD, healthy controls (HCs), and non-PD participants carrying GBA1 , LRRK2 , and/or SNCA pathogenic variants (genetic prodromals) at international sites. Study participants were chosen from PPMI enrollees based on the availability of aptamer-based CSF proteomic data, quantifying 4,071 proteins, and classified as patients with PD without GBA1 , LRRK2 , and/or SNCA pathogenic variants (nongenetic PD), HCs, patients with PD carrying the aforementioned pathogenic variants (genetic PD), or genetic prodromals. Differentially expressed protein (DEP) analysis and the least absolute shrinkage and selection operator (LASSO) were applied to the data from nongenetic PD and HCs. Signatures characteristics of nongenetic PD were quantified as a PD proteomic score (PD-ProS), validated internally and then externally using data of 1,556 CSF proteins from the LRRK2 Cohort Consortium (LCC). We further tested the PD-ProS in genetic PD and genetic prodromals and examined associations with clinical progression., Results: Data from 279 patients with nongenetic PD (mean ± SD, age 62.0 ± 9.6 years; male 67.7%) and 141 HCs (age 60.5 ± 11.9 years; male 64.5%) were used for PD-ProS derivation. From 23 DEPs, LASSO determined weights of 14 DEPs for the PD-ProS (area under the curve [AUC] 0.83, 95% CI 0.78-0.87), validated in an independent internal validation cohort of 71 patients with nongenetic PD and 35 HCs (AUC 0.81, 95% CI 0.73-0.90). In the LCC, only 5 of the 14 DEPs were also measured. Notably, these 5 DEPs still distinguished 34 patients with nongenetic PD from 31 HCs with the same weights (AUC 0.75, 95% CI 0.63-0.87). Furthermore, the PD-ProS distinguished 258 patients with genetic PD from 365 genetic prodromals. Finally, regardless of genetic status, the PD-ProS independently predicted both cognitive and motor decline in PD (dementia, adjusted hazard ratio in the highest quintile [aHR-Q5] 2.8 [95% CI 1.6-5.0]; Hoehn and Yahr stage IV, aHR-Q5 2.1 [95% CI 1.1-4.0])., Discussion: By integrating high-throughput proteomics with machine learning, we identified PD-associated CSF proteomic signatures crucial for PD development and progression., Trial Registration Information: ClinicalTrials.gov (NCT01176565). A link to the trial registry page is clinicaltrials.gov/ct2/show/NCT01141023., Classification of Evidence: This study provides Class II evidence that the CSF proteome contains clinically important information regarding the development and progression of Parkinson disease that can be deciphered by a combination of high-throughput proteomics and machine learning., (© 2023 American Academy of Neurology.)

The pathogenesis and clinical heterogeneity of Parkinson's disease (PD) have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of cerebrospinal fluid (CSF) opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in PD with 569 patients (350 idiopathic patients, 65 GBA + mutation carriers and 154 LRRK2 + mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest PD genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB, was previously reported to be upregulated in the substantia nigra of PD patients. We also compared the CSF proteomes of patients and controls. Proteome differences between GBA + patients and unaffected GBA + controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. In the LRRK2 + subcohort we found dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction/oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances. Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on previously reported protein-based risk scores.Our findings not only contribute to the identification of new therapeutic targets but also to shape personalized medicine in CNS neurodegeneration., (© 2023. The Author(s).)

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sleep is severely impaired in patients with Alzheimer's disease. Amyloid-β deposition in the brain of Alzheimer's disease patients is a key event in its pathogenesis and is associated with disrupted sleep, even before the appearance of cognitive decline. Because soluble amyloid-β oligomers are the key mediators of synaptic and cognitive dysfunction in Alzheimer's disease and impair long-term memory in rodents, the first aim of this study was to test the hypothesis that amyloid-β oligomers would directly impair sleep in mice. The cellular prion protein is a cell surface glycoprotein of uncertain function. Because cellular prion protein binds oligomeric amyloid-β with high affinity and mediates some of its neurotoxic effects, the second aim of the study was to test whether amyloid-β oligomer-induced sleep alterations were mediated by cellular prion protein. To address these aims, wild-type and cellular prion protein-deficient mice were given acute intracerebroventricular injections (on different days, at lights on) of vehicle and synthetic amyloid-β oligomers. Compared to vehicle, amyloid-β oligomers significantly reduced the amount of time spent in non-rapid eye movement sleep by wild-type mice during both the light and dark phases of the light-dark cycle. The amount of time spent in rapid eye movement sleep was reduced during the dark phase. Sleep was also fragmented by amyloid-β oligomers, as the number of transitions between states increased in post-injection hours 9-24. No such effects were observed in cellular prion protein-deficient mice. These results show that amyloid-β oligomers do inhibit and fragment sleep, and that these effects are mediated by cellular prion protein., (© 2020 European Sleep Research Society.)

