Intracellular Aβ Research Articles

Chronic cerebral hypoperfusion induces memory deficits and facilitates Aβ generation in C57BL/6J mice

Alzheimer's disease (AD) is the most common type of dementia frequently responsible for cognitive decline in the elderly. The etiology and molecular mechanism of AD pathogenesis remain inconclusive. Aging and vascular factors are important independent causes and contributors to sporadic AD. Clinical imaging studies showed that cerebral blood flow decreases before cognitive impairment in patients with AD. To investigate the effect of chronic cerebral hypoperfusion (CCH) on cognitive impairment and morphological features, we developed a new manner of CCH mouse model by narrowing bilateral common carotid arteries. Mice started to manifest spatial memory deficits 1month after the surgery and exhibited behavioral changes in a time-dependent manner. Mice also presented memory deficits accompanied with morphological changes at the neuronal and synaptic levels. CCH damaged the normal neuronal morphology and significantly reduced the expression level of PSD95. CCH activated astrocytes, increased the co-expression of GFAP and AQP4, and destroyed the blood–brain barrier (BBB). Furthermore, CCH facilitated intracellular and extracellular Aβ deposition by up-regulating γ-secretase and β-secretase levels. Our results showed good reproducibility of post-CCH pathological processes, which are characterized by neuronal apoptosis, axonal abnormalities, glial activation, BBB damage, amyloid deposition, and cognitive dysfunction; these processes may be used to decipher the complex interplay and pathological process between CCH and AD. This study provides laboratory evidence for the prevention and treatment of cognitive malfunction and AD.

Chemical modifications of amyloid-β(1-42) have a significant impact on the repertoire of brain amyloid-β(1-42) binding proteins.

The amyloid-β peptide(1-42) (Aβ) is a key player in the development and progression of Alzheimer's disease (AD). Although much attention is paid to its role in formation of extracellular amyloid plaques and protein aggregates as well as to corresponding mechanisms of their toxicity, good evidence exists that intracellular Aβ can accumulate intraneuronally and interact with intracellular target proteins. However, intracellular Aβ binding proteins as well as conditions favoring their interactions with Aβ are poorly characterized. In this study we have investigated the effect of two known pathogenic Aβ modifications, isomerization of Asp7 and phosphorylation of Ser8, on the proteomic profiles of mouse brain Aβ binding proteins. Phosphorylation of Ser8 and especially isomerization of Asp7 significantly extended the repertoire of mouse brain Aβ binding proteins. However, there were 61 proteins, common for three types of the affinity ligands. They obviously represent potential targets for direct interaction with all Aβ species. Taking into consideration spontaneous mode of Asp7 isomerization and reports on initial accumulation of phosphorylated Aβ species inside neurons it is reasonable to suggest that these modifications of intracellular Aβ peptides causing the significant increase in the repertoire of Aβ binding proteins represent a primary pathogenic effect that precedes formation of extracellular pathogenic oligomerization/aggregation of Aβ peptides well described in the literature.

Amyloid proteotoxicity initiates an inflammatory response blocked by cannabinoids

The beta amyloid (Aβ) and other aggregating proteins in the brain increase with age and are frequently found within neurons. The mechanistic relationship between intracellular amyloid, aging and neurodegeneration is not, however, well understood. We use a proteotoxicity model based upon the inducible expression of Aβ in a human central nervous system nerve cell line to characterize a distinct form of nerve cell death caused by intracellular Aβ. It is shown that intracellular Aβ initiates a toxic inflammatory response leading to the cell's demise. Aβ induces the expression of multiple proinflammatory genes and an increase in both arachidonic acid and eicosanoids, including prostaglandins that are neuroprotective and leukotrienes that potentiate death. Cannabinoids such as tetrahydrocannabinol stimulate the removal of intraneuronal Aβ, block the inflammatory response, and are protective. Altogether these data show that there is a complex and likely autocatalytic inflammatory response within nerve cells caused by the accumulation of intracellular Aβ, and that this early form of proteotoxicity can be blocked by the activation of cannabinoid receptors.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Highly potent intracellular membrane-associated Aβ seeds.

