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An active role of neuroinflammation and the NLRP3 inflammasome in Alzheimer's disease and related tauopathies is increasingly identified, supporting NLRP3 as an interesting therapeutic target. However, its effect on tau-associated neurodegeneration, a key-process in tauopathies, remains unknown. While tau pathology and neurodegeneration are closely correlated, different tau forms may act as culprits in both characteristics and NLRP3-dependent microglial processes may differently affect both processes, indicating the need to study the role of NLRP3 in both processes concomitantly. To study the role of NLRP3 on tau pathology, prion-like propagation and tau-associated neurodegeneration we generated crosses of NLRP3 deficient mice with tauP301S (PS19) transgenic mice. In this model we studied non-seeded tau pathology and hippocampal atrophy, reminiscent characteristics of tauopathies. Tau pathology in hippocampus and cortex was significantly decreased in tau.NLRP3-/- versus tau.NLRP3+/+ mice. Importantly, tau.NLRP3-/- mice also displayed significantly decreased hippocampal atrophy, indicating a role of NLRP3 in neurodegeneration. We furthermore assessed the effect of NLRP3 deficiency on tau propagation and associated hippocampal atrophy. NLRP3 deficiency significantly decreased prion-like seeding and propagation of tau pathology, reflected in decreased tau pathology in ipsi- and contralateral hippocampus and cortex in tau.NLRP3-/- following tau seeding. Most importantly, hippocampal atrophy was significantly less in tau-seeded tau.NLRP3-/- mice at 8 months. We here demonstrate for the first time that NLRP3 activation affects tau-associated neurodegeneration and seeded and non-seeded tau pathology, hence affecting key molecular processes in tauopathies. Our data thereby provide key-information in the validation of NLRP3 inflammasome as therapeutic target for AD and related tauopathies. Fonds Wetenschappelijk Onderzoek, Grant/ Award Numbers: 1259621N, G0C6819N; Stichting Alzheimer Onderzoek (SAO), Belgium; Vlaams Agentschap Innoveren en Ondernemen (VLAIO), Grant/Award Number: HBC.2017.0948 This work was supported by Vlaams Agentschap Innoveren en Ondernemen (VLAIO)—Research project No. HBC.2017.0948, Stichting Alzheimer Onderzoek (SAO) and Fonds Wetenschappelijk Onderzoek - Vlaanderen (FWO)—Research project No. G0C6819N and FWO Postdoc Fellowship No. 1259621N.

