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The function of the amyloid precursor protein (APP) is not fully understood, but its cleavage product amyloid beta (Aβ) together with neurofibrillary tangles constitute the hallmarks of Alzheimer’s disease (AD). Yet, imbalance of excitatory and inhibitory neurotransmission accompanied by loss of synaptic functions, has been reported much earlier and independent of any detectable pathological markers. Recently, soluble APP fragments have been shown to bind to presynaptic GABAB receptors (GABABRs), subsequently decreasing the probability of neurotransmitter release. In this body of work, we were able to show that overexpression of wild-type human APP in mice (hAPPwt) causes early cognitive impairment, neuronal loss, and electrophysiological abnormalities in the absence of amyloid plaques and at very low levels of Aβ. hAPPwt mice exhibited neuronal overexcitation that was evident in EEG and increased long-term potentiation (LTP). Overexpression of hAPPwt did not alter GABAergic/glutamatergic receptor components or GABA production ability. Nonetheless, we detected a decrease of GABA but not glutamate that could be linked to soluble APP fragments, acting on presynaptic GABABRs and subsequently reducing GABA release. By using a specific presynaptic GABABR antagonist, we were able to rescue hyperexcitation in hAPPwt animals. Our results provide evidence that APP plays a crucial role in regulating inhibitory neurotransmission.

Among genetic susceptibility loci associated with late-onset Alzheimer disease (LOAD), genetic polymorphisms identified in genes encoding lipid carriers led to the hypothesis that a disruption of lipid metabolism could promote disease progression. We previously reported that amyloid precursor protein (APP) involved in Alzheimer disease (AD) physiopathology impairs lipid synthesis needed for cortical networks' activity and that activation of peroxisome proliferator-activated receptor α (PPARα), a metabolic regulator involved in lipid metabolism, improves synaptic plasticity in an AD mouse model. These observations led us to investigate a possible correlation between PPARα function and full-length APP expression. Here, we report that PPARα expression and activation were inversely related to APP expression both in LOAD brains and in early-onset AD cases with a duplication of the APP gene, but not in control human brains. Moreover, human APP expression decreased PPARA expression and its related target genes in transgenic mice and in cultured cortical cells, while opposite results were observed in APP-silenced cortical networks. In cultured neurons, APP-mediated decrease or increase in synaptic activity was corrected by a PPARα-specific agonist and antagonist, respectively. APP-mediated control of synaptic activity was abolished following PPARα deficiency, indicating a key function of PPARα in this process., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

Extracellular deposition of β-amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon β-secretase–mediated cleavage of the β C-terminal fragment (β-CTF) from the Aβ precursor protein, the γ-secretase complex produces the Aβ peptides associated with AD. The familial T43I mutation within the transmembrane domain of the β-CTF (also referred to as C99) increases the ratio between the Aβ42 and Aβ40 peptides largely due to a decrease in Aβ40 formation. Aβ42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aβ42/Aβ40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. (13)C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted Aβ40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aβ40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aβ40 formation and increase the Aβ42/Aβ40 ratio.

Visual Abstract, The amyloid precursor protein (APP) has been extensively studied as the precursor of the β-amyloid (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer’s disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP−/− neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP−/− mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

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).

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

Sequential proteolysis of the amyloid precursor protein (APP) and amyloid-β peptide (Aβ) release is an upstream event in Alzheimer’s disease (AD) pathogenesis. The function of APP in neuronal physiology is still, however, poorly understood. Along with its paralog APP-like Proteins 1 and 2 (APLP1-2), APP is involved in neurite formation and synaptic function by mechanisms that are not elucidated. APP is a single-pass transmembrane protein expressed at high levels in the brain that resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell interaction processes and/or activation of intracellular pathways of signal transduction. Along this line, the APP intracellular domain (AICD) was reported to act as a transcriptional factor for targeted gene activation that mediates physiological APP functions. Here, we used an unbiased transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and upon maturation of primary cortical neurons. The transcriptome analysis did not detect any significant differences in expression of previously proposed AICD target genes. The overall transcriptional changes were subtle, but we found that genes clustered in neuronal-activity dependent pathways are dysregulated in the absence of APP. Among these genes, we found the activity-dependent Neuronal PAS domain protein 4 (NPAS4) Immediate Early Gene to be downregulated in the absence of APP. Down-regulation of NPAS4 in APP knock-out (KO) neurons is not related to AICD but to the APP ectodomain. We studied the effect of APP deficiency on GABAergic and glutamatergic transmission, and found an increased production of the inhibitory neurotransmitter GABA in APP KO neurons, along with a reduced expression of the GABA(A) receptors alpha1, suggesting an impaired GABAergic neurotransmission in the absence of APP. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Altogether, our results point out a new role for APP in the regulation of excitatory/inhibitory neurotransmission through the regulation of the activity-dependent NPAS4 gene.

