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.

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.

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.

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.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

An effector-reduced anti-ß-amyloid (Aß) antibody with unique aß binding properties promotes neuroprotection and glial engulfment of Aß.

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.

Abeta42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete Abeta42 species.

The identification of toxic Aß species and/or the process of their formation is crucial for understanding the mechanism(s) of Aß neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aß toxicity, we developed different procedures to isolate Aß species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aß42 preparations, containing a monomeric and heterogeneous mixture of Aß42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aß42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aß42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aß42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aß42 protofibrils. Together, our findings demonstrate that Aß42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aß42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aß protofibril toxicity, and important implications for designing anti-amyloid therapies.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

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.

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.

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.

Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model.

BACKGROUND: Cell transplantation offers the promise in the restoration of ventricular function after an extensive myocardial infarction, but the optimal cell type remains controversial. Human unrestricted somatic stem cells (USSCs) isolated from umbilical cord blood have great potential to differentiate into myogenic cells and induce angiogenesis. The present study evaluated the effect of USSCs on myocardial regeneration and improvement of heart function after myocardial infarction in a porcine model. METHOD AND RESULTS: The distal left anterior descending artery of Yorkshire pigs (30 to 35 kg) was occluded by endovascular implantation of a coil. Four weeks after infarction, single-photon emission computed tomography technetium 99m sestamibi scans (MIBI) and echocardiography were performed. USSCs (100 x 10(6)) or culture media were then directly injected into the infarcted region (n=8 per group). Pigs were immunosuppressed by daily administration of cyclosporin A. At 4 weeks after transplantation, MIBI and echocardiography were repeated and heart function was also assessed with a pressure-volume catheter. The infarcted myocardium and implanted cells were studied histologically. MIBI showed improved regional perfusion (P<0.05) and wall motion (P<0.05) of the infarct region in the transplant group compared with the control. Ejection fraction evaluated by both MIBI and echocardiography decreased in the control group but increased in the transplant group (P<0.01). Scar thickness of the transplant group was higher than the control. The grafted cells were detected 4 weeks after transplantation by both immunohistochemistry and in situ hybridization. CONCLUSIONS: Engrafted USSCs were detected in the infarct region 4 weeks after cell transplantation, and the implanted cells improved regional and global function of the porcine heart after a myocardial infarction. This study suggests that the USSC implantation will be efficacious for cellular cardiomyoplasty.

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.

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.