Blood-based (BB) biomarkers for A beta and tau can indicate pathological processes in the brain, in the early pathological, even pre-symptomatic stages in Alzheimer's disease. However, the relation between BB biomarkers and AD-related processes in the brain in the earliest pre-pathology stage before amyloid pathology develops, and their relation with total brain concentrations of A beta and tau, is poorly understood. This stage presents a critical window for the earliest prevention of AD. Preclinical models with well-defined temporal progression to robust amyloid and tau pathology provide a unique opportunity to study this relation and were used here to study the link between BB biomarkers with AD-related processes in pre- and pathological stages. We performed a cross-sectional study at different ages assessing the link between BB concentrations and AD-related processes in the brain. This was complemented with a longitudinal analysis and with analysis of age-related changes in a small cohort of human subjects. We found that BB-tau concentrations increased in serum, correlating with progressive development of tau pathology and with increasing tau aggregates and p-tau concentrations in brain in TauP301S mice (PS19) developing tauopathy. BB-A beta 42 concentrations in serum decreased between 4.5 and 9 months of age, correlating with the progressive development of robust amyloid pathology in APP/PS1 (5xFAD) mice, in line with previous findings. Most importantly, BB-A beta 42 concentrations significantly increased between 1.5 and 4.5 months, i.e., in the earliest pre-pathological stage, before robust amyloid pathology develops in the brain, indicating biphasic BB-A beta 42 dynamics. Furthermore, increasing BB-A beta 42 in the pre-pathological phase, strongly correlated with increasing A beta 42 concentrations in brain. Our subsequent longitudinal analysis of BB-A beta 42 in 5xFAD mice, confirmed biphasic BB-A beta 42, with an initial increase, before decreasing with progressive robust pathology. Furthermore, in human samples, BB-A beta 42 concentrations were significantly higher in old (> 60 years) compared to young (< 50 years) subjects, as well as to age-matched AD patients, further supporting age-dependent increase of A beta 42 concentrations in the earliest pre-pathological phase, before amyloid pathology. Also BB-A beta 40 concentrations were found to increase in the earliest pre-pathological phase both in preclinical models and human subjects, while subsequent significantly decreasing concentrations in the pathological phase were characteristic for BB-A beta 42. Together our data indicate that BB biomarkers reflect pathological processes in brain of preclinical models with amyloid and tau pathology, both in the pathological and pre-pathological phase. Our data indicate a biphasic pattern of BB-A beta 42 in preclinical models and a human cohort. And most importantly, we here show that BB-A beta increased and correlated with increasing concentrations of A beta in the brain, in the earliest pre-pathological stage in a preclinical model. Our data thereby identify a novel critical window for prevention, using BB-A beta as marker for accumulating A beta in the brain, in the earliest pre-pathological stage, opening new avenues for personalized early preventive strategies against AD, even before amyloid pathology develops. This project was supported by Stichting Alzheimer Onderzoek (SAO-FRA Belgium) (SAO2020022) and Fonds Wetenschappelijk Onderzoek—Vlaanderen (FWO)—Research project No. G0C6819N and BOF funding UHasselt (Methusalem fnancing).

Alzheimer's disease (AD) is characterized by a sequential progression of amyloid plaques (A), neurofibrillary tangles (T) and neurodegeneration (N), constituting ATN pathology. While microglia are considered key contributors to AD pathogenesis, their contribution in the combined presence of ATN pathologies remains incompletely understood. As sensors of the brain microenvironment, microglial phenotypes and contributions are importantly defined by the pathologies in the brain, indicating the need for their analysis in preclinical models that recapitulate combined ATN pathologies, besides their role in A and T models only. Here, we report a new tau-seed model in which amyloid pathology facilitates bilateral tau propagation associated with brain atrophy, thereby recapitulating robust ATN pathology. Single-cell RNA sequencing revealed that ATN pathology exacerbated microglial activation towards disease-associated microglia states, with a significant upregulation of Apoe as compared to amyloid-only models (A). Importantly, Colony-Stimulating Factor 1 Receptor inhibition preferentially eliminated non-plaque-associated versus plaque associated microglia. The preferential depletion of non-plaque-associated microglia significantly attenuated tau pathology and neuronal atrophy, indicating their detrimental role during ATN progression. Together, our data reveal the intricacies of microglial activation and their contributions to pathology in a model that recapitulates the combined ATN pathologies of AD. Our data may provide a basis for microglia-targeting therapies selectively targeting detrimental microglial populations, while conserving protective populations. This work was supported by Innoviris (Attract grant, BB2B 2015–2) to KM; FRA-SAO (Fondation Recherche Alzheimer—Stichting Alzheimer Onderzoek, Belgium – 20180028 & 2020022) to IDW, VLAIO (Vlaams Agentschap Innoveren en Ondernemen) and FWO (Fonds Wetenschappelijk Onderzoek—Vlaanderen—G0C6819N & 1259621 N and the fellowship of I.S.). Acknowledgements We thank Yvon Elkrim and the VIB Nucleomics core for technical assistance, VIB Tech Watch for support regarding single-cell RNA sequencing technologies.