Aβ peptides, the major components of amyloid deposits of Alzheimer’s disease, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have also been reported to contribute to APP processing. However, the respective role of each of these pathways towards APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length APPWTor the β-secretase-derived C99 fragments (β-CTFs) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aβ peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 alone is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

The current inability of clinical criteria to accurately identify the “at-risk group” for Amyotrophic Lateral Sclerosis (ALS) development as well as its unknown etiology are fueling the interest in biomarkers aimed at completing clinical approaches for the diagnosis. The Glial cell line-derived neurotrophic factor (GDNF) is a diffusible peptide critically involved in neuronal differentiation and survival. GDNF is largely studied in various neurological and neuromuscular diseases, with a great interest in the peripheral nervous system (PNS). The recent discovery of Amyloid Precursor Protein (APP)-dependent GDNF regulation driving neuro-muscular junctions' formation in APP null transgenic mice, prompts to study whether neurodegeneration relies on loss or gain of APP function and suggests that it could affect peripheral processes. Here, we explored a brand-new aspect of the loss of trophic support in ALS by measuring GDNF, APP, soluble APP fragments and Aβ peptides levels in SOD1WT or SOD1G93A transgenic mouse models of ALS and in human biological fluids [i.e. serum and cerebrospinal fluid (CSF)] from ALS patients and control subjects. Our results show that both GDNF and soluble APP fragments levels are altered at the onset of motor deficits in mice and that their levels are also modified in patient samples. This study indicates that both GDNF and soluble APPα represent possible biomarkers for ALS.

Familial mutations in C99 can increase the total level of the soluble Aβ peptides produced by proteolysis, as well as the Aβ42/Aβ40 ratio, both of which are linked to the progression of Alzheimer’s disease. We show that the extracellular sequence of C99 forms β-sheet structure upon interaction with membrane bilayers. Mutations that disrupt this structure result in a significant increase in Aβ production and, in specific cases, result in an increase in the amount of Aβ42 relative to Aβ40. Fourier transform infrared and solid-state NMR spectroscopic studies reveal a central β-hairpin within the extracellular sequence comprising Y10-E11-V12 and L17-V18-F19 connected by a loop involving H13-H14-Q15. These results suggest how familial mutations in the extracellular sequence influence C99 processing and provide a structural basis for the development of small molecule modulators that would reduce Aβ production.

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.

Mitochondrial dysfunction plays a pivotal role in the progression of Alzheimer's disease (AD), and yet the mechanisms underlying the impairment of mitochondrial function in AD remain elusive. Recent evidence suggested a role for Presenilins (PS1 or PS2) in mitochondrial function. Mutations of PSs, the catalytic subunits of the γ-secretase complex, are responsible for the majority of inherited AD cases (FAD). PSs were shown to be present in mitochondria and particularly enriched in mitochondria-associated membranes (MAM), where PS2 is involved in the calcium shuttling between mitochondria and the endoplasmic reticulum (ER). We investigated the precise contribution of PS1 and PS2 to the bioenergetics of the cell and to mitochondrial morphology in cell lines derived from wild type (PS+/+), PS1/2 double knock-out (PSdKO), PS2KO and PS1KO embryos. Our results showed a significant impairment in the respiratory capacity of PSdKO and PS2KO cells with reduction of basal oxygen consumption, oxygen utilization dedicated to ATP production and spare respiratory capacity. In line with these functional defects, we found a decrease in the expression of subunits responsible for mitochondrial oxidative phosphorylation (OXPHOS) associated with an altered morphology of the mitochondrial cristae. This OXPHOS disruption was accompanied by a reduction of the NAD+/NADH ratio. Still, neither ADP/ATP ratio nor mitochondrial membrane potential (ΔΨ) were affected, suggesting the existence of a compensatory mechanism for energetic balance. We observed indeed an increase in glycolytic flux in PSdKO and PS2KO cells. All these effects were truly dependent on PS2 since its stable re-expression in a PS2KO background led to a complete restoration of the parameters impaired in the absence of PS2. Our data clearly demonstrate here the crucial role of PS2 in mitochondrial function and cellular bioenergetics, pointing toward its peculiar role in the formation and integrity of the electron transport chain. This work was supported by a grant of the Foundation for Research on Alzheimer’s disease (PK-C), by the Interuniversity Attraction Pole Programme-Belgian Sate-Belgian Science Policy (IAP-P7/16 and IAP-P7/13) to J-NO, ID, and PK-C, by the Fondation Médicale Reine Elisabeth (FMRE) to J-NO, ID, and PK-C, by the Action de Recherche Concertée (ARC 14/19-059) to PK-C.

