Transmissible long-term neuroprotective and pro-cognitive effects of 1-42 beta-amyloid with A2T icelandic mutation in an Alzheimer's disease mouse model.

The amyloid cascade hypothesis assumes that the development of Alzheimer's disease (AD) is driven by a self-perpetuating cycle, in which ß-amyloid (Aß) accumulation leads to Tau pathology and neuronal damages. A particular mutation (A673T) of the amyloid precursor protein (APP) was identified among Icelandic population. It provides a protective effect against Alzheimer- and age-related cognitive decline. This APP mutation leads to the reduced production of Aß with A2T (position in peptide sequence) change (Aßice). In addition, Aßice has the capacity to form protective heterodimers in association with wild-type Aß. Despite the emerging interest in Aßice during the last decade, the impact of Aßice on events associated with the amyloid cascade has never been reported. First, the effects of Aßice were evaluated in vitro by electrophysiology on hippocampal slices and by studying synapse morphology in cortical neurons. We showed that Aßice protects against endogenous Aß-mediated synaptotoxicity. Second, as several studies have outlined that a single intracerebral administration of Aß can worsen Aß deposition and cognitive functions several months after the inoculation, we evaluated in vivo the long-term effects of a single inoculation of Aßice or Aß-wild-type (Aßwt) in the hippocampus of transgenic mice (APPswe/PS1dE9) over-expressing Aß1-42 peptide. Interestingly, we found that the single intra-hippocampal inoculation of Aßice to mice rescued synaptic density and spatial memory losses four months post-inoculation, compared with Aßwt inoculation. Although Aß load was not modulated by Aßice infusion, the amount of Tau-positive neuritic plaques was significantly reduced. Finally, a lower phagocytosis by microglia of post-synaptic compounds was detected in Aßice-inoculated animals, which can partly explain the increased density of synapses in the Aßice animals. Thus, a single event as Aßice inoculation can improve the fate of AD-associated pathology and phenotype in mice several months after the event. These results open unexpected fields to develop innovative therapeutic strategies against AD.

Long term worsening of amyloid pathology, cerebral function, and cognition after a single inoculation of beta-amyloid seeds with Osaka mutation.

Alzheimer's disease (AD) is characterized by intracerebral deposition of abnormal proteinaceous assemblies made of amyloid-ß (Aß) peptides or tau proteins. These peptides and proteins induce synaptic dysfunctions that are strongly correlated with cognitive decline. Intracerebral infusion of well-defined Aß seeds from non-mutated Aß1-40 or Aß1-42 peptides can increase Aß depositions several months after the infusion. Familial forms of AD are associated with mutations in the amyloid precursor protein (APP) that induce the production of Aß peptides with different structures. The Aß Osaka (Aßosa mutation (E693Δ)) is located within the Aß sequence and thus the Aßosa peptides have different structures and properties as compared to non-mutated Aß1-42 peptides (Aßwt). Here, we wondered if a single exposure to this mutated Aß can worsen AD pathology as well as downstream events including cognition, cerebral connectivity and synaptic health several months after the inoculation. To answer this question we inoculated Aß1-42-bearing Osaka mutation (Aßosa) in the dentate gyrus of APPswe/PS1dE9 mice at the age of two months. Their cognition and cerebral connectivity were analyzed at 4 months post-inoculation by behavioral evaluation and functional MRI. Aß pathology as well as synaptic density were evaluated by histology. The impact of Aßosa peptides on synaptic health was also measured on primary cortical neurons. Remarkably, the intracerebral administration of Aßosa induced cognitive and synaptic impairments as well as a reduction of functional connectivity between different brain regions, 4 months post-inoculation. It increased Aß plaque depositions and increased Aß oligomers. This is the first study showing that a single, sporadic event as Aßosa inoculation can worsen the fate of the pathology and clinical outcome several months after the event. It suggests that a single inoculation of Aß regulates a large cascade of events for a long time.

Astrocyte-neuron interplay is critical for Alzheimer's disease pathogenesis and is rescued by TRPA1 channel blockade.

