Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by the presence of extracellular plaques composed of amyloid β (Aβ) peptides and intracellular tau aggregates. The plaques are surrounded by microglia, the brain's resident immune cells, which likely participate in the clearance of Aβ by phagocytosis. The microglia that are associated with plaques display an abnormal ameboid morphology and do not respond to tissue damage, in contrast to microglia in healthy brains. Here, we used time lapse confocal microscopy to perform a detailed real-time examination of microglial motility in acute hippocampal brain slices from the 5xFAD mouse model of AD, which was crossed to Cx3cr1(GFP/GFP) mice to achieve microglia-specific GFP expression for visualization. During baseline conditions, microglia around plaques appeared hypermotile, moving the processes that were pointing away from plaques at higher speed than microglia not associated with plaques. Yet, neither plaque-associated, nor plaque-free microglia were able to extend processes toward sites of modest mechanical damage. Application of the selective adenosine A2A receptor antagonist preladenant, which restores microglial response to cellular damage in a mouse model of Parkinson's disease, reduced the hypermotility of plaque-associated microglia, but did not restore motility toward damaged cells in slices from 5xFAD mice. Our results suggest that process hypermotility and resistance to A2A antagonism during response to tissue damage may represent unique functional phenotypes of plaque-associated microglia that impair their ability to function properly in the AD brain.

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.

The glomeruli are the first synaptic relay on the olfactory pathway and play a basic role in smell perception. Glomerular degeneration occurs in humans with age and in Alzheimer's disease (AD). The glomeruli heavily express β-amyloid precursor protein (APP), β-secretase (BACE1) and γ-secretase complex. However, extracellular β-amyloid peptide (Aβ) deposition occurs fairly rarely at this location in postmortem pathological studies. We sought to explore age-related glomerular changes that might link to alteration in amyloidogenic proteins and/or plaque pathogenesis in transgenic models of AD and humans. Focally increased BACE1 immunoreactivity (IR) in the glomerular layer was identified in several transgenic models, and characterized systematically in transgenic mice harboring five familiar AD-related mutations (5XFAD). These elements were co-labeled with antibodies against APP N-terminal (22C11) and Aβ N-terminal (3D6, 6E10) and mid-sequence (4G8). They were not co-labeled with two Aβ C-terminal antibodies (Ter40, Ter42), nor associated with extracellular amyloidosis. These profiles were further characterized to be most likely abnormal olfactory nerve terminals. Reduced glomerular area was detected in 6-12-month-old 5XFAD mice relative to non-transgenic controls, and in aged humans relative to young/adult controls, more robust in AD than aged subjects without cerebral amyloid and tau pathologies. The results suggest that olfactory nerve terminals may undergo age-related dystrophic and degenerative changes in AD model mice and humans, which are associated with increased labeling for amyloidogenic proteins but not local extracellular Aβ deposition. The identified axon terminal pathology might affect neuronal signal transmission and integration at the first olfactory synaptic relay.

We have utilised laser confocal microscopy to categorise beta-amyloid plaque types that are associated with preclinical and end-stage Alzheimer's disease and to define the neurochemistry of dystrophic neurites associated with various forms of plaques. Plaques with a spherical profile were defined as either diffuse, fibrillar or dense-cored using Thioflavin S staining or immunolabelling for beta-amyloid. Confocal analysis demonstrated that fibrillar plaques had a central mass of beta-amyloid with compact spoke-like extensions leading to a confluent outer rim. Dense-cored plaques had a compacted central mass surrounded by an outer sphere of beta-amyloid. Diffuse plaques lacked a morphologically identifiable substructure, resembling a ball of homogeneous labelling. The relative proportion of diffuse, fibrillar and dense-cored plaques was 53, 22 and 25% in preclinical and 31, 49 and 20% in end-stage Alzheimer's disease cases, respectively. Plaque-associated dystrophic neurites in preclinical cases were immunolabeled for neurofilament proteins whereas, in end-stage cases, these abnormal neurites were variably labelled for tau and/or neurofilaments. Double labelling demonstrated that the proportion of diffuse, fibrillar and dense-cored plaques that were neuritic was 12, 47 and 82% and 24, 82 and 76% in preclinical and end-stage cases, respectively. Most dystrophic neurites in Alzheimer's disease cases were labelled for either neurofilaments or tau, however, confocal analysis determined that 30% of neurofilament-labelled bulb-like or elongated neurites had a core of tau immunoreactivity. These results indicate that all morphologically defined beta-amyloid plaque variants were present in both early and late stages of Alzheimer's disease. However, progression to clinical dementia was associated with both a shift to a higher proportion of fibrillar plaques that induced local neuritic alterations and a transformation of cytoskeletal proteins within associated abnormal neuronal processes. There data indicate key pathological changes that may be subject to therapeutic intervention to slow the progression of Alzheimer's disease.