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.

Rapid changes in cell volume characterize macrophage activation, but the role of water channels in inflammation remains unclear. We show here that, in vitro, aquaporin (AQP) blockade or deficiency results in reduced IL-1β release by macrophages activated with a variety of NLRP3 activators. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1β release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1β release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy.

SummaryProteolysis of the β C-terminal fragment (β-CTF) of the amyloid precursor protein generates the Aβ peptides associated with Alzheimer’s disease. Familial mutations in the β-CTF, such as the A21G Flemish mutation, can increase Aβ secretion. We establish how the Flemish mutation alters the structure of C55, the first 55 residues of the β-CTF, using FTIR and solid-state NMR spectroscopy. We show that the A21G mutation reduces β sheet structure of C55 from Leu17 to Ala21, an inhibitory region near the site of the mutation, and increases α-helical structure from Gly25 to Gly29, in a region near the membrane surface and thought to interact with cholesterol. Cholesterol also increases Aβ peptide secretion, and we show that the incorporation of cholesterol into model membranes enhances the structural changes induced by the Flemish mutant, suggesting a common link between familial mutations and the cellular environment.

Alzheimer's disease is characterized by the presence of 2 neuropathological lesions: neurofibrillary tangles, composed of tau proteins which are highly phosphorylated and phosphorylated on uncommon sites, and amyloid plaques, containing the Aß peptides generated from the amyloid precursor protein (APP). Reduction of some APP proteolytic derivatives in Alzheimer's disease such as sAPPα fragment has been reported and sAPPα has been shown to affect tau phosphorylation. To investigate in vivo the effect of absence of APP protein and its fragments on tau phosphorylation and the formation of neurofibrillary tangles, we have generated mice deleted for APP gene and overexpressing a human mutant tau protein and developing neurofibrillary tangles (APPKOTg30 mice). These APPKOTg30 mice showed more severe motor and cognitive deficits, increased tau phosphorylation, increased load of neurofibrillary tangles, and increased p25/35 ratio in the brain, compared with Tg30 mice. These data suggest that APP and/or its proteolytic derivatives interfere with the formation of neurofibrillary tangles in a transgenic mouse model that will be useful for investigating the relationship between APP and tau.

Background: Accumulation of β-amyloid peptides (Aβ) and its progressive deposition into amyloid plaques are key events in the aetiology of Alzheimer's disease (AD). To date, AD treatment is symptomatic and consists of drugs treating the cognitive decline. Objective: Identifying molecules specifically targeting Aβ production or aggregation represents a huge challenge in the development of specific AD treatments. Several molecules reported as γ-secretase inhibitors or modulators have been evaluated, but so far none of them have proven to be selective or fully efficient. We have previously investigated the potential interest of plant extracts and we reported that Pterocarpus erinaceus stem-bark extract was active on Aβ release. Our aim here was to characterize the mechanisms by which this extract reduces Aβ levels. Methods: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic β-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aβ release. We further showed that this extract inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that P. erinaceus extract is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit γ-secretase-dependent Notch intracellular domain release. Conclusion: P. erinaceus extract appears as a new potent γ-secretase inhibitor selective towards APP processing.

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

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G(29)XXXG(33) and G(38)XXXA(42) motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.

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

Background: The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer’s disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Objective: Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. Methods: We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. Results: APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. Conclusion: These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP.

