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The postsynaptic inhibition through GABA A receptors (GABA A R) relies on two mechanisms, a shunting effect due to an increase in the postsynaptic membrane conductance and, in mature neurons, a hyperpolarization effect due to an entry of chloride into postsynaptic neurons. The second effect requires the action of the K + -Cl - cotransporter KCC2 which extrudes Cl - from the cell and maintains its cytosolic concentration very low. Neuronal chloride equilibrium seems to be dysregulated in several neurological and psychiatric conditions such as epilepsy, anxiety, schizophrenia, Down syndrome, or Alzheimer's disease. In the present study, we used the KCC2 Cre-lox knockdown system to investigate the role of KCC2 in synaptic plasticity and memory formation in adult mice. Tamoxifen-induced conditional deletion of KCC2 in glutamatergic neurons of the forebrain was performed at 3  months of age and resulted in spatial and nonspatial learning impairment. On brain slices, the stimulation of Schaffer collaterals by a theta burst induced long-term potentiation (LTP). The lack of KCC2 did not affect potentiation of field excitatory postsynaptic potentials (fEPSP) measured in the stratum radiatum (dendrites) but increased population spike (PS) amplitudes measured in the CA1 somatic layer, suggesting a reinforcement of the EPSP-PS potentiation, i.e., an increased ability of EPSPs to generate action potentials. At the cellular level, KCC2 deletion induced a positive shift in the reversal potential of GABA A R-driven Cl - currents (E GABA ), suggesting an intracellular accumulation of chloride subsequent to the downregulation of KCC2. After treatment with bumetanide, an antagonist of the Na + -K + -Cl - cotransporter NKCC1, spatial memory impairment, chloride accumulation, and EPSP-PS potentiation were rescued in mice lacking KCC2. The presented results emphasize the importance of chloride equilibrium and GABA-inhibiting ability in synaptic plasticity and memory formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kreis, Issa, Yerna, Jabbour, Schakman, de Clippele, Tajeddine, Pierrot, Octave, Gualdani and Gailly.)

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 GABA B receptors (GABA B Rs), 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 (hAPP wt ) causes early cognitive impairment, neuronal loss, and electrophysiological abnormalities in the absence of amyloid plaques and at very low levels of Aβ. hAPP wt mice exhibited neuronal overexcitation that was evident in EEG and increased long-term potentiation (LTP). Overexpression of hAPP wt 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 GABA B Rs and subsequently reducing GABA release. By using a specific presynaptic GABA B R antagonist, we were able to rescue hyperexcitation in hAPP wt animals. Our results provide evidence that APP plays a crucial role in regulating inhibitory neurotransmission., (© 2021. The Author(s).)

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.

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.

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 GABA A receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABA A 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., (Copyright © 2020 Opsomer et al.)

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Mutations in genes encoding proteins involved in amyloid-β peptide (Aβ) production are responsible for inherited AD cases. The amyloid cascade hypothesis was proposed to explain the pathogeny. Despite the fact that Aβ is considered as the main culprit of the pathology, most clinical trials focusing on Aβ failed and suggested that earlier interventions are needed to influence the course of AD. Therefore, identifying risk factors that predispose to AD is crucial. Among them, the epsilon 4 allele of the apolipoprotein E gene that encodes the major brain lipid carrier and metabolic disorders such as obesity and type 2 diabetes were identified as AD risk factors, suggesting that abnormal lipid metabolism could influence the progression of the disease. Among lipids, fatty acids (FAs) play a fundamental role in proper brain function, including memory. Peroxisome proliferator-activated receptor α (PPARα) is a master metabolic regulator that regulates the catabolism of FA. Several studies report an essential role of PPARα in neuronal function governing synaptic plasticity and cognition. In this review, we explore the implication of lipid metabolism in AD, with a special focus on PPARα and its potential role in AD therapy.

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., (© 2019 Tang et al.)

Brains of Alzheimer's disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, both invariably associated with neuroinflammation. A crucial role for NLRP3-ASC inflammasome [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 identified. Aβ aggregates activate NLRP3-ASC inflammasome (Halle et al. in Nat Immunol 9:857-865, 2008) and conversely NLRP3-ASC inflammasome 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 inflammasome 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 inflammasome. We demonstrate that Tau seeds activate NLRP3-ASC-dependent inflammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of inflammasome activation in prion-like or templated seeding of Tau pathology and found significant inhibition of exogenously seeded Tau pathology by ASC deficiency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC deficiency also decreased non-exogenously seeded Tau pathology in Tau transgenic mice. Overall our findings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC inflammasome 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 inflammasome, 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 prion-like seeding of Tau pathology, Aβ pathology and neuroinflammation.

