Microglia are tissue-resident macrophages of the CNS that orchestrate local immune responses and contribute to several neurological and psychiatric diseases. Little is known about human microglia and how they orchestrate their highly plastic, context-specific adaptive responses during pathology. Here we combined two high-dimensional technologies, single-cell RNAsequencing and time-of-flight mass cytometry, to identify microglia states in the human brain during homeostasis and disease. This approach enabled us to identify and characterize a previously unappreciated spectrum of transcriptional states in human microglia. These transcriptional states are determined by their spatial distribution, and they further change with aging and brain tumor pathology. This description of multiple microglia phenotypes in the human CNS may open promising new avenues for subset-specific therapeutic interventions.

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Purpose Tau deposition is a key pathological feature of Alzheimer’s disease (AD) and other neurodegenerative disorders. The spreading of tau neurofibrillary tangles across defined brain regions corresponds to the observed level of cognitive decline in AD. Positron-emission tomography (PET) has proved to be an important tool for the detection of amyloid-beta (Aβ) aggregates in the brain, and is currently being explored for detection of pathological misfolded tau in AD and other non-AD tauopathies. Several PET tracers targeting tau deposits have been discovered and tested in humans. Limitations have been reported, especially regarding their selectivity. Methods In our screening campaign we identified pyrrolo[2,3- b :4,5- c ’]dipyridine core structures with high affinity for aggregated tau. Further characterization showed that compounds containing this moiety had significantly reduced monoamine oxidase A (MAO-A) binding compared to pyrido[4,3- b ]indole derivatives such as AV-1451. Results Here we present preclinical data of all ten fluoropyridine regioisomers attached to the pyrrolo[2,3- b :4,5- c ’]dipyridine scaffold, revealing compounds 4 and 7 with superior properties. The lead candidate [ 18 F]PI-2620 (compound 7 ) displayed high affinity for tau deposits in AD brain homogenate competition assays. Specific binding to pathological misfolded tau was further demonstrated by autoradiography on AD brain sections (Braak I-VI), Pick’s disease and progressive supranuclear palsy (PSP) pathology, whereas no specific tracer binding was detected on brain slices from non-demented donors. In addition to its high affinity binding to tau aggregates, the compound showed excellent selectivity with no off-target binding to Aβ or MAO-A/B. Good brain uptake and fast washout were observed in healthy mice and non-human primates. Conclusions Therefore, [ 18 F]PI-2620 was selected for clinical validation. Electronic supplementary material The online version of this article (10.1007/s00259-019-04397-2) contains supplementary material, which is available to authorized users.

Aggregation of amyloid-␤ (A␤)2 peptides and deposition into neuritic plaques are hallmark features of Alzheimer disease (AD) neuropathology (1, 2). Therefore, research efforts during the past 3 decades have focused on elucidating the mechanisms of A␤ fibrillization, identifying toxic species, and developing strategies to inhibit and/or reverse A␤ amyloid formation and toxicity in vivo (3,4).A␤ peptides are produced as soluble monomers (5, 6) and undergo oligomerization and amyloid fibril formation via a nucleation-dependent polymerization process (7,8). During the course of in vitro A␤ fibril formation, various nonfibrillar aggregation intermediates, collectively called soluble oligomers or protofibrils, have been shown to precede the emergence of fibrils. Increasing evidence from various sources points to A␤ oligomers/protofibrils as putative toxic species in AD pathogenesis and suggests that these species are potential therapeutic targets for treating AD (reviewed in Refs. 9, 10). Although the toxic oligomer hypothesis has emerged as one of the major current working hypotheses in AD research, the development of effective diagnostic tools and therapies on the basis of this hypothesis has yet to be realized (11-13). This is partially due to the fact that identification of a single toxic A␤ species that correlates with AD progression and severity remains elusive. Furthermore, the exact mechanisms by which these species contribute to A␤ toxicity in vivo and the nature of the toxic species are not yet fully understood. Recent evidence suggests that accelerating the process of A␤ fibrillization greatly enhances A␤ toxicity in vitro (14) and the spread of amyloid pathology in vivo (15-17).Despite significant efforts by different groups to isolate specific intermediates along the amyloid formation pathway (12, 18 -22), the inherent heterogeneity of the process and metastable nature of A␤ oligomers (11-13) have precluded the isolation of a single toxic species. Unless covalently crosslinked (23), A␤ oligomers do not exist as stable entities, i.e. they evolve into higher order aggregates and, if they are onpathway intermediates, convert into fibrils (19). Therefore, it is plausible to assume that the structural dynamics of oligomers and factors that govern their interconversion and/or growth might influence some of the disease-related cytotoxic effects of A␤. In other words, an ongoing polymerization