Adenoviral expression of human APP (hAPP), but not of hAPP deleted from its C-terminal intracellular domain, in rat cortical neurons abolishes spontaneous synchronous calcium oscillations. The intracellular domain of APP695 contains several residues that can be phosphorylated. Contrary to non-neuronal cells, a very high phosphorylation of APP on T668 is observed in neurons, which is mediated by JNK, GSK3 and Cdk5 protein kinases. JNK activity, modulated by GSK3, enhances the traffic of phosphorylated APP to nerve terminals, contrary to Cdk5. Here we show that inhibition of GSK3 and JNK restores calcium oscillations in an hAPP expressing neuronal network, whereas inhibition of Cdk5 does not. Expression of mutant hAPPT668A does not inhibit calcium oscillations, and the proportion of hAPPT668A at the plasma membrane is reduced by more than 50%. Altogether, these results indicate that the intracellular domain of APP is needed to inhibit neuronal calcium oscillations because GSK3/JNK phosphorylation of T668 controls APP trafficking at the plasma membrane.

AIM OF THE STUDY: The aim of the study was to investigate the activity on beta-amyloid peptide production of crude extracts of 9 plant species traditionally used in Benin or in Madagascar for the treatment of cognitive disorders, in order to select candidates for Alzheimer's disease treatment. MATERIALS AND METHODS: For each species, hexane, dichloromethane, ethyl-acetate and water extracts were tested, at non-toxic concentrations, on CHO cells overexpressing the human neuronal beta-amyloid peptide precursor (APP695) to measure variations of APP processing (by Western-blotting) and, for the most active, of Abeta-amyloid production (by ECLIA). RESULTS: We observed, at non-toxic concentrations, a significant increase in CTF/APP ratio with Oldenlandia affinis cyclotide-enriched fraction, Prosopis africana EtOAc extract, Pterocarpus erinaceus aqueous extract and Trichilia emetica hexane extract. We also showed that the Pterocarpus erinaceus extract significantly decreased Abeta production, displaying effects similar to those of DAPT (gamma-secretase inhibitor) on APP processing, but may act on another inhibition site. CONCLUSION: These active extracts are worth further studies to isolate the compounds responsible for the observed activities, to analyze their mode of action and determine their clinical potentials.

Extensive research efforts have been conducted over the past decades to understand the processing of the Amyloid Precursor Protein (APP). APP cleavage leads to the production of the beta-amyloid peptide (Abeta), which is the major constituent of the amyloid core of senile plaques found in the brains of patients with Alzheimer's disease (AD). Abeta is produced by the sequential cleavage of APP by beta- and gamma-secretases. Cleavage of APP by gamma-secretase also generates the APP Intracellular C-terminal Domain (AICD) peptide, which might be involved in regulation of gene transcription. Up to now, our understanding of the mechanisms controlling APP processing has been elusive. Recently, APP was found to form homo- or hetero-complexes with the APP-like proteins (APLPs), which belong to the same family and share some important structural properties with receptors having a single membrane spanning domain. Homodimerization of APP is driven by motifs present in the extracellular domain and possibly in the juxtamembrane and transmembrane (JM/TM) domains of the protein. These striking observations raise important questions about APP processing and function: How and where is APP dimerizing? What is the role of dimerization in APP processing and function? Can dimerization be targeted by small molecule therapeutics?

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

Cellular processing of the amyloid precursor protein (APP) has been extensively studied, but its precise function remains elusive. The intracellular domain of APP has been proposed to regulate expression of several genes by mechanisms that are largely unknown. We report that APP regulates expression of the aquaporin 1 (AQP1) gene in mouse embryonic fibroblasts and in transgenic mice. AQP1 mRNA and protein were down-regulated in fibroblasts lacking APP or presenilin 2 in which AQP1 expression was restored by stable expression of full-length APP or presenilin 2 but not by APP deleted from its carboxy-terminal domain. The transcriptional activity of the AQP1 gene promoter and the stability of AQP1 mRNA were identical in fibroblasts expressing or not expressing APP. Control of AQP1 expression by APP was sensitive to trichostatin A, an histone deacetylase inhibitor, and histone deacetylase activity coimmunoprecipitated with APP. Altogether, these data show that a presenilin-2-dependent gamma-secretase activity releases the intracellular domain of APP involved in the epigenetic control of AQP1 expression. Since AQP1 is found in astrocytes surrounding senile plaques, this epigenetic control of AQP1 expression could have important implications in Alzheimer disease.

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile plaque in the brain. Both disorders must be present in order to confirm a clinical diagnosis of Alzheimer's disease. Neurofibrillary tangles contain hyperphosphorylated microtubule-associated protein tau, while senile plaque contains a core of amyloidpeptide derived from its precursor. Phosphorylation of both amyloid precursor protein and tau represents a biochemical link between the two characteristic lesions of Alzheimer's disease.

The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.