An early event in Alzheimer’s disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ.

Reelin-immunoreactive neurons in entorhinal cortex layer II selectively express intracellular amyloid in early Alzheimer's disease

The onset of Alzheimer's disease (AD) is associated with subtle pathological changes including increased intracellular expression of amyloid-β (Aβ). A structure affected particularly early in the course of AD is the entorhinal cortex, where neuronal death in layer II is observed already at initial stages. Neurons in EC-layer II, particularly those that express the protein Reelin, give rise to projections to the hippocampal dentate gyrus and this projection shows severe loss of synaptic contacts during early-stage AD. Given this anatomical specificity, we sought to determine whether increased intracellular expression of Aβ is selectively associated with Reelin-immunoreactive neurons in layer II of the entorhinal cortex. Here we report that in a transgenic rat model, which mimics the onset and distribution of extracellular amyloid deposits seen in human AD subjects, expression of intracellular Aβ in entorhinal layer II selectively occurs in Reelin-immunoreactive neurons during the early, pre-plaque stage. This Reelin-Aβ association is also present in human subjects with AD-related pathological changes, even in early disease stages. These findings strongly indicate that Reelin-immunoreactive neurons in entorhinal layer II play a crucial role during the initial stages of AD, and may therefore lead to refined hypotheses concerning the origin of this devastating condition.

Intracellular Aβ and its Pathological Role in Alzheimer's Disease: Lessons from Cellular to Animal Models.

Accumulation of intraneuronal amyloid-β peptide (Aβ) appears to be an early event in Alzheimer's disease (AD), suggesting its important role in the neurodegenerative process of AD. It is indicated that intracellular Aβ originates from a portion of Aβ, which is not secreted and consequently remains intracellular, or alternatively from the secreted Aβ, which is internalized into intracellular Aβ pool. A number of cell and transgenic animal models are established to study the pathological role of intracellular Aβ, and to screen for drugs against Aβ aggregation and associated toxicity. Aβ aggregates, particularly oligomers, may lead to synaptic dysfunction and neuronal loss. Screened from high-throughput methods, a number of cellpermeable agents reduce the aggregation of intracellular Aβ and antagonize its cytotoxicity by inhibiting the formation of Aβ oligomers in vivo. The multi-functional roles of Aβ in alternate pathways and associated clinical implications for AD treatment are also discussed.

Characterization of Amyloid-β Deposits in Bovine Brains.

Amyloid-β (Aβ) deposits are seen in aged individuals of many mammalian species that possess the same aminoacid sequence as humans. This study describes Aβ deposition in 102 clinically characterized cattle brains from animals aged 0 to 20 years. Extracellular and intracellular Aβ deposition was detected with 4G8 antibody in the cortex, hippocampus, and cerebellum. X-34 staining failed to stain Aβ deposits, indicating the non β-pleated nature of these deposits. Western blot analysis and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry revealed in Tris, Triton, and formic acid fractions the presence of different Aβ peptides, characterized mainly by C-terminally truncated forms. Exploration of the genetic variability of APOE, PSEN1, and PSEN2 genes involved in Alzheimer’s disease pathogenesis revealed several previously unreported polymorphisms. This study demonstrates certain similarities between Aβ deposition patterns exhibited in cattle brains and those in the human brain in early stages of aging. Furthermore, the identification of the same Aβ peptides reported in humans, but unable to form aggregates, supports the hypothesis that cattle may be protected against amyloid plaque formation.

