Proteome-wide analysis identifies plasma immune regulators of amyloid-beta progression.

While immune function is known to play a mechanistic role in Alzheimer's disease (AD), whether immune proteins in peripheral circulation influence the rate of amyloid-ß (Aß) progression - a central feature of AD - remains unknown. In the Baltimore Longitudinal Study of Aging, we quantified 942 immunological proteins in plasma and identified 32 (including CAT [catalase], CD36 [CD36 antigen], and KRT19 [keratin 19]) associated with rates of cortical Aß accumulation measured with positron emission tomography (PET). Longitudinal changes in a subset of candidate proteins also predicted Aß progression, and the mid- to late-life (20-year) trajectory of one protein, CAT, was associated with late-life Aß-positive status in the Atherosclerosis Risk in Communities (ARIC) study. Genetic variation that influenced plasma levels of CAT, CD36 and KRT19 predicted rates of Aß accumulation, including causal relationships with Aß PET levels identified with two-sample Mendelian randomization. In addition to associations with tau PET and plasma AD biomarker changes, as well as expression patterns in human microglia subtypes and neurovascular cells in AD brain tissue, we showed that 31 % of candidate proteins were related to mid-life (20-year) or late-life (8-year) dementia risk in ARIC. Our findings reveal plasma proteins associated with longitudinal Aß accumulation, and identify specific peripheral immune mediators that may contribute to the progression of AD pathophysiology.

The Alzheimer's disease-linked protease BACE2 cleaves VEGFR3 and modulates its signaling.

The ß-secretase BACE1 is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet, little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, non-human primates and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for a safer prevention of Alzheimer's disease.

The necroptosis cell death pathway drives neurodegeneration in Alzheimer's disease.

Although apoptosis, pyroptosis, and ferroptosis have been implicated in AD, none fully explains the extensive neuronal loss observed in AD brains. Recent evidence shows that necroptosis is abundant in AD, that necroptosis is closely linked to the appearance of Tau pathology, and that necroptosis markers accumulate in granulovacuolar neurodegeneration vesicles (GVD). We review here the neuron-specific activation of the granulovacuolar mediated neuronal-necroptosis pathway, the potential AD-relevant triggers upstream of this pathway, and the interaction of the necrosome with the endo-lysosomal pathway, possibly providing links to Tau pathology. In addition, we underscore the therapeutic potential of inhibiting necroptosis in neurodegenerative diseases such as AD, as this presents a novel avenue for drug development targeting neuronal loss to preserve cognitive abilities. Such an approach seems particularly relevant when combined with amyloid-lowering drugs.

Microglia-astrocyte crosstalk in the amyloid plaque niche of an Alzheimer's disease mouse model, as revealed by spatial transcriptomics.

The amyloid plaque niche is a pivotal hallmark of Alzheimer's disease (AD). Here, we employ two high-resolution spatial transcriptomics (ST) platforms, CosMx and Spatial Enhanced Resolution Omics-sequencing (Stereo-seq), to characterize the transcriptomic alterations, cellular compositions, and signaling perturbations in the amyloid plaque niche in an AD mouse model. We discover heterogeneity in the cellular composition of plaque niches, marked by an increase in microglial accumulation. We profile the transcriptomic alterations of glial cells in the vicinity of plaques and conclude that the microglial response to plaques is consistent across different brain regions, while the astrocytic response is more heterogeneous. Meanwhile, as the microglial density of plaque niches increases, astrocytes acquire a more neurotoxic phenotype and play a key role in inducing GABAergic signaling and decreasing glutamatergic signaling in hippocampal neurons. We thus show that the accumulation of microglia around hippocampal plaques disrupts astrocytic signaling, in turn inducing an imbalance in neuronal synaptic signaling.

Alzheimer's disease-related presenilins are key to intestinal epithelial cell function and gut immune homoeostasis.

OBJECTIVE: Mutations in presenilin genes are the major cause of Alzheimer's disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis. DESIGN: Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis. RESULTS: Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation. CONCLUSION: Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

Xenografted human microglia display diverse transcriptomic states in response to Alzheimer's disease-related amyloid-ß pathology.

Microglia are central players in Alzheimer's disease pathology but analyzing microglial states in human brain samples is challenging due to genetic diversity, postmortem delay and admixture of pathologies. To circumvent these issues, here we generated 138,577 single-cell expression profiles of human stem cell-derived microglia xenotransplanted in the brain of the AppNL-G-F model of amyloid pathology and wild-type controls. Xenografted human microglia adopt a disease-associated profile similar to that seen in mouse microglia, but display a more pronounced human leukocyte antigen or HLA state, likely related to antigen presentation in response to amyloid plaques. The human microglial response also involves a pro-inflammatory cytokine/chemokine cytokine response microglia or CRM response to oligomeric Aß oligomers. Genetic deletion of TREM2 or APOE as well as APOE polymorphisms and TREM2R47H expression in the transplanted microglia modulate these responses differentially. The expression of other Alzheimer's disease risk genes is differentially regulated across the distinct cell states elicited in response to amyloid pathology. Thus, we have identified multiple transcriptomic cell states adopted by human microglia in a multipronged response to Alzheimer's disease-related pathology, which should be taken into account in translational studies.

