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[Image: see text] We recently disclosed a set of heteroaryl-fused piperazine inhibitors of BACE1 that combined nanomolar potency with good intrinsic permeability and low Pgp-mediated efflux. Herein we describe further work on two prototypes of this family of inhibitors aimed at modulating their basicity and reducing binding to the human ether-a-go-go-related gene (hERG) channel. This effort has led to the identification of compound 36, a highly potent (hAβ42 cell IC(50) = 1.3 nM), cardiovascularly safe, and orally bioavailable compound that elicited sustained Aβ(42) reduction in mouse and dog animal models.

β-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is considered to be a promising target for treating Alzheimer's disease. However, all clinical BACE1 inhibitors have failed due to lack of efficacy, and some have even led to cognitive worsening. Recent evidence points to the importance of avoiding BACE2 inhibition along with careful dose titration. In this study, we focused on the fact that the 10s loop lining the S3 pocket in BACE1 can form both "open (up)" and "closed (down)" conformations, whereas in BACE2, it prefers to adopt a "closed" form; thus, more space is available in BACE1. By leveraging the difference, we designed fused pyridine analogues that could reach the 10s loop, leading to 6 with high selectivity and significant Aβ reduction. The cocrystal structures confirmed that 6 significantly increased B-factors of the 10s loop in BACE2 relative to those in BACE1. Thus, the destabilization of BACE2 seems to offer structural insights into the reduced BACE2 potency of 6, explaining the significant improvement in BACE1 selectivity.

The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid β lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.

Purpose The accumulation of misfolded tau is a common feature of several neurodegenerative disorders, with Alzheimer’s disease (AD) being the most common. Earlier we identified JNJ-64326067, a novel isoquinoline derivative with high affinity and selectivity for tau aggregates from human AD brain. We report the dosimetry of [18F] JNJ-64326067 and results of a proof-of-concept study comparing subjects with probable Alzheimer’s disease to age-matched healthy controls. Methods [18F] JNJ-64326067 PET scans were acquired for 90 min and then from 120 to 180 min in 5 participants with [18F]-florbetapir PET amyloid positive probable AD (73 ± 9 years) and 5 [18F]-florbetapir PET amyloid negative healthy controls (71 ± 7 years). Whole-body [18F] JNJ-64326067 PET CT scans were acquired in six healthy subjects for 5.5 h in 3 scanning sessions. Brain PET scans were visually reviewed. Regional quantification included kinetic analysis of distribution volume ration (DVR) estimated by Logan graphical analysis over the entire scan and static analysis of SUVr in late frames. Both methods used ventral cerebellar cortex as a reference region. Results One of the healthy controls had focal areas of PET signal in occipital and parietal cortex underlying the site of a gunshot injury as an adolescent; the other four healthy subjects had no tau brain signal. Four of the 5 AD participants had visually apparent retention of [18F] JNJ-64326067 in relevant cortical regions. One of the AD subjects was visually negative. Cortical signal in visually positive subjects approached steady state by 120 min. Temporal and frontal cortical SUVr/DVR values in visually positive AD subjects ranged from 1.21 to 3.09/1.2 to 2.18 and from 0.92 to 1.28/0.91 to 1.16 in healthy controls. Whole-body effective dose was estimated to be 0.0257 mSv/MBq for females and 0.0254 mSv/MBq for males. Conclusions [18F] JNJ-64326067 could be useful for detection and quantitation of tau aggregates.

The hallmark pathological features of Alzheimer's disease (AD) brains are senile plaques, comprising β-amyloid (Aβ) peptides, and neuronal inclusions formed from tau protein. These plaques form 10–20 years before AD symptom onset, whereas robust tau pathology is more closely associated with symptoms and correlates with cognitive status. This temporal sequence of AD pathology development, coupled with repeated clinical failures of Aβ-directed drugs, suggests that molecules that reduce tau inclusions have therapeutic potential. Few tau-directed drugs are presently in clinical testing, in part because of the difficulty in identifying molecules that reduce tau inclusions. We describe here two cell-based assays of tau inclusion formation that we employed to screen for compounds that inhibit tau pathology: a HEK293 cell-based tau overexpression assay, and a primary rat cortical neuron assay with physiological tau expression. Screening a collection of ∼3500 pharmaceutical compounds with the HEK293 cell tau aggregation assay, we obtained only a low number of hit compounds. Moreover, these compounds generally failed to inhibit tau inclusion formation in the cortical neuron assay. We then screened the Prestwick library of mostly approved drugs in the cortical neuron assay, leading to the identification of a greater number of tau inclusion inhibitors. These included four dopamine D2 receptor antagonists, with D2 receptors having previously been suggested to regulate tau inclusions in a Caenorhabditis elegans model. These results suggest that neurons, the cells most affected by tau pathology in AD, are very suitable for screening for tau inclusion inhibitors.