In Alzheimer's disease and related dementias, amyloid beta (Aβ) and amyloid plaques can disrupt long-term synaptic plasticity, learning and memory and cognitive function. Plaque accumulation can disrupt corticocortical circuitry leading to abnormalities in sensory, motor, and cognitive processing. In this study, using 5xFAD (five Familial Alzheimer's Disease - FAD - mutations) mice, we evaluated amyloid plaque formation in different cortical areas, and whether differential amyloid accumulation across cortical fields correlates with changes in dendritic complexity of layer 3 corticocortical projection neurons and functional responses in the primary somatosensory cortex following whisker stimulation. We focused on three cortical areas: the primary somatosensory cortex (S1), the primary motor cortex (M1), and the prefrontal cortex (PFC including the anterior cingulate, prelimbic, and infralimbic subdivisions). We found that Aβ and amyloid plaque accumulation is not uniform across 5xFAD cortical areas, while there is no expression in littermate controls. We also found that there are differential layer 3 pyramidal cell dendritic complexity changes across the three areas in 5xFAD mice, compared to same age controls, with no apparent relation to differential amyloid accumulation. We used voltage-sensitive dye imaging (VSDi) to visualize neural activity in S1, M1 and PFC following whisker activation. Control mice show normal physiological responses in all three cortical areas, whereas 5xFAD mice only display physiological responses in S1. Taken together our results show that 5xFAD mutation affects the overall dendritic morphology of layer 3 pyramidal cells across sensory-motor and association cortex irrespective of the density and distribution of the Aβ amyloid proteins. Corticocortical circuitry between the sensory and motor/association areas is most likely disrupted in 5xFAD mice as cortical responses to whisker stimulation are altered.

In Alzheimer's disease (AD), numerous β-amyloid (Aβ) plaques are associated with butyrylcholinesterase (BChE) activity, the significance of which is unclear. A mouse model, containing five human familial AD genes (5XFAD), also develops Aβ plaques with BChE activity. Knock-out of BChE in this model showed diminished fibrillar Aβ plaque deposition, more so in males than females. This suggests that lack of BChE reduces deposition of fibrillar Aβ in AD and this effect may be influenced by sex.

Several studies reveal that the beneficial effects of exercise interventions are dependent on the progression of Alzheimer's disease (AD). We have previously shown that long-term treadmill exercise begun before the onset of β-amyloid (Aβ) pathology prevents the deficits of cognition and long-term potentiation (LTP) in amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mice (8 months of age) paralleled by the reduction of soluble Aβ levels and Aβ deposition in the hippocampus. In the present study, treadmill exercise was initiated at a developed Aβ deposition stage in order to further investigate whether or not treadmill exercise in this phase can delay the progression of AD in aged APP/PS1 mice (17 months of age). Our results show that 5-month treadmill exercise ameliorates the impairment of spatial learning and memory with age paralleled by synaptic plasticity enhancement in aged APP/PS1 mice. In addition, exercise-induced enhancement of synaptic plasticity was accompanied by a significant reduction of soluble Aβ levels rather than Aβ plaque deposition. Therefore, the investigation demonstrates that long-term treadmill exercise has beneficial effects on cognition and synaptic plasticity even when the brain has developed Aβ deposition, and changes in soluble Aβ levels rather than Aβ plaque deposition may contribute to exercise-induced benefits.