Complex interplay between brain function and structure during cerebral amyloidosis in APP transgenic mouse strains revealed by multi-parametric MRI comparison

Alzheimer's disease is a fatal neurodegenerative disorder affecting the aging population. Neuroimaging methods, in particular magnetic resonance imaging (MRI), have helped reveal alterations in the brain structure, metabolism, and function of patients and in groups at risk of developing AD, yet the nature of these alterations is poorly understood. Neuroimaging in mice is attractive for investigating mechanisms underlying functional and structural changes associated with AD pathology. Several preclinical murine models of AD have been generated based on transgenic insertion of human mutated APP genes. Depending on the specific mutations, mouse strains express different aspects of amyloid pathology, e.g. intracellular amyloid-β (Aβ) aggregates, parenchymal plaques, or cerebral amyloid angiopathy. We have applied multi-parametric MRI in three transgenic mouse lines to compare changes in brain function with resting-state fMRI and structure with diffusion tensor imaging and high resolution anatomical imaging. E22ΔAβ developing intracellular Aβ aggregates did not present functional or structural alterations compared to their wild-type littermates. PSAPP mice displaying parenchymal amyloid plaques displayed mild functional changes within the supplementary and barrel field cortices, and increased isocortical volume relative to controls. Extensive reduction in functional connectivity in the sensory-motor cortices and within the default mode network, as well as local volume increase in the midbrain relative to wild-type have been observed in ArcAβ mice bearing intracellular Aβ aggregates as well as parenchymal and vascular amyloid deposits. Patterns of functional and structural changes appear to be strain-specific and not directly related to amyloid deposition.

Learning impairment by minimal cortical injury in a mouse model of Alzheimer׳s disease

Brain injury accelerates amyloid-β (Aβ) deposits and exacerbates Alzheimer׳s disease (AD). Accumulation of intracellular soluble Aβ impairs cognition prior to emergence of Aβ plaques. However, it is not known whether brain injury affects learning impairment attributable to intracellular soluble Aβ. We made a small injury by injecting glutamate into the parietal cortex in 3xTg AD mice of 4–5 months old, at which age soluble Aβ is accumulated without Aβ deposits. The size of glutamate-induced lesion was significantly larger than that of saline-injected control lesion. We reduced the relative difficulty of Morris water maze (MWM) task by repeating it twice, so that saline-injected 3xTg mice could perform as well as wild-type control mice. Under this condition, glutamate-injected 3xTg mice exhibited learning deficits. DNA microarray analysis revealed that 3 genes are upregulated, with one gene downregulated, more than 2 folds in the hippocampus. These 4 genes do not appear to be involved directly in learning but may be a part of signal cascade triggered by glutamate-induced small injury. Hippocampal content of soluble Aβ1-42 was increased in the glutamate 3xTg group. Facilitation of large-conductance calcium-activated potassium (BK) channel accompanied learning recovery in the saline-control 3xTg group in agreement with our previous reports, in which learning deficits attributable to intracellular Aβ were alleviated by facilitating BK channels. However, BK channel remained suppressed in the glutamate 3xTg group. It is suggested that glutamate-induced injury worsens learning by enhancing the toxicity of soluble Aβ or increasing its content per se.

Endogenous Apolipoprotein E (ApoE) Fragmentation Is Linked to Amyloid Pathology in Transgenic Mouse Models of Alzheimer's Disease.

The epsilon 4 allele of the apolipoprotein E (ApoE4) gene is the most important risk factor implicated in Alzheimer's disease (AD) etiology. ApoE4 is more susceptible to proteolysis, and ApoE fragments have been shown to promote tau hyperphosphorylation and exert neurotoxic properties. While a plethora of studies deals with the effect of ApoE and its fragments on amyloid-β peptide (Aβ) deposition and clearance, it is largely unknown whether Aβ in turn influences human or murine ApoE expression and its proteolysis. The present study is the first to show that endogenous murine ApoE becomes proteolytically processed in a way reminiscent of human ApoE fragmentation in different AD mouse models, including APP/PS1KI or 5XFAD. Murine ApoE fragments were demonstrated to accumulate mainly in synaptic fractions in AD mouse models. In vitro experiments, as well as analysis of mouse models at different time points, suggest that the amount of total ApoE is associated with extracellular Aβ while the amount of its fragments is linked to intracellular Aβ levels. Murine ApoE fragmentation is a common feature in different AD transgenic mouse models and could be directly associated with intraneuronal Aβ accumulation. Extracellular amyloid induces an elevation in full-length ApoE expression, which might present a protective mechanism toward Aβ clearance. The demonstrated fragments of murine ApoE in vitro and in vivo might therefore play a crucial role in the progression of AD pathology in murine AD models.