Tau is most intensely studied in relation to its executive role in Tauopathies, a family of neurodegenerative disorders characterized by the accumulation of Tau aggregates [15, 21, 38, 75, 89, 111, 121, 135, 175, 176, 192]. Tau aggregation in the different Tauopathies differs in the affected cell type, the structure of aggregates and Tau isoform composition. However, in all Tauopathies, accumulation of pathological Tau in well-characterized and well-defined brain regions, correlates strongly with symptoms associated with the dysfunction of this brain region. Hence, symptoms of neurodegenerative Tauopathies can range from motoric to cognitive and behavioral symptoms, even extending to deterioration of vital functions when the disease progresses, or combinations of different symptoms governed by the affected brain regions. The most common Tauopathies are corticobasal degeneration (CBD), Pick's disease, progressive supranuclear palsy (PSP) and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). However a growing number of diseases are characterized by Tau aggregation amounting to a large family of more than 20 disorders [176]. Most Tauopathies are sporadic, and are hence linked to a combination of environmental and genetic risk factors. However, mutations in MAPT have been identified which are autosomal dominantly linked to Tauopathies, including FTDP, PSP and CBD [94, 163, 185] (Alzforum, https://www.alzforum.org/mutations/mapt ). More than 80 mutations have been identified in MAPT, both in intronic and exonic regions of the human MAPT. These mutations can be classified as missense mutations or splicing mutations. Most missense mutations cluster in or near the microtubule binding site of Tau, while most splicing mutations affect the splicing of exon 10 (encoding the R2 domain), and hence affect the 3R/4R ratio. While Alzheimer's disease (AD), is the most prevalent Tauopathy, no mutations in MAPT associated with AD have been identified. Brains of AD patients are pathologically characterized by the combined presence of amyloid plaques and neurofibrillary tangles [171]. Familial forms of AD, termed early onset familial AD (EOFAD) with clinical mutations in APP or PS1/2, have an early onset, and are invariably characterized by the combined presence of amyloid and Tau pathology [24, 80, 170]. These EOFAD cases, identify a causal link between APP/PS1 misprocessing and the development of Tau pathology and neurodegeneration [80, 170]. Furthermore, combined genetic, pathological, biomarker and in vivo modelling data, indicate that amyloid pathology precedes Tau pathology, and support a role for Aβ as initiator and Tau as executor in the pathogenetic process of AD [80, 96, 97]. Hence, AD is often considered as a secondary Tauopathy (similar as for Down syndrome patients), in contrast to the primary Tauopathies described above. Tau aggregates in Tauopathies vary with respect to the ratio of different Tau isoforms (3R/4R), to the cell types displaying Tau aggregation and the structure of the aggregates. However, in all Tauopathies a strong correlation between progressive development of pathological Tau accumulation and the loss of the respective brain functions is observed.

Brains of Alzheimer’s disease patients are characterized by the presence of amyloid plaques and neurofbrillary tangles, both invariably associated with neuroinfammation. A crucial role for NLRP3–ASC infammasome [NACHT, LRR and PYD domains-containing protein 3 (NLRP3)–Apoptosis-associated speck-like protein containing a CARD (ASC)] in amyloid-beta (Aβ)-induced microgliosis and Aβ pathology has been unequivocally identifed. Aβ aggregates activate NLRP3–ASC infammasome (Halle et al. in Nat Immunol 9:857–865, 2008) and conversely NLRP3–ASC infammasome activation exacerbates amyloid pathology in vivo (Heneka et al. in Nature 493:674–678, 2013), including by prion-like ASC-speck cross-seeding (Venegas et al. in Nature 552:355–361, 2017). However, the link between infammasome activation, as crucial sensor of innate immunity, and Tau remains unexplored. Here, we analyzed whether Tau aggregates acting as prion-like Tau seeds can activate NLRP3–ASC infammasome. We demonstrate that Tau seeds activate NLRP3–ASC-dependent infammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of infammasome activation in prion-like or templated seeding of Tau pathology and found signifcant inhibition of exogenously seeded Tau pathology by ASC defciency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC defciency also decreased nonexogenously seeded Tau pathology in Tau transgenic mice. Overall our fndings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC infammasome through the NLRP3–ASC axis, and we demonstrate an exacerbating role of the NLRP3–ASC axis on exogenously and non-exogenously seeded Tau pathology in Tau mice in vivo. The NLRP3–ASC infammasome, which is an important sensor of innate immunity and intensively explored for its role in health and disease, hence presents as an interesting therapeutic approach to target three crucial pathogenetic processes in AD, including prionlike seeding of Tau pathology, Aβ pathology and neuroinfammation. This work was supported by the Belgian Fonds National pour la Recherche Scientifque–Fonds de la Recherche Scientifque (FNRS-FRS; Qualifed Researcher—IDW), VLAIO (Vlaams Agentschap Innoveren en Ondernemen), FWO (Fonds Wetenschappelijk Onderzoek—Vlaanderen—G0C6819N) and FRA-SAO (Fondation Recherche Alzheimer—Stichting Alzheimer Onderzoek, Belgium).