The sequence of cellular dysfunctions in preclinical Alzheimer's disease must be understood if we are to plot new therapeutic routes. Hippocampal neuronal hyperactivity is one of the earliest events occurring during the preclinical stages of Alzheimer's disease in both humans and mouse models. The most common hypothesis describes amyloid-ß accumulation as the triggering factor of the disease but the effects of this accumulation and the cascade of events leading to cognitive decline remain unclear. In mice, we previously showed that amyloid-ß-dependent TRPA1 channel activation triggers hippocampal astrocyte hyperactivity, subsequently inducing hyperactivity in nearby neurons. In this work, we investigated the potential protection against Alzheimer's disease progression provided by early chronic pharmacological inhibition of the TRPA1 channel. A specific inhibitor of TRPA1 channel (HC030031) was administered intraperitoneally from the onset of amyloid-ß overproduction in the APP/PS1-21 mouse model of Alzheimer's disease. Short-, medium- and long-term effects of this chronic pharmacological TRPA1 blockade were characterized on Alzheimer's disease progression at functional (astrocytic and neuronal activity), structural, biochemical and behavioural levels. Our results revealed that the first observable disruptions in the Alzheimer's disease transgenic mouse model used correspond to aberrant hippocampal astrocyte and neuron hyperactivity. We showed that chronic TRPA1 blockade normalizes astrocytic activity, avoids perisynaptic astrocytic process withdrawal, prevents neuronal dysfunction and preserves structural synaptic integrity. These protective effects preserved spatial working memory in this Alzheimer's disease mouse model. The toxic effect of amyloid-ß on astrocytes triggered by TRPA1 channel activation is pivotal to Alzheimer's disease progression. TRPA1 blockade prevents irreversible neuronal dysfunction, making this channel a potential therapeutic target to promote neuroprotection.

CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway.

Neurodevelopmental axonal pathfinding plays a central role in correct brain wiring and subsequent cognitive abilities. Within the growth cone, various intracellular effectors transduce axonal guidance signals by remodeling the cytoskeleton. Semaphorin-3E (Sema3E) is a guidance cue implicated in development of the fornix, a neuronal tract connecting the hippocampus to the hypothalamus. Microtubule-associated protein 6 (MAP6) has been shown to be involved in the Sema3E growth-promoting signaling pathway. In this study, we identified the collapsin response mediator protein 4 (CRMP4) as a MAP6 partner and a crucial effector in Sema3E growth-promoting activity. CRMP4-KO mice displayed abnormal fornix development reminiscent of that observed in Sema3E-KO mice. CRMP4 was shown to interact with the Sema3E tripartite receptor complex within detergent-resistant membrane (DRM) domains, and DRM domain integrity was required to transduce Sema3E signaling through the Akt/GSK3 pathway. Finally, we showed that the cytoskeleton-binding domain of CRMP4 is required for Sema3E's growth-promoting activity, suggesting that CRMP4 plays a role at the interface between Sema3E receptors, located in DRM domains, and the cytoskeleton network. As the fornix is affected in many psychiatric diseases, such as schizophrenia, our results provide new insights to better understand the neurodevelopmental components of these diseases.

Effect of Aß Oligomers on Neuronal APP Triggers a Vicious Cycle Leading to the Propagation of Synaptic Plasticity Alterations to Healthy Neurons.

Alterations of excitatory synaptic function are the strongest correlate to the pathologic disturbance of cognitive ability observed in the early stages of Alzheimer's disease (AD). This pathologic feature is driven by amyloid-ß oligomers (Aßos) and propagates from neuron to neuron. Here, we investigated the mechanism by which Aßos affect the function of synapses and how these alterations propagate to surrounding healthy neurons. We used complementary techniques ranging from electrophysiological recordings and molecular biology to confocal microscopy in primary cortical cultures, and from acute hippocampal and cortical slices from male wild-type and amyloid precursor protein (APP) knock-out (KO) mice to assess the effects of Aßos on glutamatergic transmission, synaptic plasticity, and dendritic spine structure. We showed that extracellular application of Aßos reduced glutamatergic synaptic transmission and long-term potentiation. These alterations were not observed in APP KO neurons, suggesting that APP expression is required. We demonstrated that Aßos/APP interaction increases the amyloidogenic processing of APP leading to intracellular accumulation of newly produced Aßos. Intracellular Aßos participate in synaptic dysfunctions as shown by pharmacological inhibition of APP processing or by intraneuronal infusion of an antibody raised against Aßos. Furthermore, we provide evidence that following APP processing, extracellular release of Aßos mediates the propagation of the synaptic pathology characterized by a decreased spine density of neighboring healthy neurons in an APP-dependent manner. Together, our data unveil a complementary role for Aßos in AD, while intracellular Aßos alter synaptic function, extracellular Aßos promote a vicious cycle that propagates synaptic pathology from diseased to healthy neurons.SIGNIFICANCE STATEMENT Here we provide the proof that a vicious cycle between extracellular and intracellular pools of Aß oligomers (Aßos) is required for the spreading of Alzheimer's disease (AD) pathology. We showed that extracellular Aßos propagate excitatory synaptic alterations by promoting amyloid precursor protein (APP) processing. Our results also suggest that subsequent to APP cleavage two pools of Aßos are produced. One pool accumulates inside the cytosol, inducing the loss of synaptic plasticity potential. The other pool is released into the extracellular space and contributes to the propagation of the pathology from diseased to healthy neurons. Pharmacological strategies targeting the proteolytic cleavage of APP disrupt the relationship between extracellular and intracellular Aß, providing a therapeutic approach for the disease.