Hydrogen Sulfide Selectively Inhibits γ-Secretase Activity and Decreases Mitochondrial Aβ Production in Neurons from APP/PS1 Transgenic Mice.

Hydrogen sulfide (H2S) is now considered to be a gasotransmitter and may be involved in the pathological process of Alzheimer's disease (AD). A majority of APP is associated with mitochondria and is a substrate for the mitochondrial γ-secretase. The mitochondria-associated APP metabolism where APP intracellular domains (AICD) and Aβ are generated locally and may contribute to mitochondrial dysfunction in AD. Here, we aimed to investigate the ability of H2S to mediate APP processing in mitochondria and assessed the possible mechanisms underlying H2S-mediated AD development. We treated neurons from APP/PS1 transgenic mice with a range of sodium hydrosulfide (NaHS) concentrations. NaHS attenuated APP processing and decreased Aβ production in mitochondria. Meanwhile, NaHS did not changed BACE-1 and ADAM10 (a disintegrin and metalloprotease 10) protein levels, but NaHS (30μM) significantly increased the levels of presenilin 1(PS1), PEN-2, and NCT, as well as improved the γ-secretase activity, while NaHS (50μM) exhibits the opposing effects. Furthermore, the intracellular ATP and the COX IV activity of APP/PS1 neurons were increased after 30μM NaHS treatment, while the ROS level was decreased and the MMP was stabilized. The effect of NaHS differs from DAPT (a non-selective γ-secretase inhibitor), and it selectively inhibited γ-secretase in vitro, without interacting with Notch and modulating its cleavage. The results indicated that NaHS decreases Aβ accumulation in mitochondria by selectively inhibiting γ-secretase. Thus, we provide a mechanistic view of NaHS is a potential anti-AD drug candidate and it may decrease Aβ deposition in mitochondria by selectively inhibiting γ-secretase activity and therefore protecting the mitochondrial function during AD conditions.

Neuroprotective Effects of Icariin on Brain Metabolism, Mitochondrial Functions, and Cognition in Triple-Transgenic Alzheimer's Disease Mice.

This study investigated the neuroprotective properties of icariin (an effective component of traditional Chinese herbal medicine Epimedium) on neuronal function and brain energy metabolism maintenance in a triple-transgenic mouse model of Alzheimer's disease (3 × Tg-AD). 3 × Tg-AD mice as well as primary neurons were subjected to icariin treatment. Morris water maze assay, magnetic resonance spectroscopy (MRS), Western blotting, ELISA, and immunohistochemistry analysis were used to evaluate the effects of icariin administration. Icariin significantly improved spatial learning and memory retention in 3 × Tg-AD mice, promoted neuronal cell activity as identified by the enhancement of brain metabolite N-acetylaspartate level and ATP production in AD mice, preserved the expressions of mitochondrial key enzymes COX IV, PDHE1α, and synaptic protein PSD95, reduced Aβ plaque deposition in the cortex and hippocampus of AD mice, and inhibited β-site APP cleavage enzyme 1 (BACE1) expression. Icariin treatment also decreased the levels of extracellular and intracellular Aβ1-42 in 3 × Tg-AD primary neurons, modulated the distribution of Aβ along the neurites, and protected against mitochondrial fragmentation in 3 × Tg-AD neurons. Icariin shows neuroprotective effects in 3 × Tg-AD mice and may be a promising multitarget drug in the prevention/protection against AD.

Cognitive Improvement by Photic Stimulation in a Mouse Model of Alzheimer's Disease.

