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Submarine landslides have been identified in almost all ocean basins worldwide. The largest submarine landslides occur on very shallow slopes and can be far larger than any terrestrial landslide. Submarine landslides can produce tsunami whose far-reaching effects can rival those produced by earthquake-tsunamis and threaten increasingly populated coastlines. Even small landslides can damage very expensive and critically important offshore infrastructure, such as pipelines used for oil and gas recovery, and telecommunication cables that now carry over 95% of digital data traffic. A better understanding of submarine landslide processes, including triggering mechanisms, preconditioning factors, timing, and frequency as well as dynamics of submarine landslide, and their consequences are of clear societal and economic importance. Despite their importance, many fundamental submarine landslide processes are still poorly understood. We currently have many studies that have mapped and sampled submarine landslide deposits; however, in order to fill outstanding but key knowledge gaps, future studies may have to go beyond this in order to unravel processes governing submarine landslides with even more interdisciplinary approaches. This chapter provides a very short review about submarine landslide studies, with emphasis on the emerging needs in future landslide research.

Introduction There is keen interest in elucidating the biological mechanisms underlying recent failures of β-site amyloid precursor protein–cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. Methods We developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. Results Seven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid β (Aβ). Discussion Elevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such β-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aβ by dissociating β/γ secretase complexes are better candidates because they neither inhibit β- and γ-secretase nor increase BACE1 levels.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA–responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other “synucleinopathies.” In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle–like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.

The protein α-synuclein, a key player in Parkinson’s disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible “prion-like” propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a “prion-like” aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and “prion-like” aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein.

The α-synuclein protein (αS) is the major constituent of pathological neuronal inclusions both in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) with differential brain region-specific pathology patterns and clinical presentations. Two hypotheses that were recently put forward were either that of brain-region specific vulnerability or that of different αS aggregates, “strains”, that would affect different brain regions via “prion-like” spread. What governs these patterns, their hypothesized “prion-like” progression and region-specific vulnerability to αS aggregation in different synucleinopathies is still largely unknown. Data collected in the last decade suggests that αS can exhibit in different conformations under physiological and/or pathological conditions, which in turn help govern aggregation propensity. The cytosolic unfolded, monomeric form of αS (αSCU) is aggregation-prone and can misfold into soluble, toxic oligomers, protofilaments, and amyloid fibrils with self-templating properties, while the cytosolic helically folded, multimeric form (αSCH) resists disease-associated changes.

SUMMARY While misfolding of alpha-synuclein (αSyn) is central to the pathogenesis of Parkinson’s disease (PD), fundamental questions about its structure and function at the synapse remain unanswered. We examine synaptosomes from non-transgenic and transgenic mice expressing wild-type human αSyn, the E46K fPD-causing mutation, or an amplified form of E46K (“3K”). Synaptosomes from mice expressing the 3K mutant show reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, decreased SNARE complexes, and abnormal levels of certain synaptic proteins. With our intra-synaptosomal nuclear magnetic resonance (NMR) method, we reveal that WT αSyn participates in heterogeneous interactions with synaptic components dependent on endogenous αSyn and synaptosomal integrity. The 3K mutation markedly alters these interactions. The synaptic microenvironment is necessary for αSyn to reach its native conformations and establish a physiological interaction network. Its inability to populate diverse conformational ensembles likely represents an early step in αSyn dysfunction that contributes to the synaptotoxicity observed in synucleinopathies., In brief Fonseca-Ornelas et al. report the in-cell NMR behavior of αSyn at synapses. While WT αSyn participates in transient interactions with synaptic components, an αSyn variant based on an fPD mutant displays a reduced ability to do so. This results in biochemical, morphological, and functional changes that may explain αSyn-related toxicity., Graphical Abstract

α-Synuclein (αSyn) is a key player in the pathogenesis of Parkinson's disease and other synucleinopathies. Here, we report the existence of a novel soluble α-helical conformer of αSyn, obtained through transient interaction with lipid interfaces, and propose dynamic oligomerization as the mechanism underlying its stability. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might thus play crucial roles as molecular chaperones in a cellular environment.

Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies. Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases). Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted. Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet. Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models. Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival. Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. Disclosures Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other ‘synucleinopathies’. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity., Previous work suggests the existence of soluble, diffusible α-synuclein species in the brains of synucleinopathy patients. In this study, we show that soluble, non-physiological α-synuclein in dementia with Lewy bodies brains permeabilize lipid membranes in vitro compared with identically prepared extracts from control brains. In addition, we show that Lewy-associated species from these brains are neurotoxic., Graphical Abstract Graphical Abstract