Tau is most intensely studied in relation to its executive role in Tauopathies, a family of neurodegenerative disorders characterized by the accumulation of Tau aggregates [15, 21, 38, 75, 89, 111, 121, 135, 175, 176, 192]. Tau aggregation in the different Tauopathies differs in the affected cell type, the structure of aggregates and Tau isoform composition. However, in all Tauopathies, accumulation of pathological Tau in well-characterized and well-defined brain regions, correlates strongly with symptoms associated with the dysfunction of this brain region. Hence, symptoms of neurodegenerative Tauopathies can range from motoric to cognitive and behavioral symptoms, even extending to deterioration of vital functions when the disease progresses, or combinations of different symptoms governed by the affected brain regions. The most common Tauopathies are corticobasal degeneration (CBD), Pick's disease, progressive supranuclear palsy (PSP) and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). However a growing number of diseases are characterized by Tau aggregation amounting to a large family of more than 20 disorders [176]. Most Tauopathies are sporadic, and are hence linked to a combination of environmental and genetic risk factors. However, mutations in MAPT have been identified which are autosomal dominantly linked to Tauopathies, including FTDP, PSP and CBD [94, 163, 185] (Alzforum, https://www.alzforum.org/mutations/mapt ). More than 80 mutations have been identified in MAPT, both in intronic and exonic regions of the human MAPT. These mutations can be classified as missense mutations or splicing mutations. Most missense mutations cluster in or near the microtubule binding site of Tau, while most splicing mutations affect the splicing of exon 10 (encoding the R2 domain), and hence affect the 3R/4R ratio. While Alzheimer's disease (AD), is the most prevalent Tauopathy, no mutations in MAPT associated with AD have been identified. Brains of AD patients are pathologically characterized by the combined presence of amyloid plaques and neurofibrillary tangles [171]. Familial forms of AD, termed early onset familial AD (EOFAD) with clinical mutations in APP or PS1/2, have an early onset, and are invariably characterized by the combined presence of amyloid and Tau pathology [24, 80, 170]. These EOFAD cases, identify a causal link between APP/PS1 misprocessing and the development of Tau pathology and neurodegeneration [80, 170]. Furthermore, combined genetic, pathological, biomarker and in vivo modelling data, indicate that amyloid pathology precedes Tau pathology, and support a role for Aβ as initiator and Tau as executor in the pathogenetic process of AD [80, 96, 97]. Hence, AD is often considered as a secondary Tauopathy (similar as for Down syndrome patients), in contrast to the primary Tauopathies described above. Tau aggregates in Tauopathies vary with respect to the ratio of different Tau isoforms (3R/4R), to the cell types displaying Tau aggregation and the structure of the aggregates. However, in all Tauopathies a strong correlation between progressive development of pathological Tau accumulation and the loss of the respective brain functions is observed.