We previously reported that activity of the large conductance calcium-activated potassium (big-K, BK) channel is suppressed by intracellular Aβ in cortical pyramidal cells, and that this suppression was reversed by expression of the scaffold protein Homer1a in 3xTg Alzheimer's disease model mice. Homer1a is known to be expressed by physiological photic stimulation (PS) as well. The possibility thus arises that PS also reverses Aβ-induced suppression of BK channels, and thereby improves cognition in 3xTg mice. This possibility was tested here. Chronic application of 6-hour-long PS (frequency, 2 Hz; duty cycle, about 1/10; luminance, 300 lx) daily for 4 weeks improved contextual and tone-dependent fear memory in 3xTg mice and, to a lesser extent, Morris water maze performance as well. Hippocampal long-term potentiation was also enhanced after PS. BK channel activity in cingulate cortex pyramidal cells and lateral amygdalar principal cells, suppressed in 3xTg mice, were facilitated. In parallel, neuronal excitability, elevated in 3xTg mice, was recovered to the control level. Gene expression of BK channel, as well as that of the scaffold protein Homer1a, was found decreased in 3xTg mice and reversed by PS. It is known that Homer1a is an activity-dependently inducible immediate early gene product. Consistently, our previous findings showed that Homer1a induced by electrical stimulation facilitated BK channels. By using Homer1a knockouts, we showed that the present PS-induced BK channel facilitation is mediated by Homer1a expression. We thus propose that PS might be potentially useful as a non-invasive therapeutic measure against Alzheimer's disease.

β-amyloidopathy in the Pathogenesis of Age-Related Macular Degeneration in Correlation with Neurodegenerative Diseases.

Involvement of new biotechnology and genetic engineering methods to the study of the aging organism allowed to select a group of neurodegenerative diseases (NDD) which have a similar mechanism of pathogenesis including pathological processes of protein aggregation and its deposition in the structures of nerve tissue. The development of eye and brain from one embryonic germ layer, community of ethiopathogenetic and morphological manifestations of age-related macular degeneration (AMD) and Alzheimer's disease (AD), a common pathway of β-amyloid precursor protein (APP) are associated with the pathological aggregation of fibrillar β-amyloid (Aβ) protein and the development of β-amyloidopathy in structural elements of the eye and the brain. The review demonstrates the keynote of AMD and AD pathogenesis is β-amyloidopathy that is a manifestation of proteinopathy leading to cytotoxicity, neurodegeneration and the development of pathological apoptosis activated by the formation of intracellular Aβ. This view on the problem predetermines the development of new strategies for the creating of ophthalmogeriatric and neuroprotective drugs affecting the pathogenesis and including all stages of Aβ formation and pathological aggregation.

RAGE mediated intracellular Aβ uptake contributes to the breakdown of tight junction in retinal pigment epithelium.

Intracellular amyloid beta (Aβ) has been implicated in neuronal cell death in Alzheimer's disease (AD). Intracellular Aβ also contributes to tight junction breakdown of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Although Aβ is predominantly secreted from neuronal cells, the mechanism of Aβ transport into RPE remains to be fully elucidated. In this study, we demonstrated that intracellular Aβ was found concomitantly with the breakdown of tight junction in RPE after subretinal injection of Aβ into the mouse eye. We also presented evidence that receptor for advanced glycation end products (RAGE) contributed to endocytosis of Aβ in RPE. siRNA-mediated knockdown of RAGE prevented intracellular Aβ accumulation as well as subsequent tight junction breakdown in RPE. In addition, we found that RAGE-mediated p38 MAPK signaling contributed to endocytosis of Aβ. Blockade of RAGE/p38 MAPK signaling inhibited Aβ endocytosis, thereby preventing tight junction breakdown in RPE. These results implicate that intracellular Aβ contributes to the breakdown of tight junction in RPE via the RAGE/p38 MAPK-mediated endocytosis. Thus, we suggest that RAGE could be a potential therapeutic target for intracellular Aβ induced outer BRB breakdown in AMD.

The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1–transfected Chinese hamster ovary cells

Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate–conjugated Aβ1–42 was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers.

Intracellular amyloid β oligomers impair organelle transport and induce dendritic spine loss in primary neurons.