In this study, we have mapped amyloid beta (Abeta) deposition in the amygdala of five aged Japanese monkeys (from 23 to 30 years old). In brief, the aged monkey amygdala shows a topographic distribution of Abeta deposits that is subnucleus specific and exhibits a distinct temporal progression. The pattern is similar to the distribution of Abeta deposits in the human amygdala of Alzheimer's patients and of high plaque nondemented cases. The spatial distribution and temporal progression were correlated with the distribution of free zinc (Zn), which is known to mediate Abeta aggregation. For the basolateral group of subnuclei in particular, there is a clear dorsoventral gradient in the progressive distribution of Abeta. Abeta depositions first appear in the ventral division of the lateral nucleus and parvicellular division of the accessory basal nucleus, and then extend into the ventral part of the basal and paralaminar nuclei. All these nuclei are also Zn-dense. Conversely, Zn-weak nuclei, which are more dorsally situated (i.e. dorsal division of lateral nucleus and magnocellular division of basal nucleus) showed only a low level of Abeta deposits, even in brains with the greatest Abeta burden. In contrast to the basolateral group, the central and medial nuclei and cortical group had Abeta deposits only at later stages. In the central and medial nuclei, we identified a lateromedial gradient of Abeta deposits, again similar to the gradient of Zn-distribution. In the cortical group, Abeta deposits are densest in the deep layer, where Zn is also densest. Thus, we suggest the macaque amygdala, with its clear topographic distribution of Abeta deposits, may be an effective model for examining the complex mechanisms of vulnerability to Abeta deposits. A primate model would be advantageous for experimental interventions geared toward therapeutic protection from Alzheimer's disease, including by microarray analysis and genetic manipulation.

Administration of non-steroidal anti-inflammatory agents reduces the risk of developing Alzheimer's disease in normal aging populations, an effect that may occur from inhibition of the cyclooxygenases, the rate-limiting enzymes in the formation of prostaglandins. In this study, we investigated whether increased activity of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, potentiates disease progression in a transgenic mouse model of Alzheimer's disease. To study the functional effects of COX-2 activity, male and female bigenic mice (amyloid precursor protein with Swedish mutation [APPswe]-presenilin-1 protein with deletion of exon 9 [PS1dE9] and trigenic COX-2/APPswe-PS1dE9) were behaviorally tested +/-administration of the selective COX-2 inhibitor celecoxib. Behavioral testing included a three-trial Y maze that measures spatial working and recognition memories and an open field task that tested levels of hyperactivity. Overexpression of COX-2 in APPswe-PS1dE9 mice resulted in specific deficits in spatial working memory in female but not male mice. These sex-specific deficits were abolished by pharmacological inhibition of COX-2 activity. Importantly, COX-2-associated deficits were dependent on co-expression of all three transgenes since COX-2 single transgenic and APPswe-PS1dE9 bigenic mice showed normal memory. Quantification of amyloid plaque load and total Abeta 40 and 42 peptides did not reveal significant differences in trigenic versus bigenic mice treated with either vehicle or celecoxib. Taken together, these data indicate an interaction between the effects of COX-2 and Abeta peptides on cognition that occurs in a sex-specific manner in the absence of significant changes in amyloid burden. These findings suggest that pathological activation of COX-2 may potentiate the toxicity of Abeta peptides, particularly in females, without significantly affecting Abeta accumulation.

Presenilin 1 (PS1), a causative molecule of familial Alzheimer's disease (AD), is known to be an unprimed substrate of glycogen synthase kinase 3 β (GSK3β) [Twomey and McCarthy (2006) FEBS Lett 580:4015-4020] and is phosphorylated at serine 353, 357 residues in its cytoplasmic loop region [Kirschenbaum et al. (2001) J Biol Chem 276:7366-7375]. In this report, we investigated the effect of PS1 phosphorylation on AD pathophysiology and obtained two important results--PS1 phosphorylation increased amyloid β (Aβ) 42/40 ratio, and PS1 phosphorylation was enhanced in the human AD brains. Interestingly, we demonstrated that PS1 phosphorylation promoted insulin receptor (IR) cleavage and the IR intracellular domain (IR ICD) generated by γ-secretase led to a marked transactivation of Akt (PKB), which down-regulated GSK3β activity. Thus, the cleavage of IR by γ-secretase can inhibit PS1 phosphorylation in the long run. Taken together, our findings indicate that PS1 phosphorylation at serine 353, 357 residues can play a pivotal role in the pathology of AD and that the dysregulation of this mechanism may be causally associated with its pathology.