Misfolded, aggregated forms of alpha-synuclein (αSyn) are the principal protein component of Lewy bodies, the defining lesions of Parkinson’s disease (PD) and dementia with Lewy bodies. Increased copy number, missense mutations, and specific 5’ and 3’ regulatory elements of αSyn can all cause familial PD. Although αSyn participates in the regulation of synaptic vesicles, fundamental questions about the relationship between its structure and function in the presynaptic terminal are unanswered. Here, we systematically examined brain synaptosomes from non-transgenic (NTG) mice and transgenic mice expressing wild-type (WT) human αSyn or an amplified form of the E46K PD mutation (called 3K) that impairs the normal α-helical tetramerization of WT αSyn and increases its membrane affinity. Synaptosomes from transgenic mice expressing the 3K mutant had reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, impaired SNARE-complex assembly, and abnormal levels of select proteins involved in synaptic activity compared with NTG and WT human αSyn mice. We also developed a method to perform intra-synaptosomal NMR that revealed that WT αSyn participates in heterogeneous interactions with synaptic components. We show that these interactions depend on the presence of endogenous αSyn and that the 3K mutation diminishes the interplay between αSyn and the synapse. We conclude that the microenvironment inside presynaptic terminals is necessary for the proper conformation and interaction network of αSyn, where unfolded monomers of αSyn no longer represent the dominant conformation. We speculate that the inability to populate dynamically different conformational ensembles likely represents the early steps in αSyn misfunction that result in the neurotoxicity observed in PD and related synucleinopathies. With the advance of techniques such as cryo-electron tomography, in-cell single-molecule FRET, and in-cell NMR, in situ approaches like that presented here have the potential to address questions about the physiology of proteins such as αSyn that show diverse and changing conformations in their native, highly complex environments.

α-Synuclein's physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimetry (ITC) are established techniques for the study of binding equilibria in biological systems and, especially when used in combination, allow a thorough characterization of the protein-lipid interplay.Here we provide general guidelines and describe some common pitfalls of these experiments. This protocol describes the preparation of small unilamellar vesicles (SUVs), mimicking the curved bilayers α-synuclein normally interacts with, the CD-monitored titration of α-synuclein with SUVs, the ITC (lipid-into-protein) experiment, and the subsequent data analysis using an n independent binding site model.

α-Synuclein's physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimetry (ITC) are established techniques for the study of binding equilibria in biological systems and, especially when used in combination, allow a thorough characterization of the protein-lipid interplay.Here we provide general guidelines and describe some common pitfalls of these experiments. This protocol describes the preparation of small unilamellar vesicles (SUVs), mimicking the curved bilayers α-synuclein normally interacts with, the CD-monitored titration of α-synuclein with SUVs, the ITC (lipid-into-protein) experiment, and the subsequent data analysis using an n independent binding site model.

N-terminal acetylation is one of the most common post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular α-synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely Nα-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely Nα-acetylate our model substrate α-synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.

α-synuclein (αSyn) is one of the key players in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies. Its misfolding and subsequent aggregation into intracellular inclusions are the pathological hallmark of these diseases and may also play a central role in the molecular cascade leading to neurodegeneration. In this work, we report the existence of a novel soluble α-helical conformer of αSyn, an archetypal “intrinsically disordered protein” (IDP), obtained through transient interaction with lipid interfaces. We describe how the stability of this conformer is highly dependent on the continuous, dynamic oligomerization of the folded species. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might play crucial roles as molecular chaperones for cytosolic species in a cellular environment, as they do in the case of this previously unreported structure.Significance StatementBoth genetic and histopathologic evidence tie α-synuclein (αSyn) to the pathogenesis of Parkinson’s disease (PD), a widespread neurodegenerative disorder. Lipids play a central role in the dynamics of αSyn in physiology and disease. αSyn undergoes a coil-to-helix transition when binding to lipid vesicles and it is involved in the regulation of synaptic vesicle trafficking. Furthermore, recently discovered α-helical, aggregation-resistant “multimers” of αSyn could constitute a protective conformational pathway. We report the existence of a folded, lipid-unbound αSyn conformer that forms upon transient interaction with lipids and is stabilized by dynamic homooligomerization, suggesting that synaptic activity could modulate resistance towards aggregation. Our results are therefore important both for the molecular pathology of PD and the structural biology of intrinsically disordered proteins.

The microtubule-associated protein Tau is a natively unfolded protein, involved in a number of neurodegenerative disorders, collectively called tauopathies, aggregating in neurofibrillary tangles (NFT). It is an open question how the conversion from a microtubule (MT) bound molecule to an aggregation-prone Tau species occurs and, also, if and how tauopathy-related mutations affect its behavior in the cell. To address these points, we exploited a genetically encoded FRET sensor based on the full length Tau protein, to monitor in real time Tau conformational changes in different conditions in live cells. By studying the FRET signal we found that soluble Tau molecules, detached from MTs, display an unfolded structure. On the contrary, we observed an increased FRET signal generated by Tau monomers bound to MT, indicating that the association with MTs induced a folding of Tau protein, decreasing the distance between its N and C termini. We exploited the FRET sensor to investigate the impact of FTDP-17 mutations and of phosphorylation-site mutations on Tau folding and mobility in live cells. We demonstrate that the FTDP-17 Tau mutations weaken the interaction of Tau with cellular MTs, shifting the equilibrium towards the soluble pool while, conversely, phosphorylation site mutations shift the equilibrium of Tau towards the MT-bound state and a more closed conformation.