BACE1 is an attractive target for disease-modifying treatment of Alzheimer's disease. BACE2, having high homology around the catalytic site, poses a critical challenge to identifying selective BACE1 inhibitors. Recent evidence indicated that BACE2 has various roles in peripheral tissues and the brain, and therefore, the chronic use of nonselective inhibitors may cause side effects derived from BACE2 inhibition. Crystallographic analysis of the nonselective inhibitor verubecestat identified explicit water molecules with different levels of free energy in the S2' pocket. Structure-based design targeting them enabled the identification of propynyl oxazine 3 with improved selectivity. Further optimization efforts led to the discovery of compound 6 with high selectivity. The cocrystal structures of 7, a close analogue of 6, bound to BACE1 and BACE2 confirmed that one of the explicit water molecules is displaced by the propynyl group, suggesting that the difference in the relative water displacement cost may contribute to the improved selectivity.

In Alzheimer’s disease, the density and spread of aggregated tau protein track well with neurodegeneration and cognitive decline, making the imaging of aggregated tau a compelling biomarker. A structure–activity relationship exploration around an isoquinoline hit, followed by an exploration of tolerated fluorination positions, allowed us to identify 9 (JNJ-64326067), a potent and selective binder to aggregated tau with a favorable pharmacokinetic profile and no apparent off-target binding. This was confirmed in rat and monkey positron emission tomography studies using [18F]9.

In previous studies, the introduction of electron withdrawing groups to 1,4-oxazine BACE1 inhibitors reduced the pKa of the amidine group, resulting in compound 2 that showed excellent in vivo efficacy, lowering Aβ levels in brain and CSF. However, a suboptimal cardiovascular safety margin, based on QTc prolongation, prevented further progression. Further optimization resulted in the replacement of the 2-fluoro substituent by a CF3-group, which reduced hERG inhibition. This has led to compound 3, with an improved cardiovascular safety margin and sufficiently safe in GLP toxicity studies to progress into clinical trials.

Synaptic dysfunction and disconnectivity are core deficits in Alzheimer’s disease (AD), preceding clear changes in histopathology and cognitive functioning. Here, the early and late effects of tau pathology induction on functional network connectivity were investigated in P301L mice. Multichannel EEG oscillations were used to compute (1) coherent activity between the prefrontal cortex (PFC) and hippocampus (HPC) CA1-CA3 networks; (2) phase-amplitude cross frequency coupling (PAC) between theta and gamma oscillations, which is instrumental in adequate cognitive functioning; (3) information processing as assessed by auditory evoked potentials and oscillations in the passive oddball mismatch negativity-like (MMN) paradigm. At the end, the density of tau aggregation and GABA parvalbumin (PV+) interneurons were quantified by immunohistochemistry. Early weakening of EEG theta oscillations and coherent activity were revealed between the PFC and HPC CA1 and drastic impairments in theta–gamma oscillations PAC from week 2 onwards, while PV+ interneurons count was not altered. Moreover, the tau pathology disrupted the MMN complex amplitude and evoked gamma oscillations to standard and deviant stimuli suggesting altered memory formation and recall. The induction of intracellular tau aggregation by tau seed injection results in early altered connectivity and strong theta–gamma oscillations uncoupling, which may be exploited as an early electrophysiological signature of dysfunctional neuronal networks.

Purpose The accumulation of misfolded tau is a common feature of several neurodegenerative disorders, with Alzheimer’s disease (AD) being the most common. Earlier we identified JNJ-64326067, a novel isoquinoline derivative with high affinity and selectivity for tau aggregates from human AD brain. We report the dosimetry of [18F] JNJ-64326067 and results of a proof-of-concept study comparing subjects with probable Alzheimer’s disease to age-matched healthy controls. Methods [18F] JNJ-64326067 PET scans were acquired for 90 min and then from 120 to 180 min in 5 participants with [18F]-florbetapir PET amyloid positive probable AD (73 ± 9 years) and 5 [18F]-florbetapir PET amyloid negative healthy controls (71 ± 7 years). Whole-body [18F] JNJ-64326067 PET CT scans were acquired in six healthy subjects for 5.5 h in 3 scanning sessions. Brain PET scans were visually reviewed. Regional quantification included kinetic analysis of distribution volume ration (DVR) estimated by Logan graphical analysis over the entire scan and static analysis of SUVr in late frames. Both methods used ventral cerebellar cortex as a reference region. Results One of the healthy controls had focal areas of PET signal in occipital and parietal cortex underlying the site of a gunshot injury as an adolescent; the other four healthy subjects had no tau brain signal. Four of the 5 AD participants had visually apparent retention of [18F] JNJ-64326067 in relevant cortical regions. One of the AD subjects was visually negative. Cortical signal in visually positive subjects approached steady state by 120 min. Temporal and frontal cortical SUVr/DVR values in visually positive AD subjects ranged from 1.21 to 3.09/1.2 to 2.18 and from 0.92 to 1.28/0.91 to 1.16 in healthy controls. Whole-body effective dose was estimated to be 0.0257 mSv/MBq for females and 0.0254 mSv/MBq for males. Conclusions [18F] JNJ-64326067 could be useful for detection and quantitation of tau aggregates. Electronic supplementary material The online version of this article (10.1007/s00259-020-04880-1) contains supplementary material, which is available to authorized users.