Synaptotoxicity in Alzheimer's Disease Involved a Dysregulation of Actin Cytoskeleton Dynamics through Cofilin 1 Phosphorylation.

Amyloid-ß (Aß) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer's disease (AD), but how Aß affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aß oligomers (Aßo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aßo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aßo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aßo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aßo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aß-induced synaptic impairment.SIGNIFICANCE STATEMENT We report that amyloid-ß oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aßo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer's disease.

Involvement of CRF2 signaling in enterocyte differentiation.

AIM: To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS: For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS: CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION: Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

TRPA1 channels promote astrocytic Ca2+ hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-ß peptide.

BACKGROUND: Excessive synaptic loss is thought to be one of the earliest events in Alzheimer's disease (AD). However, the key mechanisms that maintain plasticity of synapses during adulthood or initiate synapse dysfunction in AD remain unknown. Recent studies suggest that astrocytes contribute to functional changes observed during synaptic plasticity and play a major role in synaptic dysfunction but astrocytes behavior and involvement in early phases of AD remained largely undefined. METHODS: We measure astrocytic calcium activity in mouse CA1 hippocampus stratum radiatum in both the global astrocytic population and at a single cell level, focusing in the highly compartmentalized astrocytic arbor. Concurrently, we measure excitatory post-synaptic currents in nearby pyramidal neurons. RESULTS: We find that application of soluble Aß oligomers (Aßo) induced fast and widespread calcium hyperactivity in the astrocytic population and in the microdomains of the astrocyte arbor. We show that astrocyte hyperactivity is independent of neuronal activity and is repaired by transient receptor potential A1 (TRPA1) channels blockade. In return, this TRPA1 channels-dependent hyperactivity influences neighboring CA1 neurons triggering an increase in glutamatergic spontaneous activity. Interestingly, in an AD mouse model (APP/PS1-21 mouse), astrocyte calcium hyperactivity equally takes place at the beginning of Aß production, depends on TRPA1 channels and is linked to CA1 neurons hyperactivity. CONCLUSIONS: Our experiments demonstrate that astrocytes contribute to early Aßo toxicity exhibiting a global and local Ca2+ hyperactivity that involves TRPA1 channels and is related to neuronal hyperactivity. Together, our data suggest that astrocyte is a frontline target of Aßo and highlight a novel mechanism for the understanding of early synaptic dysregulation induced by soluble Aßo species.

Nicotine-induced cellular stresses and autophagy in human cancer colon cells: A supportive effect on cell homeostasis via up-regulation of Cox-2 and PGE(2) production.

Nicotine, one of the active components in cigarette smoke, has been described to contribute to the protective effect of smoking in ulcerative colitis (UC) patients. Furthermore, the nicotinic acetylcholine receptor α7 subunit (α7nAChR) expressed on immune cells, is an essential regulator of inflammation. As intestinal epithelial cells also express α7nAChR, we investigated how nicotine could participate in the homeostasis of intestinal epithelial cells. First, using the human adenocarcinoma cell line HT-29, we revealed that nicotine, which triggers an influx of extracellular Ca(2+) following α7nAChR stimulation, induces mitochondrial reactive oxygen species (ROS) production associated with a disruption of the mitochondrial membrane potential and endoplasmic reticulum stress. This results in caspase-3 activation, which in turn induces apoptosis. Additionally, we have shown that nicotine induces a PI3-K dependent up-regulation of cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 (PGE2) production. In this context, we suggest that this key mediator participates in the cytoprotective effects of nicotine against apoptosis by stimulating autophagy in colon cancer cells. Our results provide new insight into one potential mechanism by which nicotine could protect from UC and suggest an anti-inflammatory role for the cholinergic pathway at the epithelial cell level.

CRF2 signaling is a novel regulator of cellular adhesion and migration in colorectal cancer cells.

