Lewis D. B. Evans

η-Secretase processing of APP inhibits neuronal activity in the hippocampus

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide. Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-β fragments generated by the α- and β-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (β-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504–505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-β). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

An escort mechanism for cycling of export chaperones during flagellum assembly

Assembly of the bacterial flagellar filament requires a type III export pathway for ordered delivery of structural subunits from the cytosol to the cell surface. This is facilitated by transient interaction with chaperones that protect subunits and pilot them to dock at the membrane export ATPase complex. We reveal that the essential export protein FliJ has a novel chaperone escort function in the pathway, specifically recruiting unladen chaperones for the minor filament-class subunits of the filament cap and hook-filament junction substructures. FliJ did not recognize unchaperoned subunits or chaperone-subunit complexes, and it associated with the membrane ATPase complex, suggesting a function postdocking. Empty chaperones that were recruited by FliJ in vitro were efficiently captured from FliJ-chaperone complexes by cognate subunits. FliJ and subunit bound to the same region on the target chaperone, but the cognate subunit had a ≈700-fold greater affinity for chaperone than did FliJ. The data show that FliJ recruits chaperones and transfers them to subunits, and indicate that this is driven by competition for a common binding site. This escort mechanism provides a means by which free export chaperones can be cycled after subunit release, establishing a new facet of the secretion process. As FliJ does not escort the chaperone for the major filament subunit, cycling may offer a mechanism for export selectivity and thus promote assembly of the junction and cap substructures required for initiation of flagellin polymerization.

APP Metabolism Regulates Tau Proteostasis in Human Cerebral Cortex Neurons

Summary Accumulation of Aβ peptide fragments of the APP protein and neurofibrillary tangles of the microtubule-associated protein tau are the cellular hallmarks of Alzheimer’s disease (AD). To investigate the relationship between APP metabolism and tau protein levels and phosphorylation, we studied human-stem-cell-derived forebrain neurons with genetic forms of AD, all of which increase the release of pathogenic Aβ peptides. We identified marked increases in intracellular tau in genetic forms of AD that either mutated APP or increased its dosage, suggesting that APP metabolism is coupled to changes in tau proteostasis. Manipulating APP metabolism by β-secretase and γ-secretase inhibition, as well as γ-secretase modulation, results in specific increases and decreases in tau protein levels. These data demonstrate that APP metabolism regulates tau proteostasis and suggest that the relationship between APP processing and tau is not mediated solely through extracellular Aβ signaling to neurons.

A chain mechanism for flagellum growth

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip.

Building a flagellum outside the bacterial cell

Highlights • Cryoelectron tomography reveals details of the intact flagellar export machinery.• Mechanistic studies reveal discrete stages of the flagellar subunit export pathway.• Unanticipated chain mechanism for constant rate of flagellum growth.

Selective binding of virulence type III export chaperones by FliJ escort orthologues InvI and YscO

Abstract Bacteria secrete flagella subunits and deliver virulence effectors via type III export systems. During flagellar filament assembly, a chaperone escort mechanism has been proposed to enhance the export of early, minor flagellar filament components by selectively binding and cycling their chaperones. Here we identify virulence orthologues of the flagellar chaperone escort FliJ and show that the orthologues Salmonella InvI and Yersinia YscO are, like FliJ, essential for their type III export pathway and similarly, do not bind export substrates. Like FliJ, they recognize a subset of export chaperones, in particular those of the host membrane translocon components required for subsequent effector delivery.

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

The frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency.

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways

Summary In Alzheimer’s disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.

Interactions of Flagellar Structural Subunits with the Membrane Export Machinery

During assembly of the bacterial flagellum, structural subunits synthesized inside the cell must be exported across the cytoplasmic membrane before they can crystallize into the nascent flagellar structure. This export process is facilitated by a specialized Flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. Here, we describe three methods-isothermal titration calorimetry, photo-crosslinking using unnatural amino acids, and a subunit capture assay-used to investigate the interactions of flagellar structural subunits with the membrane export machinery component FlhB.