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain. ispartof: NATURE NEUROSCIENCE vol:22 issue:6 pages:1021-1035 ispartof: location:United States status: published

BACE1 inhibitors hold potential as agents in disease-modifying treatment for Alzheimer's disease. BACE2 cleaves the melanocyte protein PMEL in pigment cells of the skin and eye, generating melanin pigments. This role of BACE2 implies that nonselective and chronic inhibition of BACE1 may cause side effects derived from BACE2. Herein, we describe the discovery of potent and selective BACE1 inhibitors using structure-based drug design. We targeted the flap region, where the shape and flexibility differ between these enzymes. Analysis of the cocrystal structures of an initial lead 8 prompted us to incorporate spirocycles followed by its fine-tuning, culminating in highly selective compounds 21 and 22. The structures of 22 bound to BACE1 and BACE2 revealed that a relatively high energetic penalty in the flap of the 22-bound BACE2 structure may cause a loss in BACE2 potency, thereby leading to its high selectivity. These findings and insights should contribute to responding to the challenges in exploring selective BACE1 inhibitors.

In this study, we have synthesized and evaluated 18F-JNJ64349311, a tracer with high affinity for aggregated tau (inhibition constant value, 8 nM) and high (≥500×) in vitro selectivity for tau over β-amyloid, in comparison with the benchmark compound 18F-AV1451 (18F-T807) in mice, rats, and a rhesus monkey. Methods: In vitro binding characteristics were determined for Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration patient brain tissue slices using autoradiography studies. Ex vivo biodistribution studies were performed in mice. Radiometabolites were quantified in the brain and plasma of mice and in the plasma of a rhesus monkey using high-performance liquid chromatography. Dynamic small-animal PET studies were performed in rats and a rhesus monkey to evaluate tracer pharmacokinetics in the brain. Results: Mouse biodistribution studies showed moderate initial brain uptake and rapid brain washout. Radiometabolite analyses after injection of 18F-JNJ64349311 in mice showed the presence of a polar radiometabolite in plasma, but not in the brain. Semiquantitative autoradiography studies on postmortem tissue sections of human Alzheimer's disease brains showed highly displaceable binding to tau-rich regions. No specific binding was, however, found on human progressive supranuclear palsy and corticobasal degeneration brain slices. Small-animal PET scans of Wistar rats revealed moderate initial brain uptake (SUV, ∼1.5 at 1 min after injection) and rapid brain washout. Gradual bone uptake was, however, also observed. Blocking and displacement did not affect brain time-activity curves, suggesting no off-target specific binding of the tracer in the healthy rat brain. A small-animal PET scan of a rhesus monkey revealed moderate initial brain uptake (SUV, 1.9 at 1 min after injection) with a rapid washout. In the monkey, no bone uptake was detected during the 120-min scan. Conclusion: This biologic evaluation suggests that 18F-JNJ64349311 is a promising tau PET tracer candidate, with a favorable pharmacokinetic profile, as compared with 18F-AV1451.

A mini-HTS on 4000 compounds selected using 2D fragment-based similarity and 3D pharmacophoric and shape similarity to known selective tau aggregate binders identified N-(6-methylpyridin-2-yl)quinolin-2-amine 10 as a novel potent binder to human AD aggregated tau with modest selectivity versus aggregated β-amyloid (Aβ). Initial medicinal chemistry efforts identified key elements for potency and selectivity, as well as suitable positions for radiofluorination, leading to a first generation of fluoroalkyl-substituted quinoline tau binding ligands with suboptimal physicochemical properties. Further optimization toward a more optimal pharmacokinetic profile led to the discovery of 1,5-naphthyridine 75, a potent and selective tau aggregate binder with potential as a tau PET tracer.