In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P 2 in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers., Competing Interests: The authors declare no conflict of interest.

Close appositions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are a general feature of all cells and are abundant in neurons. A function of these appositions is lipid transport between the two adjacent bilayers via tethering proteins that also contain lipid transport modules. However, little is known about the properties and dynamics of these proteins in neurons. Here we focused on TMEM24/C2CD2L, an ER-localized SMP domain containing phospholipid transporter expressed at high levels in the brain, previously shown to be a component of ER-PM contacts in pancreatic β-cells. TMEM24 is enriched in neurons versus glial cells and its levels increase in parallel with neuronal differentiation. It populates ER-PM contacts in resting neurons, but elevations of cytosolic Ca 2+ mediated by experimental manipulations or spontaneous activity induce its transient redistribution throughout the entire ER. Dissociation of TMEM24 from the plasma membrane is mediated by phosphorylation of an array of sites in the C-terminal region of the protein. These sites are only partially conserved in C2CD2, the paralogue of TMEM24 primarily expressed in nonneuronal tissues, which correspondingly display a much lower sensitivity to Ca 2+ elevations. ER-PM contacts in neurons are also sites where Kv2 (the major delayed rectifier K + channels in brain) and other PM and ER ion channels are concentrated, raising the possibility of a regulatory feedback mechanism between neuronal excitability and lipid exchange between the ER and the PM., Competing Interests: The authors declare no conflict of interest.

The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca 2+ levels accompanied by an enhanced basal activity of Ca 2+ /calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca 2+ homeostasis and neurotransmitter release.

Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications., Competing Interests: The authors declare no conflict of interest.

More than a trillion nerve terminals interconnect neurons in the human brain. These terminals are fundamental for signal transmission and nerve cell communication. Among other techniques, the isolation of nerve terminals [or synaptosomes (Whittaker et al. Biochem J, 90(2):293-303, 1964)] has been fundamental to study the biochemistry and the physiology of the nervous system. This chapter describes the isolation and purification of intact synaptosomes from rodent brain tissue that can be used to further characterize synaptic structure and function and to examine the molecular mechanisms of neurotransmission.

Insulin is released by β cells in pulses regulated by calcium and phosphoinositide signaling. Here, we describe how transmembrane protein 24 (TMEM24) helps coordinate these signaling events. We showed that TMEM24 is an endoplasmic reticulum (ER)-anchored membrane protein whose reversible localization to ER-plasma membrane (PM) contacts is governed by phosphorylation and dephosphorylation in response to oscillations in cytosolic calcium. A lipid-binding module in TMEM24 transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] precursor phosphatidylinositol between bilayers, allowing replenishment of PI(4,5)P 2 hydrolyzed during signaling. In the absence of TMEM24, calcium oscillations are abolished, leading to a defect in triggered insulin release. Our findings implicate direct lipid transport between the ER and the PM in the control of insulin secretion, a process impaired in patients with type II diabetes., (Copyright © 2017, American Association for the Advancement of Science.)

Background: Amyloid-β oligomers (AβO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AβO-mediated mouse model of AD, we explored whether the memory alteration AβOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved., Methods: C57 naïve mice were given a single intracerebroventricular injection of synthetic AβO-containing solution (1μM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1β receptor antagonist) to prevent impairment in the novel object recognition task, and compared AβO's effects in TLR4 knockout mice., Results: A single AβO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AβO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AβO's action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation., Conclusions: These data provide new information on AβO's mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well., (Copyright © 2016. Published by Elsevier Inc.)

