Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

Mapping microglia states in the human brain through the integration of high-dimensional techniques.

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 RNA-sequencing 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.

Discovery and preclinical characterization of [18F]PI-2620, a next-generation tau PET tracer for the assessment of tau pathology in Alzheimer's disease and other tauopathies.

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 (Aß) 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 [18F]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 Aß or MAO-A/B. Good brain uptake and fast washout were observed in healthy mice and non-human primates. CONCLUSIONS: Therefore, [18F]PI-2620 was selected for clinical validation.

Calpain-mediated tau fragmentation is altered in Alzheimer's disease progression.

The aggregation of intracellular tau protein is a major hallmark of Alzheimer's disease (AD). The extent and the stereotypical spread of tau pathology in the AD brain are correlated with cognitive decline during disease progression. Here we present an in-depth analysis of endogenous tau fragmentation in a well-characterized cohort of AD and age-matched control subjects. Using protein mass spectrometry and Edman degradation to interrogate endogenous tau fragments in the human brain, we identified two novel proteolytic sites, G323 and G326, as major tau cleavage events in both normal and AD cortex. These sites are located within the sequence recently identified as the structural core of tau protofilaments, suggesting an inhibitory mechanism of fibril formation. In contrast, a different set of novel cleavages showed a distinct increase in late stage AD. These disease-associated sites are located outside of the protofilament core sequence. We demonstrate that calpain 1 specifically cleaves at both the normal and diseased sites in vitro, and the site selection is conformation-dependent. Monomeric tau is predominantly cleaved at G323/G326 (normal sites), whereas oligomerization increases cleavages at the late-AD-associated sites. The fragmentation patterns specific to disease and healthy states suggest novel regulatory mechanisms of tau aggregation in the human brain.

Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects.

Tau pathology is associated with cognitive decline in Alzheimer's disease, and missense tau mutations cause frontotemporal dementia. Hyperphosphorylation and misfolding of tau are considered critical steps leading to tauopathies. Here, we determine how motifs controlling conformational changes in the microtubule-binding domain determine tau pathology in vivo. Human tau was overexpressed in the adult mouse forebrain to compare variants carrying residues that modulate tau propensity to acquire a ß-sheet conformation. The P301S mutation linked to frontotemporal dementia causes tau aggregation and rapidly progressing motor deficits. By comparison, wild-type tau becomes heavily hyperphosphorylated, and induces behavioral impairments that do not progress over time. However, the behavioral defects caused by wild-type tau can be suppressed when ß-sheet breaking proline residues are introduced in the microtubule-binding domain of tau. This modification facilitates tau interaction with microtubules, as shown by lower levels of phosphorylation, and by the enhanced protective effects of mutated tau against the severing of the cytoskeleton in neurons exposed to vinblastine. Altogether, motifs that are critical for tau conformation determine interaction with microtubules and subsequent pathological modifications, including phosphorylation and aggregation.

Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of ß-amyloid-42 aggregation for the treatment of Alzheimer's disease.

The aggregation of amyloid-ß peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-ß. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope.

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer's disease.

Structure of Crenezumab Complex with Aß Shows Loss of ß-Hairpin.

Accumulation of amyloid-ß (Aß) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aß remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aß monoclonal IgG4 that binds multiple forms of Aß, with higher affinity for aggregated forms, and that blocks Aß 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 Aß. 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 Aß, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aß and breaks an essential salt-bridge characteristic of the ß-hairpin conformation, eliminating features characteristic of the basic organization in Aß 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 Aß oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

Antibody-Mediated Targeting of Tau In Vivo Does Not Require Effector Function and Microglial Engagement.

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.

Synthesis and structure-activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of ß-amyloid-42 aggregation.

It is assumed that amyloid-ß aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the ß-sheet conformation of Aß via donor-acceptor-donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aß. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aß aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aß aggregation.

An Anti-ß-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome.

In Down syndrome (DS) or trisomy of chromosome 21, the ß-amyloid (Aß) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aß as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aß is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer's disease (AD). At present, no treatment targets Aß-related pathogenesis in people with DS. Herein we used a vaccine containing the Aß 1-15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aß vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aß IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aß without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aß levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aß as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aß immunotherapeutic approach may act to target Aß-related pathology in a mouse model of DS.

BACE-1 is expressed in the blood-brain barrier endothelium and is upregulated in a murine model of Alzheimer's disease.

Endothelial cells of the blood-brain barrier form a structural and functional barrier maintaining brain homeostasis via paracellular tight junctions and specific transporters such as P-glycoprotein. The blood-brain barrier is responsible for negligible bioavailability of many neuroprotective drugs. In Alzheimer's disease, current treatment approaches include inhibitors of BACE-1 (ß-site of amyloid precursor protein cleaving enzyme), a proteinase generating neurotoxic ß-amyloid. It is known that BACE-1 is highly expressed in endosomes and membranes of neurons and glia. We now provide evidence that BACE-1 is expressed in blood-brain barrier endothelial cells of human, mouse, and bovine origin. We further show its predominant membrane localization by 3D-dSTORM super-resolution microscopy, and by biochemical fractionation that further shows an abluminal distribution of BACE-1 in brain microvessels. We confirm its functionality in processing APP in primary mouse brain endothelial cells. In an Alzheimer's disease mouse model we show that BACE-1 is upregulated at the blood-brain barrier compared to healthy controls. We therefore suggest a critical role for BACE-1 at the blood-brain barrier in ß-amyloid generation and in vascular aspects of Alzheimer's disease, particularly in the development of cerebral amyloid angiopathy.

Engineering amyloid-like assemblies from unstructured peptides via site-specific lipid conjugation.

Aggregation of amyloid beta (Aß) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of ß-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of ß-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.

Cognitive enhancers (Nootropics). Part 3: drugs interacting with targets other than receptors or enzymes. Disease-modifying drugs. Update 2014.

Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers, are very productive. The review "Drugs interacting with Targets other than Receptors or Enzymes. Disease-modifying Drugs" was accepted in October 2012. In the last 20 months, new targets for the potential treatment of Alzheimer's disease were identified. Enormous progress was realized in the pharmacological characterization of natural products with cognitive enhancing properties. This review covers the evolution of research in this field through May 2014.

Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes. Update 2014.

Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review on Drugs interacting with Enzymes was accepted in August 2012. However, this field is very dynamic. New potential targets for the treatment of Alzheimer's disease were identified. This update describes drugs interacting with 60 enzymes versus 43 enzymes in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through April 2014.

Cognitive enhancers (Nootropics). Part 1: drugs interacting with receptors. Update 2014.

Scientists working in the fields of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review "Cognitive enhancers (nootropics): drugs interacting with receptors" was accepted for publication in July 2012. Since then, new targets for the potential treatment of Alzheimer's disease were identified. This update describes drugs interacting with 42 receptors versus 32 receptors in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through March 2014.

Increased efflux of amyloid-ß peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.

The formation and accumulation of toxic amyloid-ß peptides (Aß) in the brain may drive the pathogenesis of Alzheimer's disease. Accordingly, disease-modifying therapies for Alzheimer's disease and related disorders could result from treatments regulating Aß homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aß or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aß burden in AßPPPS1, hAßPPSL, and AßPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aß peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aß in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aß in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aß peptide in pre-plaque mhAßPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aß peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aß transport across the Blood Brain Brain (BBB). Thus increased Aß clearance through the BBB may contribute to reduced Aß burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects.

Human genome-guided identification of memory-modulating drugs.

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory--a trait central to posttraumatic stress disorder--and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand-receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand-receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.