Filamentous tau aggregates, the hallmark lesions of Alzheimer disease (AD), play key roles in neurodegeneration. Activation of protein degradation systems has been proposed to be a potential strategy for removing pathological tau, but it remains unclear how effectively tau aggregates can be degraded by these systems. By applying our previously established cellular model system of AD-like tau aggregate induction using preformed tau fibrils, we demonstrate that tau aggregates induced in cells with regulated expression of full-length mutant tau can be gradually cleared when soluble tau expression is suppressed. This clearance is at least partially mediated by the autophagy-lysosome pathway, although both the ubiquitin-proteasome system and the autophagy-lysosome pathway are deficient in handling large tau aggregates. Importantly, residual tau aggregates left after the clearance phase leads to a rapid reinstatement of robust tau pathology once soluble tau expression is turned on again. Moreover, we succeeded in generating monoclonal cells persistently carrying tau aggregates without obvious cytotoxicity. Live imaging of GFP-tagged tau aggregates showed that tau inclusions are dynamic structures constantly undergoing "fission" and "fusion," which facilitate stable propagation of tau pathology in dividing cells. These findings provide a greater understanding of cell-to-cell transmission of tau aggregates in dividing cells and possibly neurons.

The protease β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is required for the production of the amyloid-β (Aβ) peptide, which is central to the pathogenesis of Alzheimer's disease (AD). Chronic inhibition of this protease may temper amyloid production and cure or prevent AD. However, while BACE1 inhibitors are being pushed forward as drug candidates, a remarkable gap in knowledge on the physiological functions of BACE1 and its close homolog BACE2 becomes apparent. Here we discuss the major discoveries of the past 3 years concerning BACE1 biology and to what extent these could limit the use of BACE1 inhibitors in the clinic.

In Alzheimer's disease, the density and spread of aggregated tau protein track well with neurodegeneration and cognitive decline, making the imaging of aggregated tau a compelling biomarker. A structure-activity relationship exploration around an isoquinoline hit, followed by an exploration of tolerated fluorination positions, allowed us to identify 9 (JNJ-64326067), a potent and selective binder to aggregated tau with a favorable pharmacokinetic profile and no apparent off-target binding. This was confirmed in rat and monkey positron emission tomography studies using [

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain.

Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.

Background: Positive allosteric modulators of AMPA receptors (AMPA-PAMs) are small molecules that keep the AMPA receptor in an active state by decreasing deactivation or desensitization. They have been proposed to treat cognitive decline in dementias and Alzheimer disease (AD). S 47445 is a novel AMPA-PAM. Here, the mechanisms by which S 47445 could improve synaptic strengthen and connectivity were studied and compared between young (3-months old) and middle-aged freely moving mice (14-months old). Methods: LTP was evoked at the CA3-CA1 synapse using high-frequency stimulation (HFS) protocol at two occasions on day 1 and on day 13 after the first administration of S 47445 (3mg/kg or 10mg/kg). Assessment of drug effect on LTP was performed after either acute or chronic treatment. The drug effect on micro-cytoarchitecture in hippocampal (CA1 and CA3) and cortical tissue (L2-3 and L5-6) was also evaluated by confocal mi-croscopy by assessing number and size of positive particles for the presynaptic vesicular glutamate transporter VGlut1 and the post-synaptic protein spinophilin. Results: Middle-aged control mice displayed a marked significant deficit of LTP as compared to young control mice following the first presentation of the HFS protocol. After acute administration of S 47445 at 10mg/kg in middle-aged mice, a complete reversion of the LTP deficit was observed on day 1. Then, chronic treatment with S 47445 at 10mg/kg in middle aged animals significantly counteracted the aged deficit of LTP. During the second HFS presentation, LTP was much smaller than during the previous acute experiment in each mice group, suggesting a loss of LTP-evoked phenomenon. Interestingly, the chronic administration of S 47445 at 10mg/kg in middle-aged mice induced significantly larger LTP values as compared to young control and middle-aged control mice. Moreover, chronic treatment with S 47445 at 10mg/kg significantly prevented the decrease in number and size of VGlut1 and spinophilin in CA1-CA3 and in the cortex of middle-aged mice. Conclusions: Collectively, by its different effects observed, S 47445 is able to modulate both the structure and function of hippocampal excitatory synapses known to be involved in learning and memory processes and could provide beneficial effects in dementias such as AD. Background: In AD, Tau pathology progresses in a stereotypic spatiotemporal pattern, strongly correlating with progression of disease symptoms. Prion-like propagation of Tau-pathology, or propagation of Tau-misfolding between cells and functionally connected brain regions, provides a compelling mechanism for this ste-reotypic progression of Tau-pathology, and hence an attractive therapeutic target. Methods: To test and validate novel therapeutic targets capable of inhibiting prion-like progression of Tau-pathology , we have generated combined in vitro and in vivo models reca-pitulating prion-like seeding and propagation of Tau-pathology. Seeding of pre-aggregated Tau in Tau expressing primary neurons and Tau transgenic mice recapitultes strong induction of Tau-pathology and propagation to the contralateral side. Results: Using these models we have analysed and evaluated the modifying potential of different targets on Tau pathology and propagation of Tau pathology. Targets under evaluation encompass Tau-interactome based Tau interacting proteins, including OTUB1 and other identified Tau-interacting proteins, as well as hypothesis based analysis. Within this analysis we identified OTUB1 as Tau deubiquitinase, involved in the accumulation of pathological forms of Tau. We furthermore identified novel targets with modifying effect on prion-like seeding and propagation of Tau-pathology. Conclusions: We here present the identification of different targets with a modu-latory effect in vitro and in vivo on Tau-pathology and prion-like propagation of Tau-pathology.