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-3-hydroxy-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., (© 2019 Pierrot et al.)

Aβ peptides, the major components of Alzheimer's disease (AD) amyloid deposits, 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 been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the β-secretase-derived C99 fragment (β-CTF) 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 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 SOD1 WT or SOD1 G93A 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.

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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

The amyloid precursor protein (APP) modulates synaptic activity, resulting from the fine tuning of excitatory and inhibitory neurotransmission. GABAergic inhibitory neurotransmission is affected by modifications in intracellular chloride concentrations regulated by Na + -K + -2Cl - cotransporter 1 (NKCC1) and neuronal K + -Cl - cotransporter 2 (KCC2), allowing entrance and efflux of chloride, respectively. Modifications in NKCC1 and KCC2 expression during maturation of cortical cells induce a shift in GABAergic signaling. Here, we demonstrated that APP affects this GABA shift. Expression of APP in cortical cells decreased the expression of KCC2, without modifying NKCC1, eliciting a less inhibitory GABA response. Downregulation of KCC2 expression by APP was independent of the APP intracellular domain, but correlated with decreased expression of upstream stimulating factor 1 (USF1), a potent regulator of Slc12a5 gene expression (encoding KCC2). KCC2 was also downregulated in vivo following APP expression in neonatal mouse brain. These results argue for a key role of APP in the regulation of GABAergic neurotransmission.

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.

Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation., (© FASEB.)

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.

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.

Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

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.

Ethnopharmacological Relevance: The aqueous decoction of Pterocarpus erinaceus has been traditionally used in Benin against memory troubles., Aim of the Study: New strategies are needed against Alzheimer׳s disease (AD), for, to date, AD treatment is symptomatic and consists in drugs treating the cognitive decline. An interesting target is the β-amyloid peptide (Aβ), whose accumulation and progressive deposition into amyloid plaques are key events in AD aetiology. Identifying new and more selective γ-secretase inhibitors or modulators (none of the existing has proven so far to be selective or fully efficient) appears in this respect of particular interest. We studied the activity and mechanisms of action of Pterocarpus erinaceus kino aqueous extract, after the removal of catechic tannins (KAST)., Methods and Results: We tested KAST at non-toxic concentrations on cells expressing the human Amyloid Precursor Protein (APP695), as well as on primary neurons. Pterocarpus erinaceus extract was found to inhibit Aβ release in both models. We further showed that KAST inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that KAST is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit the cleavage of Notch, another γ-secretase substrate responsible for major detrimental side effects observed with γ-secretase inhibitors. Epicatechin was further identified in KAST by HPLC-MS., Conclusion: Pterocarpus erinaceus kino extract appears therefore as a new γ-secretase inhibitor selective towards APP processing., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aβ peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: It is suspected that excess of brain cholesterol plays a role in Alzheimer's disease (AD). Membrane-associated cholesterol was shown to be increased in the brain of individuals with sporadic AD and to correlate with the severity of the disease. We hypothesized that an increase of membrane cholesterol could trigger sporadic AD early phenotypes., Results: We thus acutely loaded the plasma membrane of cultured neurons with cholesterol to reach the 30% increase observed in AD brains. We found changes in gene expression profiles that are reminiscent of early AD stages. We also observed early AD cellular phenotypes. Indeed we found enlarged and aggregated early endosomes using confocal and electron microscopy after immunocytochemistry. In addition amyloid precursor protein vesicular transport was inhibited in neuronal processes, as seen by live-imaging. Finally transient membrane cholesterol loading lead to significantly increased amyloid-β42 secretion., Conclusions: Membrane cholesterol increase in cultured neurons reproduces most early AD changes and could thus be a relevant model for deciphering AD mechanisms and identifying new therapeutic targets.

We previously demonstrated that APP epigenetically regulates Egr1 expression both in cultured neurons and in vivo. Since Egr1 is an immediate early gene involved in memory formation, we wondered whether other early genes involved in memory were regulated by APP and we studied molecular mechanisms involved. By comparing prefrontal (PF) cortex from wild type (APP+/+) and APP knockout mice (APP-/-), we observed that APP down regulates expression of four immediate early genes, Egr1, c-Fos, Bdnf and Arc. Down regulation of Egr1, c-Fos and Bdnf transcription resulted from a decreased enrichment of acetylated histone H4 on the corresponding gene promoter. Further characterization of H4 acetylation at Egr1 and c-Fos promoters revealed increased acetylation of H4K5 and H4K12 residues in APP-/- mice. Whereas APP affected Egr1 promoter activity by reducing access of the CREB transcription factor, its effect on c-Fos appeared to depend on increased recruitment of HDAC2 histone deacetylase to the gene promoter. The physiological relevance of the epigenetic regulation of Egr1 and c-Fos gene transcription by APP was further analyzed following exposure of mice to novelty. Although transcription of Egr1 and c-Fos was increased following exposure of APP+/+ mice to novelty, such an induction was not possible in APP-/- mice with a high basal level of expression of these immediate early genes. Altogether, these results demonstrate that APP-mediated regulation of c-Fos and Egr1 by different epigenetic mechanisms is needed for their induction during exposure to novelty.