Synapsins (Syns) are synaptic vesicle-associated phosphoproteins involved in neuronal development and neurotransmitter release. While Syns are implicated in the regulation of brain-derived neurotrophic factor (BDNF)-induced neurotransmitter release, their role in the BDNF developmental effects has not been fully elucidated. By using primary cortical neurons from Syn I knockout (KO) and Syn I/II/III KO mice, we studied the effects of BDNF and nerve growth factor (NGF) on axonal growth. While NGF had similar effects in all genotypes, BDNF induced significant differences in Syn KO axonal outgrowth compared to wild type (WT), an effect that was rescued by the re-expression of Syn I. Moreover, the significant increase of axonal branching induced by BDNF in WT neurons was not detectable in Syn KO neurons. The expression analysis of BDNF receptors in Syn KO neurons revealed a significant decrease of the full length TrkB receptor and an increase in the levels of the truncated TrkB.t1 isoform and p75 NTR associated with a marked reduction of the BDNF-induced MAPK/Erk activation. By using the Trk inhibitor K252a, we demonstrated that these differences in BDNF effects were dependent on a TrkB/p75 NTR imbalance. The data indicate that Syn I plays a pivotal role in the BDNF signal transduction during axonal growth.

Pentraxins are a superfamily of fluid phase pattern recognition molecules conserved in evolution and characterized by a cyclic multimeric structure. C-reactive protein (CRP) and serum amyloid P component (SAP) constitute the short pentraxin arm of the superfamily. CRP and SAP are produced in the liver in response to IL-6 and are acute phase reactants in humans and mice respectively. In addition SAP has been shown to affect tissue remodelling and fibrosis by stabilizing all types of amyloid fibrils and by regulating monocyte to fibrocyte differentiation. Pentraxin 3 (PTX3) is the prototype of the long pentraxin arm. Gene targeted mice and genetic and epigenetic studies in humans suggest that PTX3 plays essential non-redundant roles in innate immunity and inflammation as well as in tissue remodelling. Recent studies have revealed the role of PTX3 as extrinsic oncosuppressor, able to tune cancer-related inflammation. In addition, at acidic pH PTX3 can interact with provisional matrix components promoting inflammatory matrix remodelling. Thus acidification during tissue repair sets PTX3 in a tissue remodelling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Alzheimer disease (AD) is the most prevalent form of dementia. Loss of hippocampal synapses is the first neurodegenerative event in AD. Synaptic loss has been associated with the accumulation in the brain parenchyma of soluble oligomeric forms of amyloid β peptide (Aβ1-42wt). Clinical observations have shown that a mutation in the APP protein (A673V) causes an early onset AD-type dementia in homozygous carriers while heterozygous carriers are unaffected. This mutation leads to the formation of mutated Aβ peptides (Aβ1-42A2V) in homozygous patients, while in heterozygous subjects both Aβ1-42wt and Aβ1-42A2V are present. To better understand the impact of the A673V mutation in AD, we analyzed the synaptotoxic effect of oligomers formed by aggregation of different Aβ peptides (Aβ1-42wt or Aβ1-42A2V) and the combination of the two Aβ1-42MIX (Aβ1-42wt and Aβ1-42A2V) in an in vitro model of synaptic injury. We showed that Aβ1-42A2V oligomers are more toxic than Aβ1-42wt oligomers in hippocampal neurons, confirming the results previously obtained in cell lines. Furthermore, we reported that oligomers obtained by the combination of both wild type and mutated peptides (Aβ1-42MIX) did not exert synaptic toxicity. We concluded that the combination of Aβ1-42wt and Aβ1-42A2V peptides hinders the toxicity of Aβ1-42A2V and counteracts the manifestation of synaptopathy in vitro. Finally we took advantage of this finding to generate a cell-permeable peptide for clinical application, by fusing the first six residues of the Aβ1-42A2V to the TAT cargo sequence (Aβ1-6A2VTAT(D)). Noteworthy, the treatment with Aβ1-6A2VTAT(D) confers neuroprotection against both in vitro and in vivo synaptopathy models. Therefore Aβ1-6A2VTAT(D) may represent an innovative therapeutic tool to prevent synaptic degeneration in AD., (Copyright © 2015. Published by Elsevier Inc.)