The excitatory neurotransmitter l -glutamate is transported into synaptic vesicles by vesicular glutamate transporters (VGluTs) to transmit glutamatergic signals. Changes in their expression have been linked to various brain disorders including schizophrenia, Parkinson's, and Alzheimer's disease. Deleting either the VGluT1 or VGluT2 gene leads to profound developmental and neurological complications and early death, but mice heterozygous for VGluT1 or VGluT2 are viable and thrive. Acquisition, retention and extinction of conditioned visuospatial and emotional responses were compared between VGluT1 +/− and VGluT2 +/− mice, and their wildtype littermates, using different water maze procedures, appetitive scheduled conditioning, and conditioned fear protocols. The distinct brain expression profiles of the VGluT1 and -2 isoforms particularly in telencephalic structures, such as neocortex, hippocampus and striatum, are reflected in very specific behavioral changes. VGluT2 +/− mice were unimpaired in spatial learning tasks and fear extinction. Conversely, VGluT1 +/− mice displayed spatial extinction learning deficits and markedly impaired fear extinction. These data indicate that VGluT1, but not VGluT2, plays a role in the neural processes underlying inhibitory learning.

Dysregulated proteostasis is a key feature of a variety of neurodegenerative disorders. In Alzheimer's disease (AD), progression of symptoms closely correlates with spatiotemporal progression of Tau aggregation, with "early" oligomeric Tau forms rather than mature neurofibrillary tangles (NFTs) considered to be pathogenetic culprits. The ubiquitin-proteasome system (UPS) controls degradation of soluble normal and abnormally folded cytosolic proteins. The UPS is affected in AD and is identified by genomewide association study (GWAS) as a risk pathway for AD. The UPS is determined by balanced regulation of ubiquitination and deubiquitination. In this work, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based Tau interactome mapping to gain unbiased insight into Tau pathophysiology and to identify novel Tau-directed therapeutic targets. Focusing on Tau deubiquitination, we here identify Otub1 as a Tau-deubiquitinating enzyme. Otub1 directly affected Lys48-linked Tau deubiquitination, impairing Tau degradation, dependent on its catalytically active cysteine, but independent of its noncanonical pathway modulated by its N-terminal domain in primary neurons. Otub1 strongly increased AT8-positive Tau and oligomeric Tau forms and increased Tau-seeded Tau aggregation in primary neurons. Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2 months in Tau transgenic mice in vivo, including AT8-positive Tau and oligomeric Tau forms. Taken together, we here identified Otub1 as a Tau deubiquitinase in vitro and in vivo, involved in formation of pathological Tau forms, including small soluble oligomeric forms. Otub1 and particularly Otub1 inhibitors, currently under development for cancer therapies, may therefore yield interesting novel therapeutic avenues for Tauopathies and AD. This work was supported by the Belgian Fonds National pour la Recherche Scientifique-Fonds de la Recherche Scientifique (FNRS-FRS; Qualified Researcher, Impulse Financing, Research Credits), by Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, by the Belgian Fonds de la Recherche Scientifique Medicale, by the Queen Elisabeth Medical Foundation of Belgium (QEMF-FMRE), by the Stichting Alzheimer Onderzoek (SAO), by the Institute for the Promotion of Innovation by Science and Technology (IWT) in Flanders (IWT O&O, currently FWO), Belgium, and by Stellar funding of Janssen Research Foundation. We would like to thank Markus Boesche, Carola Doce, Frank Fischer, and Melanie Jundt for their technical expertise.