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

We investigated the therapeutic effects of two different versions of A␤ 1-15 (16) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetrapalmitoylated amyloid 1-15 peptide (palmA␤ 1-15), or with amyloid 1-16 peptide (PEG-A␤ 1-16) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmA␤ 1-15 liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-A␤ 1-16 had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmA␤ 1-15, whereas those elicited by PEG-A␤ 1-16 were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly ␤-sheet conformation of palmA␤1-15 and random coil of PEG-A␤ 1-16. We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a ''conformational'' disease, implying that antibodies against amyloid sequences in the ␤-sheet conformation are preferred as potential therapeutic agents.Alzheimer's disease ͉ ␤-amyloid ͉ vaccine C linical manifestations of Alzheimer's disease (AD) include progressive memory loss, cognitive impairment, confusion, and personality changes. The major neuropathological changes in the brains of AD patients are senile plaques and neurofibrillar tangles causing progressive neuronal dysfunction. These pathological alterations are likely causally involved in eventual neuronal death, particularly in brain regions related to memory and cognition (1-4). Senile plaques are formed predominantly by the ␤-amyloid peptide A␤ 1-42 that is coiled and ␣-helical in its soluble form but, upon conformational transition, aggregates into ␤-sheeted multimers. The monomeric A␤ peptide is a physiological metabolite of the large amyloid precursor protein (APP, 695-770 aa) that undergoes processing by several sequential proteolytic steps (5). The A␤ 1-42 aggregates are proposed to play the key role in the pathogenesis of AD (6). In transgenic animals that overexpress mutant human APP, anti-A␤-specific antibodies decreased the A␤ burden and improved memory after either passive (7-11) or active (12-18) immunization.We previously demonstrated that i.p. inoculation of tetrapalmitoylated A␤1-16 reconstituted in liposomes to transgenic NORBA mice elicited significant titers of anti-A␤ antibodies, that solubilized amyloid fibers in vitro and pancreatic A␤ plaques in vivo (19). To circumvent T cell-mediated immune responses known to be causatively involved in the adverse events of meningoencephalitis of AD patients immunized with A␤1-42 (20-22), the A␤1-16 and 1-15 sequences were used for immunization of APPxPS1 double-transgenic mice (23) because strong T cell epitopes are located more toward the C-te...

Accumulation of amyloid-β (Aβ) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer’s disease (AD). The precise pathological species of Aβ remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aβ monoclonal IgG4 that binds multiple forms of Aβ, with higher affinity for aggregated forms, and that blocks Aβ aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aβ. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab’s versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aβ, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aβ and breaks an essential salt-bridge characteristic of the β-hairpin conformation, eliminating features characteristic of the basic organization in Aβ oligomers and fibrils, and explains crenezumab’s inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab’s unique mechanism of action, particularly regarding Aβ oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

The spread of tau pathology correlates with cognitive decline in Alzheimer's disease. In vitro, tau antibodies can block cell-to-cell tau spreading. Although mechanisms of anti-tau function in vivo are unknown, effector function might promote microglia-mediated clearance. In this study, we investigated whether antibody effector function is required for targeting tau. We compared efficacy in vivo and in vitro of two versions of the same tau antibody, with and without effector function, measuring tau pathology, neuron health, and microglial function. Both antibodies reduced accumulation of tau pathology in Tau-P301L transgenic mice and protected cultured neurons against extracellular tau-induced toxicity. Only the full-effector antibody enhanced tau uptake in cultured microglia, which promoted release of proinflammatory cytokines. In neuron-microglia co-cultures, only effectorless anti-tau protected neurons, suggesting full-effector tau antibodies can induce indirect toxicity via microglia. We conclude that effector function is not required for efficacy, and effectorless tau antibodies may represent a safer approach to targeting tau.