IntroductionSynaptic dysfunction and intracellular transport defects are early events in Alzheimer’s disease (AD). Extracellular amyloid β (Aβ) oligomers cause spine alterations and impede the transport of proteins and organelles such as brain-derived neurotrophic factor (BDNF) and mitochondria that are required for synaptic function. Meanwhile, intraneuronal accumulation of Aβ precedes its extracellular deposition and is also associated with synaptic dysfunction in AD. However, the links between intracellular Aβ, spine alteration, and mechanisms that support synaptic maintenance such as organelle trafficking are poorly understood.ResultsWe compared the effects of wild-type and Osaka (E693Δ)-mutant amyloid precursor proteins: the former secretes Aβ into extracellular space and the latter accumulates Aβ oligomers within cells. First we investigated the effects of intracellular Aβ oligomers on dendritic spines in primary neurons and their tau-dependency using tau knockout neurons. We found that intracellular Aβ oligomers caused a reduction in mushroom, or mature spines, independently of tau. We also found that intracellular Aβ oligomers significantly impaired the intracellular transport of BDNF, mitochondria, and recycling endosomes: cargoes essential for synaptic maintenance. A reduction in BDNF transport by intracellular Aβ oligomers was also observed in tau knockout neurons.ConclusionsOur findings indicate that intracellular Aβ oligomers likely contribute to early synaptic pathology in AD and argue against the consensus that Aβ-induced spine loss and transport defects require tau.

Amyloid-β Oligomers May Impair SNARE-Mediated Exocytosis by Direct Binding to Syntaxin 1a.

Alzheimer's disease (AD) is closely associated with synaptic dysfunction, and thus current treatments often aim to stimulate neurotransmission to improve cognitive impairment. Whereas the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic transmission, the correlation between SNAREs and AD neuropathology is unknown. Here, we report that intracellular amyloid-β (Aβ) oligomers directly inhibit SNARE-mediated exocytosis by impairing SNARE complex formation. We observe abnormal reduction of SNARE complex levels in the brains of APP/PS1 transgenic (TG) mice compared to age-matched wild-types. We demonstrate that Aβ oligomers block SNARE complex assembly through the direct interaction with a target membrane (t)-SNARE syntaxin 1a invitro. Furthermore, the results of the invitro single-vesicle content-mixing assay reveal that Aβ oligomers inhibit SNARE-mediated fusion pores. Thus, our study identifies apotential molecular mechanism by which intracellular Aβ oligomers hamper SNARE-mediated exocytosis, likely leading to AD-associated synaptic dysfunctions.

Nilotinib and bosutinib modulate pre-plaque alterations of blood immune markers and neuro-inflammation in Alzheimer’s disease models

Alzheimer’s disease (AD) brains exhibit plaques and tangles in association with inflammation. The non-receptor tyrosine kinase Abl is linked to neuro-inflammation in AD. Abl inhibition by nilotinib or bosutinib facilitates amyloid clearance and may decrease inflammation. Transgenic mice that express Dutch, Iowa and Swedish APP mutations (TgAPP) and display progressive Aβ plaque deposition were treated with tyrosine kinase inhibitors (TKIs) to determine pre-plaque effects on systemic and CNS inflammation using milliplex® ELISA. Plaque Aβ was detected at 4months in TgAPP and pre-plaque intracellular Aβ accumulation (2.5months) was associated with changes of cytokines and chemokines prior to detection of glial changes. Plaque formation correlated with increased levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1α, IL-1β) and markers of immunosuppressive and adaptive immunity, including, IL-4, IL-10, IL-2, IL-3, Vascular Endothelial Growth Factor (VEGF) and IFN-γ. An inverse relationship of chemokines was observed as CCL2 and CCL5 were lower than WT mice at 2months and significantly increased after plaque appearance, while soluble CX3CL1 decreased. A change in glial profile was only robustly detected at 6months in Tg-APP mice and TKIs reduced astrocyte and dendritic cell number with no effects on microglia, suggesting alteration of brain immunity. Nilotinib decreased blood and brain cytokines and chemokines and increased CX3CL1. Bosutinib increased brain and blood IL-10 and CX3CL1, suggesting a protective role for soluble CX3CL1. Taken together these data suggest that TKIs regulate systemic and CNS immunity and may be useful treatments in early AD through dual effects on amyloid clearance and immune modulation.