In this study, we have synthesized and evaluated

Background: Identification of Tau mutations linked to neurodegen-erative Tauopathies have indicated that Tau-dysfunction is causally linked to neurodegenerative processes. Accumulating evidence furthermore corroborates a crucial executive role for Tau in the pathogenesis of AD, designating Tau as an important therapeutic target. Identification of modifiers of Tau that can modulate Tau-pathology and Tau-related phenotypic features and neurodegenerative processes observed in preclinical models has become an important research objective. Methods: A Tau-interactome mapping has been performed to identify proteins binding to Tau in an unbiased way. To further identify Tau-modifying potential of the identified interacting proteins, an AAV-based approach was used. AAV injections, driving neuronal expression of the protein of interest, were performed at P0 in previously characterized Tau transgenic mice, and in primary neuronal cultures. Biochemical and IHC analysis was performed to analyze the modifying potential of the protein of interest on Tau and Tau-pathology in vitro and in vivo. Results: Using this approach we demonstrate Tau-modifying potential for several novel proteins involved in protein degradation, more particularly in the ubiquitin proteasome degradation. Background: Although tau pathology, behavioral deficits, and neuronal loss are each observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Methods: Here we found that rTg4510 mice, overexpressing human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity, which correlates with neurofi-brillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To investigate whether attenuating the tau pathology in this animal model can prevent the progression of the behavioral deficits, we treated the rTg4510 mice with either doxy

tissue homogenates with exogenous Ab1-42 under multiple conditions. Samples were analyzed using acid urea gels followed by Western blotting. Results:The PDAPP mice study revealed Ab42 to Ab40 conversion over time reaching equilibrium by 72hr. Acid urea gel analyses demonstrated that over half of the Ab1-42 peptide administered was converted to Ab1-40. Furthermore, ELISA results from rat studies showed similar conversion rates from Ab42 to Ab40 regardless of the route of administration, centrally or peripherally. Ex-vivo studies using rat tissue homog-enates incubated with exogenous Ab1-42 peptide also exhibited Ab conversion. This conversion was present in all tissues tested, cortex, kidney, liver, pancreas, and spleen, and was exacerbated when the pH was lowered to pH5 from pH7. The rate of conversion to Ab40 was diminished when a c-terminal antibody or pro-tease inhibitor was incorporated into the in-vivo/ex-vivo studies. Conclusions: We have identified in-vivo processing of the carboxyl-terminus of Ab in rodents. The extent of in-vivo processing is exacerbated when an Ab antibody extends the half-life of the peptide. The conversion of Ab1-42 to Ab1-40 occurs both centrally and peripherally. Although the potential for this conversion in human is unknown, these results suggest additional biology after secretase liberation of the Ab from the APP may be important for the overall Ab ratios being measured in CNS and periphery. Background: Combined genetic, pathological and clinical data provided the basis for the amyloid cascade hypothesis, which is further supported by biomarker data and remains the major hypothesis for development of therapeutic strategies. In vitro and preclinical in vivo models have robustly recapitulated amyloid induced Tau-pathology providing support to the amyloid cascade hypothesis and providing tools to understand this event, generally considered to be crucial in the pathogenesis of AD. Methods: We previously reported a preclinical model with robust amyloid induced Tau-pathology , providing an experimental window for analysis of mechanisms of ABeta-induced Tau-pathology in vivo. In this model, we demonstrated the induction of Tau-pathology along functional connections , in regions relatively spared of amyloid pathology. The striking similarity between abeta-induced Tau-pathology in pre-clinical models and in our recently reported Tau-seeding model, provided the basis for our current analysis of Abeta-induced Tau-fi-brillization by cross-seeding and its subsequent propagating potential , in vitro and in vivo. Cell-free assays were used to analyze Tau

Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) show largely complementary distribution in the mature rodent brain and tend to segregate to synapses with different physiological properties. In the hippocampus, VGLUT1 is the dominate subtype in adult animals, whereas VGLUT2 is transiently expressed during early postnatal development. We generated and characterized VGLUT1 knockout mice in order to examine the functional contribution of this transporter to hippocampal synaptic plasticity and hippocampus-dependent spatial learning. Because complete deletion of VGLUT1 resulted in postnatal lethality, we used heterozygous animals for analysis. Here, we report that deletion of VGLUT1 resulted in impaired hippocampal long-term potentiation (LTP) in the CA1 region in vitro. In contrast, heterozygous VGLUT2 mice that were investigated for comparison did not show any changes in LTP. The reduced ability of VGLUT1-deficient mice to express LTP was accompanied by a specific deficit in spatial reversal learning in the water maze. Our data suggest a functional role of VGLUT1 in forms of hippocampal synaptic plasticity that are required to adapt and modify acquired spatial maps to external stimuli and changes.