The role of first-stage β-amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β-amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment.

Genetic defects in myelin formation and maintenance cause leukodystrophies, a group of white matter diseases whose mechanistic underpinnings are poorly understood. Hypomyelination and congenital cataract (HCC), one of these disorders, is caused by mutations in FAM126A, a gene of unknown function. We show that FAM126A, also known as hyccin, regulates the synthesis of phosphatidylinositol 4-phosphate (PtdIns(4)P), a determinant of plasma membrane identity. HCC patient fibroblasts exhibit reduced PtdIns(4)P levels. FAM126A is an intrinsic component of the plasma membrane phosphatidylinositol 4-kinase complex that comprises PI4KIIIα and its adaptors TTC7 and EFR3 (refs 5,7). A FAM126A-TTC7 co-crystal structure reveals an all-α-helical heterodimer with a large protein-protein interface and a conserved surface that may mediate binding to PI4KIIIα. Absence of FAM126A, the predominant FAM126 isoform in oligodendrocytes, destabilizes the PI4KIIIα complex in mouse brain and patient fibroblasts. We propose that HCC pathogenesis involves defects in PtdIns(4)P production in oligodendrocytes, whose specialized function requires massive plasma membrane expansion and thus generation of PtdIns(4)P and downstream phosphoinositides. Our results point to a role for FAM126A in supporting myelination, an important process in development and also following acute exacerbations in multiple sclerosis.

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin., (© 2015 Nakatsu et al.)

To assess whether NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that mediates the activation of caspase-1 (CASP-1) and pro-inflammatory cytokines IL-18 and IL-1β, could be involved in the chronic inflammatory state observed in chronic kidney disease patients undergoing hemodialysis treatment (CKD-HD), we employed several biomolecular techniques including RT-PCR, western blot, FACS analysis, confocal microscopy and microarray. Interestingly, peripheral blood mononuclear cells from 15 CKD-HD patients showed higher mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18 and P2X7 receptor compared to 15 healthy subjects. Western blotting analysis confirmed the above results. In particular, active forms of CASP-1, IL1-β and IL-18 resulted significantly up-regulated in CKD-HD versus controls. Additionally, elevated mitochondrial ROS level, colocalization of NLRP3/ASC/mitochondria in peripheral blood mononuclear cells from CKD-HD patients and down-regulation of CASP-1, IL1-β and IL-18 protein levels in immune-cells of CKD-HD patients stimulated with LPS/ATP in presence of mitoTEMPO, inhibitor of mitochondrial ROS production, suggested a possible role of this organelle in the aforementioned CKD-associated inflammasome activation. Then, microarray analysis confirmed, in an independent microarray study cohort, that NLRP3 and CASP-1, along with other inflammasome-related genes, were up-regulated in 17 CKD-HD patients and they were able to clearly discriminate these patients from 5 healthy subjects. All together these data showed, for the first time, that NLRP3 inflammasome was activated in uremic patients undergoing dialysis treatment and they suggested that this unphysiological condition could be possibly induced by mitochondrial dysfunction.

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aβ1-42, PrP106-126, and ataxin-3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan-3-ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti-amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We recently reported a novel Aβ precursor protein mutation (A673V), corresponding to position 2 of Aβ1-42 peptides (Aβ1-42A2V), that caused an early onset AD-type dementia in a homozygous individual. The heterozygous relatives were not affected as an indication of autosomal recessive inheritance of this mutation. We investigated the folding kinetics of native unfolded Aβ1-42A2V in comparison with the wild type sequence (Aβ1-42WT) and the equimolar solution of both peptides (Aβ1-42MIX) to characterize the oligomers that are produced in the early phases. We carried out the structural characterization of the three preparations using electron and atomic force microscopy, fluorescence emission, and x-ray diffraction and described the soluble oligomer formation kinetics by laser light scattering. The mutation promoted a peculiar pathway of oligomerization, forming a connected system similar to a polymer network with hydrophobic residues on the external surface. Aβ1-42MIX generated assemblies very similar to those produced by Aβ1-42WT, albeit with slower kinetics due to the difficulties of Aβ1-42WT and Aβ1-42A2V peptides in building up of stable intermolecular interaction., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits., (Copyright © 2014, Messa et al.)