Stress has been proposed to be a tumor promoting factor through the secretion of specific neuromediators, such as Urocortin2 and 3 (Ucn2/3), however its role in colorectal cancer (CRC) remains elusive. We observed that Ucn2/3 and their receptor the Corticotropin Releasing Factor receptor 2 (CRF2) were up-regulated in high grade and poorly differentiated CRC. This suggests a role for CRF2 in the loss of cellular organization and tumor progression. Using HT-29 and SW620 cells, two CRC cell lines differing in their abilities to perform cell-cell contacts, we found that CRF2 signals through Src/ERK pathway to induce the alteration of cell-cell junctions and the shuttle of p120ctn and Kaiso in the nucleus. In HT-29 cells, this signaling pathway also leads to the remodeling of cell adhesion by i) the phosphorylation of Focal Adhesion Kinase and ii) a modification of actin cytoskeleton and focal adhesion complexes. These events stimulate cell migration and invasion. In conclusion, our findings indicate that CRF2 signaling controls cellular organization and may promote metastatic potential of human CRC cells through an epithelial-mesenchymal transition like process. This contributes to the comprehension of the tumor-promoting effects of stress molecules and designates Ucn2/3-CRF2 tandem as a target to prevent CRC progression and aggressiveness.

Enterocytic differentiation is modulated by lipid rafts-dependent assembly of adherens junctions.

Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation. We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.

Cyclin-dependent kinase 2/cyclin E complex is involved in p120 catenin (p120ctn)-dependent cell growth control: a new role for p120ctn in cancer.

Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy.

Increased proteolysis of diphtheria toxin by human monocytes after heat shock: a subsidiary role for heat-shock protein 70 in antigen processing.

The expression of heat-shock proteins (hsp) increases after exposure to various stresses including elevated temperatures, oxidative injury, infection and inflammation. As molecular chaperones, hsp have been shown to participate in antigen processing and presentation, in part through increasing the stability and expression of major histocompatibility complex molecules. Heat shock selectively increases human T-cell responses to processed antigen, but does not affect T-cell proliferation induced by non-processed antigens. Here, we have analysed the mechanisms by which stress such as heat shock, and the ensuing hsp over-expression affect the processing of diphtheria toxin (DT) in peripheral blood monocytes. We found that heat shock increased DT proteolysis in endosomes and lysosomes while the activities of the cathepsins B and D, classically involved in DT proteolysis, were decreased. These effects correlated with the heat-shock-mediated increase in hsp 70 expression observed in endosomes and lysosomes. Actinomycin D or blocking anti-hsp 70 antibodies abolished the heat-shock-mediated increase in DT proteolysis. These data indicate that the increased expression of hsp 70 constitutes a subsidiary mechanism that facilitates antigen proteolysis in stressed cells. Confirming these data, presentation by formaldehyde-fixed cells of DT proteolysates that were obtained with endosomes and lysosomes from heat-shocked peripheral blood monocytes showed higher stimulation of T cells than those generated with endosomes and lysosomes from control peripheral blood monocytes.

Laminin-5-integrin interaction signals through PI 3-kinase and Rac1b to promote assembly of adherens junctions in HT-29 cells.

Human intestinal cell differentiation is mediated by signaling pathways that remain largely undefined. We and others have shown that cell migration and differentiation along the crypt-villus axis is associated with temporal and spatial modulations of the repertoire, as well as with the function of integrins and E-cadherins and their substrates. Cross-talk between integrin and cadherin signaling was previously described and seems to coordinate this differentiation process. Here, we report that engagement of alpha6 and, to a lesser extent, alpha3 integrin subunits after HT-29 cell adhesion on laminin 5 increases the expression of E-cadherin, which then organizes into nascent adherens junctions. We further identify that phosphoinositide 3-kinase (PI 3-kinase) activation plays a key role in this cross-talk. Indeed, integrin-dependent adhesion on laminin 5 stimulates PI 3-kinase activity. Immunofluorescence and immunoprecipitation experiments revealed that activated PI 3-kinase is recruited at cell-cell contacts. Using LY294002, an inhibitor of PI 3-kinase activity, we found that this activation is essential for E-cadherin connection with the cytoskeleton and for biogenesis of adherens junctions. Finally, we demonstrated that PI 3-kinase could signal through Rac1b activation to control adherens junction assembly. Our results provide a mechanistic insight into integrin-cadherin cross-talk and identify a novel role for PI 3-kinase in the establishment of adherens junctions.