Alzheimer's disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of β-amyloid (Aβ) plaques, which are the other histological hallmark of AD, does not follow the same strict spatiotemporal pattern, and it correlates poorly with cognitive decline. Instead, soluble Aβ oligomers have received increasing attention as probable inducers of pathogenesis. In this study, we use microinjections into electrophysiologically defined primary hippocampal rat neurons to demonstrate the direct neuron-to-neuron transfer of soluble oligomeric Aβ. Additional studies conducted in a human donor–acceptor cell model show that this Aβ transfer depends on direct cellular connections. As the transferred oligomers accumulate, acceptor cells gradually show beading of tubulin, a sign of neurite damage, and gradual endosomal leakage, a sign of cytotoxicity. These observations support that intracellular Aβ oligomers play a role in neurodegeneration, and they explain the manner in which Aβ can drive disease progression, even if the extracellular plaque load is poorly correlated with the degree of cognitive decline. Understanding this phenomenon sheds light on the pathophysiological mechanism of AD progression. Additional elucidation will help uncover the detailed mechanisms responsible for the manner in which AD progresses via anatomical connections and will facilitate the development of new strategies for stopping the progression of this incapacitating disease.

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

The hallmarks of Alzheimer disease are amyloid-β plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-β1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different β-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C₂mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern. Video S1: Video depicting motion of single particles of SNAP-CD44-1 GFP labeled with two different dyes (coded in green, JF549-cpSNAP ligand, and red, JF646-SNAP ligand) acquired for 400 frames at 16ms exposure time. Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s1.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd56827777e574c0020c9975ac006d333b0fc5d8a3bdf4a58d9642095d14efdf4722b8e104ba117e006fc97f8265 Video S2: Video depicting a track of two differently labeled SNAP-CD44-GFP (coded in green, JF549-cpSNAP ligand, and red, JF646-SNAP ligand) showing co-localization of the protein over multiple frames. Download Original Video (.2 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s2.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd55827731dd00e5d8e270eb1a277ab80b2d6e07f4bf3a64e0a46b9c10da7ff92a5a65609c0acbad41f31b7cd99f Video S3: Video depicting motion of single particles of SNAP-CD44-GFP acquired at ˜60fps (3) and an example individual track (4). Download Original Video (1.4 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s3.,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b49252dfe02d1f87e44537fa094ec05070714f8be30f4b85a6204a2bce0d6e685dc5c0685a106b9e5908621039e5898952b0cec40822e73afff2dd2522c7dcdaafbf6dc390666d0fc2a2bb719d5cf570637e70f759e1830fac2efe6eef8327c323af1840ed Video S4: Video depicting motion of single particles of SNAP-CD44-GFP acquired at ˜60fps (3) and an example individual track (4). Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s4.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518512abb28b22067a1f9884d85997ddab96149b9477c71571c229bbd7b1ea6e1e798c3f82e72b1b4002e6517d9153759c9a032a9739c89529d759d2c5032763fe3f7fba7042c7f8b08b6e8e1fdc38b4326600e69ba8cc6d8657ded8991be4c48e038c31c8713035497a3e2f7cc05fb2d1ef75c840aa9077ac4ae63dba6ff0cea30e766ab940a0a704010cb0e3775ae4886049b8473be4b956a0d7a1 Video S5: Video depicting motion of single particles of SNAP-CD44TmICD-GFP acquired at ˜60fps. Download Original Video (4.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s5.,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b49252dfe02d1f87e44537fa094ec05070714f8be30f4b85a6204a2bce0d6e685dc5c0685a106b9e5908621039e5898954b0ce211bc15be0d1dd62af8ded514c9e5af927c71a0ef396c2fc1e5ada373c37ef836f320753db56a6dba92fcdcc4eec61b76fc7 Video S6: Video depicting motion of single particles of SNAP-CD44Tm-GFP acquired at ˜60fps. Download Original Video (4.6 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-11-0715/20190930/media/mc-e18-11-0715-video_s6.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518414abb28b3a1288f73c3055a5474bbf090edb6c3233559d1b16ff38c592a8e8e15ae303e79c682b0a95bc4fab8cf97b06cfef7c50cf0c1b69293f95cd7f96d691bf74a72a469b46a0db6715c651a10df44453b3bd9b5708e458009ea1194808b2590e81ed08680c73b1aa626dcdfc8b32dd518277bf465b1f0ff7119a74c08313cf581f53c42895d8a6145d39aec619edd173d56d5d60122c99e71eb8c6f1