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles ('bulk' endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity., (Copyright © 2014, Wu et al.)

Modulation of synaptic vesicle retrieval is considered to be potentially important in steady-state synaptic performance. Here we show that at physiological temperature endocytosis kinetics at hippocampal and cortical nerve terminals show a bi-phasic dependence on electrical activity. Endocytosis accelerates for the first 15-25 APs during bursts of action potential firing, after which it slows with increasing burst length creating an optimum stimulus for this kinetic parameter. We show that activity-dependent acceleration is only prominent at physiological temperature and that the mechanism of this modulation is based on the dephosphorylation of dynamin 1. Nerve terminals in which dynamin 1 and 3 have been replaced with dynamin 1 harboring dephospho- or phospho-mimetic mutations in the proline-rich domain eliminate the acceleration phase by either setting endocytosis at an accelerated state or a decelerated state, respectively. DOI:http://dx.doi.org/10.7554/eLife.00845.001.

A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23-31 and 95-105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD.

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

APP mutations cause Alzheimer disease (AD) with virtually complete penetrance. We found a novel APP mutation (A673V) in the homozygous state in a patient with early-onset AD-type dementia and in his younger sister showing initial signs of cognitive decline. It is noteworthy that the heterozygous relatives were not affected, suggesting that this mutation is inherited as an autosomal recessive trait. Studies on molecular events for the recessive mutation in causing disease revealed a double synergistic effect: the A673V APP variant shifts APP processing towards the amyloidogenic pathway with increased production of Aβ peptides and it markedly enhances the aggregation and fibrillogenic properties of both Aβ1-40 and Aβ1-42. However, co-incubation of mutated and wild-type (wt) Aβ species resulted in inhibition of amyloidogenesis, consistent with the observation that heterozygous carriers do not develop the disease. The opposite effects of the A673V mutation in the homozygous and heterozygous state on amyloidogenesis account for the autosomal recessive pattern of inheritance, revealing a new scenario in AD genetics and pathogenesis. The anti-amyloidogenic properties of this novel human Aβ variant may offer grounds for the development of therapeutic strategies for AD based on modified Aβ peptides. Indeed, the interaction between mutated Aβ1-6 and wt full-length Aβ prevents amyloid fibril formation. The anti-amyloidogenic effect is further amplified by the use of a mutated six-mer peptide, constructed entirely from D-amino acids to increase the its stability in vivo. Here we reviewed the studies on pathogenic mechanisms associated with the A673V mutation and the first experimental steps toward the development of a novel disease-modifying therapy for AD., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The pathogenesis of Alzheimer's disease (AD) is only partially understood. β-amyloid (Aβ) is physiologically generated by sequential cleavage of its precursor protein by the β- and the γ-secretase and it is normally disposed of. In Alzheimer's disease, Aβ is excessively produced or less dismissed, but the hypothesis on its physiological and pathological role are heterogeneous and often discordant. It has been described a positive feedback loop from the γ- to the β-secretase cleavages of Aβ precursor protein, which is activated by mutations of Presenilin 1 (PS1), the catalytic core of the γ-secretase. These findings show that Aβ precursor protein as well the activity of the γ-secretase are required to obtain the up-regulation of β-secretase which is induced by Presenilin 1 mutations. Then, Aβ 1-42 is the Aβ precursor protein derivative that up-regulates the expression of β-secretase, and c-jun N-terminal kinase (JNK)/c-Jun and ERK1/2 are involved. Here, we describe the activation of β-secretase and c-jun N-terminal kinase related proteins by monomeric Aβ 1-42, defining the conditions that most efficiently strike the described signaling without producing toxicity. Taken together these data imply that monomeric Aβ 1-42, at non-toxic concentrations and time frames, are able to induce a signaling pathway that leads to transcriptional activation of β-secretase., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.