Mechanisms driving cognitive improvements following nuclear receptor activation are poorly understood. The peroxisome proliferator–activated nuclear receptor alpha (PPARα) forms heterodimers with the nuclear retinoid X receptor (RXR). We report that PPARα mediates the improvement of hippocampal synaptic plasticity upon RXR activation in a transgenic mouse model with cognitive deficits. This improvement results from an increase in GluA1 subunit expression of the alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, eliciting an AMPA response at the excitatory synapses. Associated with a two times higher PPARα expression in males than in females, we show that male, but not female, PPARα null mutants display impaired hippocampal long-term potentiation. Moreover, PPARα knockdown in the hippocampus of cognition-impaired mice compromises the beneficial effects of RXR activation on synaptic plasticity only in males. Furthermore, selective PPARα activation with pemafibrate improves synaptic plasticity in male cognition-impaired mice, but not in females. We conclude that striking sex differences in hippocampal synaptic plasticity are observed in mice, related to differences in PPARα expression levels., SCOPUS: ar.j, info:eu-repo/semantics/published

Synaptic dysfunction is an early pathological feature of neurodegenerative diseases associated with Tau, including Alzheimer's disease. Interfering with early synaptic dysfunction may be therapeutically beneficial to prevent cognitive decline and disease progression, but the mechanisms underlying synaptic defects associated with Tau are unclear. In disease conditions, Tau mislocalizes into pre- and postsynaptic compartments; here we show that, under pathological conditions, Tau binds to presynaptic vesicles in Alzheimer's disease patient brain. We define that the binding of Tau to synaptic vesicles is mediated by the transmembrane vesicle protein Synaptogyrin-3. In fly and mouse models of Tauopathy, reduction of Synaptogyrin-3 prevents the association of presynaptic Tau with vesicles, alleviates Tau-induced defects in vesicle mobility, and restores neurotransmitter release. This work therefore identifies Synaptogyrin-3 as the binding partner of Tau on synaptic vesicles, revealing a new presynapse-specific Tau interactor, which may contribute to early synaptic dysfunction in neurodegenerative diseases associated with Tau.

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.

Animal models recapitulate characteristic pathological features of Alzheimer’s disease and related tauopathies, in addition to several phenotypic traits reminiscent of the disease. These animal models have been used as a central tool in the identification of therapeutic targets and their preclinical validation. A major focus has been on therapies targeting amyloid-β (Aβ). However, clinical trials aiming at Aβ have led to the general view that once the disease process has started, multitargeted combined therapies are required for disease-modifying therapies, including downstream targets in the amyloid cascade. These include not only tau as a crucial executor of the pathogenesis, but also mechanisms involved in spreading of tau-pathology and in Aβ-induced tau-pathology. In this work, we review the use of preclinical models for identification of targets for disease-modifying therapies. We thereby focus on the initial validation of targets currently tested in clinical trials, their constraints and subsequent fine-tuning, and on new venues for future therapies currently under study in preclinical models.

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.

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

TNFα varies in inverse proportion to IL-1β. Astrocytes primed with 1 μg/ml LPS for 3 h were treated with 10 μM Aβ42 for 3 h. The concentration of TNFα or IL-1β released in culture supernatants were then measured by specific ECLIA. Results are the mean ± SEM, and statistical differences were determined using an ANOVA analysis and post Bonferroni multiple comparison test (n = 4 in duplicate). ***P

Path length (cm) measured during the probe trial in the Morris water maze (MWM). Mice were let in the MWM for a 1-min free swimming test. Total distances traveled were quantified and showed no difference between all tested groups (15 F− A+/+, 14 F+ A+/+, and 11 F+ A+/−).

Neutralization of CCL3 by a specific antibody. Astrocytes primed with 1 μg/ml LPS for 3 h were treated with 10 μM Aβ42 for 3 h. Specific antibody against CCL3 was added 45 min after Aβ42. The concentration of CCL3 in culture supernatants was then measured by ECLIA. Results are the mean ± SEM derived from three experiments in duplicate, and statistical differences were determined using a Mann-Whitney test. ***P