Background and purpose: The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice. Experimental approach: The effect of obestatin on 6-h cumulative food intake was studied in fasted wildtype (ghrelin+/+) and ghrelin knockout (ghrelin−/−) mice. In both genotypes, the effect of obestatin and/or ghrelin was studied in vivo on gastric emptying measured with the 14C-octanoic acid breath test and in vitro on neural responses elicited by electrical field stimulation (EFS) of fundic smooth muscle strips. Key results: Administration of obestatin did not influence fasting-induced hyperphagia or gastric emptying in both genotypes. Injection of ghrelin accelerated gastric emptying in ghrelin+/+ and ghrelin−/− mice but the effect was not reversed by co-injection with obestatin. In fundic strips from ghrelin+/+ and ghrelin−/− mice, ghrelin increased EFS-induced contractions, but obestatin was without effect. However, co-administration with obestatin tended to reduce the excitatory effect of ghrelin in both genotypes. Conclusions and implications: In ghrelin−/− mice, obestatin failed to affect food intake and gastric motility. These results suggest that endogenous ghrelin does not mask the effect of obestatin and confirm that obestatin administered peripherally is not a major regulator of satiety signalling or gut motility. British Journal of Pharmacology (2008) 153, 1550–1557; doi:10.1038/sj.bjp.0707683; published online 21 January 2008

In this study the ATP-induced (P2X) currents in isolated peritoneal macrophages of wild type (WT) and P2X(4) knockout (P2X(4)(-/-)) mice were studied by means of whole-cell patch clamp in order to (1) survey the P2X currents of native macrophages and (2) to investigate the expression of P2X(4)-like currents in the WT versus P2X(4)(-/-) mice. Three types of currents were observed in the isolated macrophages: (1) in approximately 10% of both WT and P2X(4)(-/-) macrophages a fast activating and inactivating P2X1-like current was recorded with low concentrations (0.1-1 microM) of ATP; (2) 85% of wild type and 100% of P2X(4)(-/-) macrophages exhibited a non-desensitizing P2X(7)-like current activated at high concentrations of ATP (10mM). The identity of the P2X(7) current was confirmed using the specific blocker A-740003; (3) 88.6% of the WT but none of the P2X(4)(-/-) macrophages showed a small P2X(4)-like current that desensitized slowly upon ATP application at intermediate concentrations (3-300 microM). Several observations indicated that the slowly desensitizing current in WT macrophages was P2X(4). The EC50 value of 5.3 microM ATP was as expected for P2X(4) and the current induced by 3-300 microM ATP was absent in P2X(4)(-/-) mice. Upon application of 3 microM ivermectin, a P2X(4)-selective modulator, the amplitude of this current was increased and the desensitization was inhibited in WT cells. In addition, this current was facilitated by 10 microM Zn(2+) but inhibited by Cu(2+) (in contrast to P2X(2)). We conclude that the P2X(4) and P2X(7) currents are functionally expressed in recruited peritoneal macrophages of WT mice and that the P2X(4)-like current is absent in P2X(4)(-/-) mice.

Cannabinoid 2 (CB2) receptors have bothantinociceptive and antihypersensitivity effects, alth-oughtheprecisemechanismsofactionarestillunclear.In this study, the modulatory role of CB2 receptors onthe mesenteric afferent response to the endogenousimmunogenic agent bradykinin (BK) was investigated.Mesenteric afferent recordings were obtained fromanaesthetized wild-type and CB2 )/ mice using con-ventional extracellular recording techniques. Controlresponses to BK were obtained in all experiments priorto administration of either CB2 receptor agonistAM1241, or AM1241 plus the CB2 receptor antagonistAM630. Bradykinin consistently evoked activation ofmesentericafferents(n ¼ 32).AM1241inhibitedtheBKresponseinadosedependentmanner.InthepresenceofAM630 (10 mg kg )1 ), however, AM1241 (10 mg kg )had no significant effect on the BK response. Moreover,AM1241 had also no significant effect on the BK re-sponse in CB2 )/) mice. Activation of the CB2 receptorinhibits the BK response in mesenteric afferents, dem-onstrating that the CB2 receptor is an important regu-lator of neuroimmune function. This may be amechanism of action for the antinociceptive and anti-hypersensitive effects of CB2 receptor agonists.Keywords afferent, cannabinoid, immune, mast cell,neuron, sensory.

