Aberrant Excitatory–Inhibitory Synaptic Mechanisms in Entorhinal Cortex Microcircuits During the Pathogenesis of Alzheimer’s Disease

Synaptic dysfunction is widely proposed as an initial insult leading to the neurodegeneration observed in Alzheimer’s disease (AD). We hypothesize that the initial insult originates in the lateral entorhinal cortex (LEC) due to deficits in key interneuronal functions and synaptic signaling mechanisms, in particular, Wnt (Wingless/integrated). To investigate this hypothesis, we utilized the first knock-in mouse model of AD (App^{NL/NL-F}, expressing a mutant form of human amyloid-β (Aβ) precursor protein. This model shows an age-dependent accumulation of Aβ, neuroinflammation, and neurodegeneration. Prior to the typical AD pathology, we showed a decrease in canonical Wnt signaling activity first affecting the LEC in combination with synaptic hyperexcitation and severely disrupted excitatory–inhibitory inputs onto principal cells. This synaptic imbalance was consistent with a reduction in the number of parvalbumin-containing (PV) interneurons, and a reduction in the somatic inhibitory axon terminals in the LEC compared with other cortical regions. However, targeting GABA_{A} receptors on PV cells using allosteric modulators, diazepam, zolpidem, or a nonbenzodiazepine, L-838,417 (modulator of α2/3 subunit-containing GABAA receptors), restored the excitatory–inhibitory imbalance observed at principal cells in the LEC. These data support our hypothesis, providing a rationale for targeting the synaptic imbalance in the LEC for early stage therapeutic intervention to prevent neurodegeneration in AD

Selective Modulation of α5 GABAA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in APP Mouse Model of Alzheimer’s Disease

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABAA receptors (GABAARs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer’s disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABAARs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABAAR subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD (APPNL−F/NL−F), which expresses a mutant form of human amyloid-β (Aβ), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged APPNL−F/NL−F mice, selective preservation of α5 GABAARs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the APPNL−F/NL−F mouse model, here we show that this excessive inhibition was “normalized” to control values with bath-applied α5-SOP002 (1 μM). However, α5-SOP002, further impaired inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a deficit of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABAAR NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABAAR is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABAARs

Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation.

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.This work was supported by the UK BBSRC and the Wellcome Trust (CMD, TPJK, MV), the Frances and Augustus Newman Foundation (TPJK), Magdalene College, Cambridge (AKB) , St John’s College, Cambridge (TCTM), the Cambridge Home and EU Scholarship Scheme (GM), Elan Pharmaceuticals (CMD, TPJK, MV, CG) and the Leverhulme Trust (AKB).This is the accepted manuscript. The final version is available from NPG at http://www.nature.com/nchembio/journal/v11/n3/abs/nchembio.1750.htm

The ELBA Force Field for Coarse-Grain Modeling of Lipid Membranes

A new coarse-grain model for molecular dynamics simulation of lipid membranes is presented. Following a simple and conventional approach, lipid molecules are modeled by spherical sites, each representing a group of several atoms. In contrast to common coarse-grain methods, two original (interdependent) features are here adopted. First, the main electrostatics are modeled explicitly by charges and dipoles, which interact realistically through a relative dielectric constant of unity (). Second, water molecules are represented individually through a new parametrization of the simple Stockmayer potential for polar fluids; each water molecule is therefore described by a single spherical site embedded with a point dipole. The force field is shown to accurately reproduce the main physical properties of single-species phospholipid bilayers comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) in the liquid crystal phase, as well as distearoylphosphatidylcholine (DSPC) in the liquid crystal and gel phases. Insights are presented into fundamental properties and phenomena that can be difficult or impossible to study with alternative computational or experimental methods. For example, we investigate the internal pressure distribution, dipole potential, lipid diffusion, and spontaneous self-assembly. Simulations lasting up to 1.5 microseconds were conducted for systems of different sizes (128, 512 and 1058 lipids); this also allowed us to identify size-dependent artifacts that are expected to affect membrane simulations in general. Future extensions and applications are discussed, particularly in relation to the methodology's inherent multiscale capabilities

Nuclear astrophysics with radioactive ions at FAIR

The nucleosynthesis of elements beyond iron is dominated by neutron captures in the s and r processes. However, 32 stable, proton-rich isotopes cannot be formed during those processes, because they are shielded from the s-process flow and r-process, β-decay chains. These nuclei are attributed to the p and rp process. For all those processes, current research in nuclear astrophysics addresses the need for more precise reaction data involving radioactive isotopes. Depending on the particular reaction, direct or inverse kinematics, forward or time-reversed direction are investigated to determine or at least to constrain the desired reaction cross sections. The Facility for Antiproton and Ion Research (FAIR) will offer unique, unprecedented opportunities to investigate many of the important reactions. The high yield of radioactive isotopes, even far away from the valley of stability, allows the investigation of isotopes involved in processes as exotic as the r or rp processes

M1 and E2 transition rates from core-excited states in semi-magic 94Ru

Lifetimes of high-spin states have been measured in the semi-magic (N= 50) nucleus 94Ru. Excited states in 94Ru were populated in the 58Ni(40Ca, 4p)94Ru* fusion-evaporation reaction at the Grand Accélérateur National d’Ions Lourds (GANIL) accelerator complex. DSAM lifetime analysis was performed on the Doppler broadened line shapes in energy spectra obtained from γ-rays emitted while the residual nuclei were slowing down in a thick 6mg/cm^2 metallic 58Ni target. In total eight excited-state lifetimes in the angular momentum range I= (13 - 20) ℏ have been measured, five of which were determined for the first time. The corresponding B(M1) and B(E2) reduced transition strengths are discussed within the framework of large-scale shell model calculations to study the contribution of different particle-hole configurations, in particular for analyzing contributions from core-excited configurations

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field