The amyloid cascade hypothesis has been the prevailing hypothesis in Alzheimer's Disease research, although the final and most wanted proof i.e. fully successful anti-amyloid clinical trials in patients, is still lacking. This may require a better in depth understanding of the cascade. Particularly, the exact toxic forms of Aβ and Tau, the molecular link between them and their respective contributions to the disease process need to be identified in detail. Although the lack of final proof has raised substantial criticism on the hypothesis per se, accumulating experimental evidence in in vitro models, in vivo models and from biomarkers analysis in patients supports the amyloid cascade and particularly Aβ-induced Tau-pathology, which is the focus of this review. We here discuss available models that recapitulate Aβ-induced Tau-pathology and review some potential underlying mechanisms. The availability and diversity of these models that mimic the amyloid cascade partially or more complete, provide tools to study remaining questions, which are crucial for development of therapeutic strategies for Alzheimer's Disease. We thank the Belgian Fonds National pour la Recherche Scientifique –Fonds de la Recherche Scientifique (FNRS-FRS; Qualified Researcher, Impulse Financing, Research Credits), the Institute for the Promotion of Innovation by Science and Technology (IWT) (Belgium), the European Commission (AgedBrainSYSBIO, FP7, ECGA No305299) and the Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, for financial support. We thank F. Van Leuven for his contribution to this review.

Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aβ) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer's Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aβ-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aβ, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aβ can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aβ-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aβ provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aβ, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aβ-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in 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 the Stellar project (Johnson and Johnson pharmaceutical companies), by Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, by the Fondation Médicale Reine Elisabeth (F.M.R.E.), by the Institute for the Promotion of Innovation and by Science and Technology (IWT) in Flanders (IWTO&O), Belgium.

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants. Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants. 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 ProgrammeBelgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, by the Institute for the Promotion of Innovation by Science and Technology (IWT) in Flanders (IWT O&O), Belgium.

Background The proinflammatory cytokine interleukin-1β (IL-1β) is overexpressed in Alzheimer disease (AD) as a key regulator of neuroinflammation. Amyloid-β (Aβ) peptide triggers activation of inflammasomes, protein complexes responsible for IL-1β maturation in microglial cells. Downregulation of NALP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome has been shown to decrease amyloid load and rescue cognitive deficits in a mouse model of AD. Whereas activation of inflammasome in microglial cells has been described in AD, no data are currently available concerning activation of inflammasome in astrocytes, although they are involved in inflammatory response and phagocytosis. Here, by targeting the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain), we investigated the influence of activation of the inflammasome on the phagocytic activity of astrocytes. Methods We used an ASC knockout mouse model, as ASC is a central protein in the inflammasome, acting as an adaptor and stabilizer of the complex and thus critical for its activation. Lipopolysaccharide (LPS)-primed primary cultures of astrocytes from newborn mice were utilized to evaluate Aβ-induced inflammasome activation by measuring IL-1β release by ECLIA (electro-chemiluminescence immunoassay). Phagocytosis efficiency was measured by incorporation of bioparticles, and the release of the chemokine CCL3 (C-C motif ligand 3) was measured by ECLIA. ASC mice were crossbred with 5xFAD (familial Alzheimer disease) mice and tested for spatial reference memory using the Morris water maze (MWM) at 7–8 months of age. Amyloid load and CCL3 were quantified by thioflavine S staining and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Results Cultured astrocytes primed with LPS and treated with Aβ showed an ASC-dependent production of IL-1β resulting from inflammasome activation mediated by Aβ phagocytosis and cathepsin B enzymatic activity. ASC+/− astrocytes displayed a higher phagocytic activity as compared to ASC+/+ and ASC −/− cells, resulting from a higher release of the chemokine CCL3. A significant decrease in amyloid load was measured in the brain of 7–8-month-old 5xFAD mice carrying the ASC +/− genotype, correlated with an increase in CCL3 gene expression. In addition, the ASC +/− genotype rescued spatial reference memory deficits observed in 5xFAD mice. Conclusions Our results demonstrate that Aβ is able to activate astrocytic inflammasome. Downregulation of inflammasome activity increases phagocytosis in astrocytes due to the release of CCL3. This could explain why downregulation of inflammasome activity decreases amyloid load and rescues memory deficits in a mouse model of AD. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0477-y) contains supplementary material, which is available to authorized users.