Tau alterations are now considered an executor of neuronal demise and cognitive dysfunction in Alzheimer's disease (AD). Mouse models combining amyloidosis and tauopathy and their parental counterparts are important tools to further investigate the interplay of abnormal amyloid-β (Aβ) and Tau species in pathogenesis, synaptic and neuronal dysfunction, and cognitive decline. Here, we crossed APP/PS1 mice with 5 early-onset familial AD mutations (5xFAD) and TauP301S (PS19) transgenic mice, denoted F(+)/T(+) mice, and phenotypically compared them to their respective parental strains, denoted F(+)/T(-) and F(-)/T(+) respectively, as controls. We found dramatically aggravated tauopathy (~10-fold) in F(+)/T(+) mice compared to the parental F(-)/T(+) mice. In contrast, amyloidosis was unaltered compared to the parental F(+)/T(-) mice. Tauopathy was invariably and very robustly aggravated in hippocampal and cortical brain regions. Most important, F(+)/T(+) displayed aggravated cognitive deficits in a hippocampus-dependent spatial navigation task, compared to the parental F(+)/T(-) strain, while parental F(-)/T(+) mice did not display cognitive impairment. Basal synaptic transmission was impaired in F(+)/T(+) mice compared to nontransgenic mice and the parental strains (≥40%). Finally, F(+)/T(+) mice displayed a significant hippocampal atrophy (~20%) compared to nontransgenic mice, in contrast to the parental strains. Our data indicate for the first time that pathological Aβ species (or APP/PS1) induced changes in Tau contribute to cognitive deficits correlating with synaptic deficits and hippocampal atrophy in an AD model. Our data lend support to the amyloid cascade hypothesis with a role of pathological Aβ species as initiator and pathological Tau species as executor., (© FASEB.)

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proteolysis 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Neurofibrillary degeneration in transgenic models of tauopathies has been observed to be enhanced when these models are crossed with transgenic models developing an Aβ pathology. The mechanisms leading to this enhanced tau pathology are not well understood. We have performed a detailed analysis of tau misprocessing in a new transgenic mouse model combining APP, PS1 and tau mutations (5xFAD×Tg30 mice) by comparison with littermates expressing only a FTD mutant tau (Tg30 mice). These 5xFAD×Tg30 mice showed a more severe deficient motor phenotype than Tg30 mice and developed with age a dramatically accelerated NFT load in the brain compared to Tg30 mice. Insoluble tau in 5xFAD×Tg30 mice compared to insoluble tau in Tg30 mice showed increased phosphorylation, enhanced misfolding and truncation changes mimicking more closely the post-translational changes characteristic of PHF-tau in Alzheimer's disease. Endogenous wild-type mouse tau was recruited at much higher levels in insoluble tau in 5xFAD×Tg30 than in Tg30 mice. Extracellular amyloid load, Aβ40 and Aβ42, β-CTFs and β-CTF phosphorylation levels were lower in 5xFAD×Tg30 mice than in 5xFAD mice. Despite this reduction of Aβ, a significant hippocampal neuronal loss was observed in 5xFAD×Tg30 but not in 5xFAD mice indicating its closer association with increased tau pathology. This 5xFAD×Tg30 model thus mimics more faithfully tau pathology and neuronal loss observed in AD and suggests that additional post-translational changes in tau and self-recruitment of endogenous tau drive the enhanced tau pathology developing in the presence of Aβ pathology., (© 2013.)