The interplay between anatomical connectivity and dynamics in neural networks plays a key role in the functional properties of the brain and in the associated connectivity changes induced by neural diseases. However, a detailed experimental investigation of this interplay at both cellular and population scales in the living brain is limited by accessibility. Alternatively, to investigate the basic operational principles with morphological, electrophysiological and computational methods, the activity emerging from large in vitro networks of primary neurons organized with imposed topologies can be studied. Here, we validated the use of a new bio-printing approach, which effectively maintains the topology of hippocampal cultures in vitro and investigated, by patch-clamp and MEA electrophysiology, the emerging functional properties of these grid-confined networks. In spite of differences in the organization of physical connectivity, our bio-patterned grid networks retained the key properties of synaptic transmission, short-term plasticity and overall network activity with respect to random networks. Interestingly, the imposed grid topology resulted in a reinforcement of functional connections along orthogonal directions, shorter connectivity links and a greatly increased spiking probability in response to focal stimulation. These results clearly demonstrate that reliable functional studies can nowadays be performed on large neuronal networks in the presence of sustained changes in the physical network connectivity.

Curcumin derivatives with high chemical stability, improved solubility and carrying a functionalized appendage for the linkage to other entities, have been synthesized in a straightforward manner. All compounds retained Curcumin ability to bind Aβ peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aβ deposits., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function.

The ability of dopamine receptors to interact with other receptor subtypes may provide mechanisms for modulating dopamine-related functions and behaviors. In particular, there is evidence suggesting that the trace amine-associated receptor 1 (TAAR1) affects the dopaminergic system by regulating the firing rate of dopaminergic neurons or by altering dopamine D2 receptor (D2R) responsiveness to ligands. TAAR1 is a Gα(s) protein-coupled receptor that is activated by biogenic amines, "trace amines," such as β-phenylethylamine (β-PEA) and tyramine that are normally found at low concentrations in the mammalian brain. In the present study, we investigated the biochemical mechanism of interaction between TAAR1 and D2R and the role this interaction plays in D2R-related signaling and behaviors. Using a bioluminescence resonance energy transfer biosensor for cAMP, we demonstrated that the D2R antagonists haloperidol, raclopride, and amisulpride were able to enhance selectively a TAAR1-mediated β-PEA increase of cAMP. Moreover, TAAR1 and D2R were able to form heterodimers when coexpressed in human embryonic kidney 293 cells, and this direct interaction was disrupted in the presence of haloperidol. In addition, in mice lacking TAAR1, haloperidol-induced striatal c-Fos expression and catalepsy were significantly reduced. Taken together, these data suggest that TAAR1 and D2R have functional and physical interactions that could be critical for the modulation of the dopaminergic system by TAAR1 in vivo.

The existence of neuron-specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. Although lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Accumulation of β-sheet-rich peptide (Aβ) is strongly associated with Alzheimer's disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aβ species are potent neurotoxins, however the molecular mechanism responsible for Aβ toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aβ 1-40 and Aβ 1-42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aβ toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aβ receptors in N2a cells, triggering a multi factorial toxicity.

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-gamma(35)S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50 = 0.15 microM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90alpha inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.

Synapsins are synaptic vesicle (SV)-associated phosphoproteins involved in the regulation of neurotransmitter release. Synapsins reversibly tether SVs to the cytoskeleton and their phosphorylation by serine/threonine kinases increases SV availability for exocytosis by impairing their association with SVs and/or actin. We recently showed that synapsin I, through SH3- or SH2-mediated interactions, activates Src and is phosphorylated by the same kinase at Tyr301. Here, we demonstrate that, in contrast to serine phosphorylation, Src-mediated tyrosine phosphorylation of synapsin I increases its binding to SVs and actin, and increases the formation of synapsin dimers, which are both potentially involved in SV clustering. Synapsin I phosphorylation by Src affected SV dynamics and was physiologically regulated in brain slices in response to depolarization. Expression of the non-phosphorylatable (Y301F) synapsin I mutant in synapsin-I-knockout neurons increased the sizes of the readily releasable and recycling pools of SVs with respect to the wild-type form, which is consistent with an increased availability of recycled SVs for exocytosis. The data provide a mechanism for the effects of Src on SV trafficking and indicate that tyrosine phosphorylation of synapsins, unlike serine phosphorylation, stimulates the reclustering of recycled SVs and their recruitment to the reserve pool.