New precision medicine avenues to the prevention of Alzheimer's disease from insights into the structure and function of γ-secretases.

Two phase-III clinical trials with anti-amyloid peptide antibodies have met their primary goal, i.e. slowing of Alzheimer's disease (AD) progression. However, antibody therapy may not be the optimal therapeutic modality for AD prevention, as we will discuss in the context of the earlier small molecules described as "γ-secretase modulators" (GSM). We review here the structure, function, and pathobiology of γ-secretases, with a focus on how mutations in presenilin genes result in early-onset AD. Significant progress has been made in generating compounds that act in a manner opposite to pathogenic presenilin mutations: they stabilize the proteinase-substrate complex, thereby increasing the processivity of substrate cleavage and altering the size spectrum of Aß peptides produced. We propose the term "γ-secretase allosteric stabilizers" (GSAS) to distinguish these compounds from the rather heterogenous class of GSM. The GSAS represent, in theory, a precision medicine approach to the prevention of amyloid deposition, as they specifically target a discrete aspect in a complex cell biological signalling mechanism that initiates the pathological processes leading to Alzheimer's disease.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.

The γ-secretase substrate proteome and its role in cell signaling regulation.

γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell.

Discovery of brain permeable 2-Azabicyclo[2.2.2]octane sulfonamides as a novel class of presenilin-1 selective γ-secretase inhibitors.

This paper describes the rational design, synthesis, structure-activity relationship (SAR), and biological profile of presenilin-1 (PSEN-1) complex selective γ-secretase inhibitors, assessed for selectivity using a unique set of four γ-secretase subtype complexes. A set of known PSEN-1 selective γ-Secretase inhibitors (GSIs) was analyzed to understand the pharmacophoric features required for selective inhibition. Conformational modeling suggests that a characteristic 'U' shape orientation between aromatic sulfone/sulfonamide and aryl ring is crucial for PSEN-1 selectivity and potency. Using these insights, a series of brain-penetrant 2-azabicyclo[2,2,2]octane sulfonamides was devised and synthesized as a new class of PSEN-1 selective inhibitors. Compounds 13c and 13k displayed high potency towards PSEN1-APH1B complex but moderate selectivity towards PSEN2 complexes. However, compound (+)-13b displayed low nanomolar potency towards the PSEN1-APH1B complex, little (∼4-fold) selectivity towards PSEN1-APH1A, and high selectivity (>350-fold) versus PSEN2 complexes. Excellent brain penetration, no significant CYP inhibition, or cardiotoxicity, good solubility, and permeability make (+)-13b an excellent candidate for further lead optimization.

MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer's disease.

Neuronal cell loss is a defining feature of Alzheimer's disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD.

Intracerebral Hemorrhage Among Blood Donors and Their Transfusion Recipients.