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., (2013 S. Karger AG, Basel)

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Following transcriptome comparison of primary cultures isolated from brain of mice expressing or not the amyloid precursor protein APP, we found transcription of the EGR-1 gene to be regulated by APP. In primary cultures of cortical neurons, APP significantly down regulated EGR-1 expression at both mRNA and protein levels in a γ-secretase independent manner. The intracellular domain of APP did not interact with EGR-1 gene promoter, but enrichment of acetylated histone H4 at the EGR-1 promoter region was measured in APP-/- neurons, as well as in brain of APP-/- mice, in which increase in EGR-1 expression was also measured. These results argue for an important function of APP in the epigenetic regulation of EGR-1 gene transcription both in vitro and in vivo. In APP-/- mice, constitutive overexpression of EGR-1 in brain impaired epigenetic induction of this early transcriptional regulator during exposure to novelty. Altogether, these results indicate an important function of APP in the epigenetic regulation of the transcription of EGR-1, known to be important for memory formation.

Despite intensive studies of the secretase-mediated processing of the amyloid precursor protein (APP) to form the amyloid β-peptide (Aβ), in relation to Alzheimer's disease (AD), no new therapeutic agents have reached the clinics based on reducing Aβ levels through the use of secretase inhibitors or immunotherapy. Furthermore, the normal neuronal functions of APP and its various metabolites still remain under-investigated and unclear. Here, we highlight emerging areas of APP function that may provide new insights into synaptic development, cognition, and gene regulation. By modulating expression levels of endogenous APP in primary cortical neurons, the frequency and amplitude of calcium oscillations is modified, implying a key role for APP in maintaining neuronal calcium homeostasis essential for synaptic transmission. Disruption of this homeostatic mechanism predisposes to aging and AD. Synaptic spine loss is a feature of neurogeneration resulting in learning and memory deficits, and emerging evidence indicates a role for APP, probably mediated via one or more of its metabolites, in spine structure and functions. The intracellular domain of APP (AICD) has also emerged as a key epigenetic regulator of gene expression controlling a diverse range of genes, including APP itself, the amyloid-degrading enzyme neprilysin, and aquaporin-1. A fuller understanding of the physiological and pathological actions of APP and its metabolic network could provide new opportunities for therapeutic intervention in AD., (© 2013 International Society for Neurochemistry.)

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

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., (Copyright © 2012 S. Karger AG, Basel.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aim of the Study: The aim of the study was to investigate the activity on β-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 β-amyloid peptide precursor (APP695) to measure variations of APP processing (by Western-blotting) and, for the most active, of Aβ-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 Aβ production, displaying effects similar to those of DAPT (γ-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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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 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?

Unlabelled: Recent reports have drawn attention to dysfunctions of intrinsic neuronal excitability and network activity in Alzheimer disease (AD). Here we review the possible causes of these basic dysfunctions and implications for AD, based on in vitro and in vivo findings. We then review the current therapeutic approaches particularly linked to the issue of neuronal excitability in AD., Conclusion: AD is a complex, neurodegenerative disorder. Hippocampal synaptic dysfunction is an early feature of the degenerative process that is clearly linked to memory impairment, the first and major symptom of AD. A growing body of evidence points toward a dysfunction of neuronal networks. Intrinsic neuronal excitability, mainly through profound dysregulation of calcium homeostasis, appears to be largely affected. Consequently, neuronal communication is disturbed. Such cellular defects might underlie cognitive manifestations like fluctuations in cognitive impairment and might also explain several observations obtained with EEG, MEG, MRI, or PET studies, leading to the concept of a disconnection syndrome in AD.

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.

Synchronous calcium oscillations are observed in primary cultures of rat cortical neurons when mature networks are formed. This spontaneous neuronal activity needs an accurate control of calcium homeostasis. Alteration of intraneuronal calcium concentration is described in many neurodegenerative disorders, including Alzheimer disease (AD). Although processing of amyloid precursor protein (APP) that generates Abeta peptide has critical implications for AD pathogenesis, the neuronal function of APP remains unclear. Here, we report that expression of human APP (hAPP) in rat cortical neurons increases L-type calcium currents, which stimulate SK channels, calcium-dependent K(+) channels responsible for medium afterhyperpolarization (mAHP). In a neuronal network, increased mAHP in some neurons expressing hAPP leads to inhibition of calcium oscillations in all the cells of the network. This inhibition is independent of production and secretion of Abeta and other APP metabolites. In a neuronal network, reduction of endogenous APP expression using shRNA increases the frequency and reduces the amplitude of calcium oscillations. Altogether, these data support a key role for APP in the control of neuronal excitability.

Processing of amyloid precursor protein (APP) by gamma-secretase is the last step in the formation of the Abeta peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we show that the TM domain forms an alpha-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the epsilon-cleavage site. This helix-to-coil transition is required for gamma-secretase processing; mutations that extend the TM alpha-helix inhibit epsilon cleavage, leading to a low production of Abeta peptides and an accumulation of the alpha- and beta-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM alpha-helix.