The development, connectivity, and structural plasticity of neuronal networks largely depend on the directional growth of axonal growth cones (GCs). The morphology and 3-D profile of axons and GCs of primary hippocampal neurons, grown onto glass surfaces coated with poly-D-lysine (PDL) and micropatterned with stripes of the adhesion molecule L1 by using the indirect microcontact printing, were investigated. Neurons were fixed at early stages (one to seven days) of in vitro development prior to synapse formation, and analyzed by fluorescence and atomic force microscopy. The latter technique allowed us to investigate the 3-D morphology of the GCs, and detect their morphological rearrangements during axon outgrowth and during contact with the underlying substrate. We found that axons decreased their height-to-width ratio over development in culture, and that this value became particularly low when the axon and the GC proceeded onto a surface containing attracting cues such as L1 with respect to GCs growing onto a nonspecific adhesion substrate such as PDL. Along with this shape change of the axons, GCs lying onto L1 tracks displayed a flattened shape, ideal for sensing and progression, whereas GCs onto areas of nonspecific adhesion displayed more prominent shapes and steeper edges.

Investigations of amyloidogenic diseases use synthetic peptides for cell-free and in vitro studies. However, amyloidogenic peptides often show intrinsic variability that markedly affects the reproducibility of experiments. Proof of physicochemical and biological variability with different batches of amyloidogenic peptides have been reported in literature. Here, we show that differences can be observed even within the same batch of Abeta1-42 peptide after storing lyophilised samples at -20 degrees C. This change (referred to as 'peptide aging') was reproduced with Abeta1-40 peptide samples by using a series of lyophilisation cycles, showing that lyophilisation, rather than preserving the physicochemical and biological features of Abeta peptides, introduces wide variability. To counteract synthetic peptide aging, we set up a procedure involving the sequential use of trifluoroacetic acid, formic acid and sodium hydroxide solutions that disaggregate preformed seeds and enriched Abeta peptide solutions into monomers and low-molecular-weight oligomers. This procedure enabled us to obtain reproducible physicochemical and biological features of Abeta peptides, irrespective of their age.

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Acid generation and elimination processes were compared with total base (bicarbonate plus metabolizable anions) turnover in 18 anuric patients undergoing post-dilutional haemofiltration. The study was conducted during the second haemodialysis session of the week by means of a whole-body base balance technique. The results showed that the mean rates of base loss and base gain during dialysis did not differ (i.e. the dialysis base balance approximated to zero). The concurrent mean rate of intestinal base absorption was 66+/-26 mmol/2 days, as calculated from the whole-body balance of the various inorganic cation and anion differences in a metabolic steady state. This level of intestinal base absorption would be capable of neutralizing the 59+/-21 mmol of H(+) ions/2 days that is contributed by sulphuric acid, which is the most important endogenous acid produced in anuric patients. In spite of the fact that intestinal base supply was adequate to neutralize endogenous acid production completely, our patients presented with pre-dialysis non-carbonic acidosis. The depression of plasma bicarbonate levels could not, however, be explained by increased concentrations of the anion gap and organic acids, which were within normal limits both before and after dialysis. We suggest as an alternative hypothesis that this pre-dialysis acidosis may represent an isotonic dilution acidosis that is induced by the ingestion of base-free tap water in order for plasma tonicity to be protected from the accumulation of impermeant dietary solutes, which takes place during the interdialysis period in anuric patients.

An improvement of regional and global left ventricle dysfunction can be achieved in patients with coronary artery disease either by coronary revascularization with percutaneous transluminal coronary angioplasty or coronary artery bypass grafting. Several techniques have been developed to identify dysfunctional but viable myocardium. In the last decade, positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) has been used for the detection of hibernating myocardium. It can accurately predict the recovery of abnormal wall motion in hibernating segments prior to surgery. In particular, assessment of viability with 18F-FDG is indicated in high-risk surgical candidates being considered for revascularization or transplantation. Moreover, patient selection criteria and economic consideration are also relevant for cost-effective use of the technique.