Importance: Recent reports have suggested that cerebral amyloid angiopathy, a common cause of multiple spontaneous intracerebral hemorrhages (ICHs), may be transmissible through parenteral injection of contaminated cadaveric pituitary hormone in humans. Objective: To determine whether spontaneous ICH in blood donors after blood donation is associated with development of spontaneous ICH in transfusion recipients. Design, Setting, and Participants: Exploratory retrospective cohort study using nationwide blood bank and health register data from Sweden (main cohort) and Denmark (validation cohort) and including all 1 089 370 patients aged 5 to 80 years recorded to have received a red blood cell transfusion from January 1, 1970 (Sweden), or January 1, 1980 (Denmark), until December 31, 2017. Exposures: Receipt of red blood cell transfusions from blood donors who subsequently developed (1) a single spontaneous ICH, (2) multiple spontaneous ICHs, or (3) no spontaneous ICH. Main Outcomes and Measures: Spontaneous ICH in transfusion recipients; ischemic stroke was a negative control outcome. Results: A total of 759 858 patients from Sweden (median age, 65 [IQR, 48-73] years; 59% female) and 329 512 from Denmark (median age, 64 [IQR, 50-73] years; 58% female) were included, with a median follow-up of 5.8 (IQR, 1.4-12.5) years and 6.1 (IQR, 1.5-11.6) years, respectively. Patients who underwent transfusion with red blood cell units from donors who developed multiple spontaneous ICHs had a significantly higher risk of a single spontaneous ICH themselves, compared with patients receiving transfusions from donors who did not develop spontaneous ICH, in both the Swedish cohort (unadjusted incidence rate [IR], 3.16 vs 1.12 per 1000 person-years; adjusted hazard ratio [HR], 2.73; 95% CI, 1.72-4.35; P < .001) and the Danish cohort (unadjusted IR, 2.82 vs 1.09 per 1000 person-years; adjusted HR, 2.32; 95% CI, 1.04-5.19; P = .04). No significant difference was found for patients receiving transfusions from donors who developed a single spontaneous ICH in the Swedish cohort (unadjusted IR, 1.35 vs 1.12 per 1000 person-years; adjusted HR, 1.06; 95% CI, 0.84-1.36; P = .62) nor the Danish cohort (unadjusted IR, 1.36 vs 1.09 per 1000 person-years; adjusted HR, 1.06; 95% CI, 0.70-1.60; P = .73), nor for ischemic stroke as a negative control outcome. Conclusions and Relevance: In an exploratory analysis of patients who received red blood cell transfusions, patients who underwent transfusion with red blood cells from donors who later developed multiple spontaneous ICHs were at significantly increased risk of spontaneous ICH themselves. This may suggest a transfusion-transmissible agent associated with some types of spontaneous ICH, although the findings may be susceptible to selection bias and residual confounding, and further research is needed to investigate if transfusion transmission of cerebral amyloid angiopathy might explain this association.

A fully automated home cage for long-term continuous phenotyping of mouse cognition and behavior.

Automated home-cage monitoring systems present a valuable tool for comprehensive phenotyping of natural behaviors. However, current systems often involve complex training routines, water or food restriction, and probe a limited range of behaviors. Here, we present a fully automated home-cage monitoring system for cognitive and behavioral phenotyping in mice. The system incorporates T-maze alternation, novel object recognition, and object-in-place recognition tests combined with monitoring of locomotion, drinking, and quiescence patterns, all carried out over long periods. Mice learn the tasks rapidly without any need for water or food restrictions. Behavioral characterization employs a deep convolutional neural network image analysis. We show that combined statistical properties of multiple behaviors can be used to discriminate between mice with hippocampal, medial entorhinal, and sham lesions and predict the genotype of an Alzheimer's disease mouse model with high accuracy. This technology may enable large-scale behavioral screening for genes and neural circuits underlying spatial memory and other cognitive processes.

Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics.

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aß1-40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals.

Early alterations in the MCH system link aberrant neuronal activity and sleep disturbances in a mouse model of Alzheimer's disease.

Early Alzheimer's disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in AppNL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in AppNL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in AppNL-G-F mice. AppNL-G-F mice spend less time in rapid eye movement (REM) sleep. AppNL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions.

microRNA-132 regulates gene expression programs involved in microglial homeostasis.

microRNA-132 (miR-132), a known neuronal regulator, is one of the most robustly downregulated microRNAs (miRNAs) in the brain of Alzheimer's disease (AD) patients. Increasing miR-132 in AD mouse brain ameliorates amyloid and Tau pathologies, and also restores adult hippocampal neurogenesis and memory deficits. However, the functional pleiotropy of miRNAs requires in-depth analysis of the effects of miR-132 supplementation before it can be moved forward for AD therapy. We employ here miR-132 loss- and gain-of-function approaches using single-cell transcriptomics, proteomics, and in silico AGO-CLIP datasets to identify molecular pathways targeted by miR-132 in mouse hippocampus. We find that miR-132 modulation significantly affects the transition of microglia from a disease-associated to a homeostatic cell state. We confirm the regulatory role of miR-132 in shifting microglial cell states using human microglial cultures derived from induced pluripotent stem cells.

Selective inhibitors of the PSEN1-gamma-secretase complex.

Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.

Neurodegeneration cell per cell.

The clinical definition of neurodegenerative diseases is based on symptoms that reflect terminal damage of specific brain regions. This is misleading as it tells little about the initial disease processes. Circuitry failures that underlie the clinical symptomatology are themselves preceded by clinically mostly silent, slowly progressing multicellular processes that trigger or are triggered by the accumulation of abnormally folded proteins such as Aß, Tau, TDP-43, and α-synuclein, among others. Methodological advances in single-cell omics, combined with complex genetics and novel ways to model complex cellular interactions using induced pluripotent stem (iPS) cells, make it possible to analyze the early cellular phase of neurodegenerative disorders. This will revolutionize the way we study those diseases and will translate into novel diagnostics and cell-specific therapeutic targets, stopping these disorders in their early track before they cause difficult-to-reverse damage to the brain.