Filamentous tau aggregates, the hallmark lesions of Alzheimer disease (AD), play key roles in neurodegeneration. Activation of protein degradation systems has been proposed to be a potential strategy for removing pathological tau, but it remains unclear how effectively tau aggregates can be degraded by these systems. By applying our previously established cellular model system of AD-like tau aggregate induction using preformed tau fibrils, we demonstrate that tau aggregates induced in cells with regulated expression of full-length mutant tau can be gradually cleared when soluble tau expression is suppressed. This clearance is at least partially mediated by the autophagy-lysosome pathway, although both the ubiquitin-proteasome system and the autophagy-lysosome pathway are deficient in handling large tau aggregates. Importantly, residual tau aggregates left after the clearance phase leads to a rapid reinstatement of robust tau pathology once soluble tau expression is turned on again. Moreover, we succeeded in generating monoclonal cells persistently carrying tau aggregates without obvious cytotoxicity. Live imaging of GFP-tagged tau aggregates showed that tau inclusions are dynamic structures constantly undergoing “fission” and “fusion,” which facilitate stable propagation of tau pathology in dividing cells. These findings provide a greater understanding of cell-to-cell transmission of tau aggregates in dividing cells and possibly neurons.

Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX-R currents are available, this study employs Na(v)1.8 and Na(v)1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na(v)1.9 and Na(v)1.8 null, wild-type and naive mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20-24 post-infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na(v)1.9 and Na(v)1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 +/- 0.3, infected = 3.6 +/- 0.7 APs at 2x rheobase, P = 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability was maintained in Na(v)1.9(-/-) mice, but hyperexcitability was absent and APs were blunted in Na(v)1.8(-/-) mice. This study identifies a critical role for Na(v)1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na(v)1.9. Nb infection-induced hyperexcitability is not observed in Na(v)1.8(-/-) mice, but is still present in Na(v)1.9(-/-) mice. It is not clear whether hyperexcitability is due to a change in the function of Na(v)1.8 channels or a change in the number of Na(v)1.8 channels.

Uptake ofl-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1–VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons andin vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

We have reported transgenic mice with neuronal overexpression of the clinical mutant beta-amyloid precursor protein (APP) known as London, which develop an AD-related phenotype [Moechers, Dewachter, Lorent, Reversé, Baekelandt, Nadiu, Tesseur, Spittaels, Van den Haute, Checler, et al. (1999) J. Biol. Chem. 274, 6483-6492]. Characterized early symptoms (3-9 months) include disturbed behaviour, neophobia, aggression, hypersensitivity to kainic acid, hyposensitivity to N-methyl-D-aspartate, defective cognition and memory, and decreased long-term potentiation. Late in life, at 12-15 months, amyloid plaques develop in the brain and correlate with increased levels of beta-amyloid (A beta)40/42 (the 40- and 42-amino-acid forms of A beta). The formation of amyloid plaques is dissociated in time from and not involved in the early phenotype. Hyperphosphorylated protein tau is present but no tangle pathology is observed. In double-transgenic mice, i.e. APP/London x Presenilin 1, the increased production of A beta 42 results in amyloid plaques developing by the age of 6 months. Transgenic mice with overexpression of either human apolipoprotein E4 (ApoE4) or human protein tau in central neurons develop severe axonopathy in the brain and spinal cord. Progressive degeneration of nerves and muscles is demonstrated by motor problems, wasting and premature death. Tau is hyperphosphorylated but there is no formation of filaments or neurofibrillary tangles. The tangle aspect of AD pathology is still missing from all current transgenic amyloid models. Its implementation will require insight into the cellular signalling pathways which regulate the microtubule-stabilizing function by phosphorylation of neuronal tau.

Epidemiological studies have established that the epsilon 4 allele of the ApoE gene (ApoE4) constitutes an important risk factor for Alzheimer’s disease and might influence the outcome of central nervous system injury. The mechanism by which ApoE4 contributes to the development of neurodegeneration remains unknown. To test one hypothesis or mode of action of ApoE, we generated transgenic mice that overexpressed human ApoE4 in different cell types in the brain, using four distinct gene promoter constructs. Many transgenic mice expressing ApoE4 in neurons developed motor problems accompanied by muscle wasting, loss of body weight, and premature death. Overexpression of human ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau. In three independent transgenic lines from two different promoter constructs, increased phosphorylation of protein tau was correlated with ApoE4 expression levels. Hyperphosphorylation of protein tau increased with age. In the hippocampus, astrogliosis and ubiquitin-positive inclusions were demonstrated. These findings demonstrate that expression of ApoE in neurons results in hyperphosphorylation of protein tau and suggests a role for ApoE in neuronal cytoskeletal stability and metabolism.