Multiple cellular systems exist to prevent uncontrolled inflammation in brain tissues; the suppressor of cytokine signaling (SOCS) proteins have key roles in these processes. SOCS proteins are involved in restricting cellular signaling pathways by enhancing the degradation of activated receptors and removing the stimuli for continued activation. There are eight separate SOCS genes that code for proteins with similar structures and properties. All SOCS proteins can reduce signaling of activated transcription factors Janus kinase (JAK) and signal transducer and activator of transcription (STAT), but they also regulate many other signaling pathways. SOCS-1 and SOCS-3 have particular roles in regulating inflammatory processes. Chronic inflammation is a key feature of the pathology present in Alzheimer's disease (AD)-affected brains resulting from responses to amyloid plaques or neurofibrillary tangles, the pathological hallmarks of AD. The goal of this study was to examine SOCS gene expression in human non-demented (ND) and AD brains and in human brain-derived microglia to determine if AD-related pathology resulted in a deficit of these critical molecules. We demonstrated that SOCS-1, SOCS-2, SOCS-3 and cytokine-inducible SH2 containing protein (CIS) mRNA expression was increased in amyloid beta peptide (Aβ)- and inflammatory-stimulated microglia, while SOCS-6 mRNA expression was decreased by both types of treatments. Using human brain samples from the temporal cortex from ND and AD cases, SOCS-1 through SOCS-7 and CIS mRNA and SOCS-1 through SOCS-7 protein could be detected constitutively in ND and AD human brain samples. Although, the expression of key SOCS genes did not change to a large extent as a result of AD pathology, there were significantly increased levels of SOCS-2, SOCS-3 and CIS mRNA and increased protein levels of SOCS-4 and SOCS-7 in AD brains. In summary, there was no evidence of a deficit of these key inflammatory regulating proteins in aged or AD brains.

Sleep disturbances are a common early symptom of neurodegenerative diseases, including Alzheimer's disease (AD) and other age-related dementias, and emerging evidence suggests that poor sleep may be an important contributor to development of amyloid pathology. Of the causes of sleep disturbances, it is estimated that 10-20% of adults in the United States have sleep-disordered breathing (SDB) disorder, with obstructive sleep apnea accounting for the majority of the SBD cases. The clinical and epidemiological data clearly support a link between sleep apnea and AD; yet, almost no experimental research is available exploring the mechanisms associated with this correlative link. Therefore, we exposed an AD-relevant mouse model (APP/PS1 KI) to chronic intermittent hypoxia (IH) (an experimental model of sleep apnea) to begin to describe one of the potential mechanisms by which SDB could increase the risk of dementia. Previous studies have found that astrogliosis is a contributor to neuropathology in models of chronic IH and AD; therefore, we hypothesized that a reactive astrocyte response might be a contributing mechanism in the neuroinflammation associated with sleep apnea. To test this hypothesis, 10-11-month-old wild-type (WT) and APP/PS1 KI mice were exposed to 10 hours of IH, daily for four weeks. At the end of four weeks brains were analyzed from amyloid burden and astrogliosis. No effect was found for chronic IH exposure on amyloid-beta levels or plaque load in the APP/PS1 KI mice. A significant increase in GFAP staining was found in the APP/PS1 KI mice following chronic IH exposure, but not in the WT mice. Profiling of genes associated with different phenotypes of astrocyte activation identified GFAP, CXCL10, and Ggta1 as significant responses activated in the APP/PS1 KI mice exposed to chronic IH.

β-Site APP-cleaving enzyme 1 (BACE1) initiates the generation of amyloid-β (Aβ), thus representing a prime therapeutic target for Alzheimer's disease (AD). Previous work including ours has used BACE1 haploinsufficiency (BACE1(+/-); i.e., 50% reduction) as a therapeutic relevant model to evaluate the efficacy of partial β-secretase inhibition. However, it is unclear whether the extent of Aβ reductions in amyloid precursor protein (APP) transgenic mice with BACE1(+/-) gene ablation may vary with sex or disease progression. Here, we compared the impacts of BACE1 haploinsufficiency on Aβ concentrations and APP processing in 5XFAD Alzheimer mice (1) between males and females and (2) between different stages with moderate and robust Aβ accumulation. First, male and female 5XFAD mice at 6-7 months of age showed equivalent levels of Aβ, BACE1, full-length APP and its metabolites. BACE1 haploinsufficiency significantly lowered soluble Aβ oligomers, total Aβ42 levels and plaque burden in 5XFAD mouse brains irrespective of sex. Furthermore, there was no sex difference in reductions of β-cleavage products of APP (C99 and sAPPβ) found in BACE1(+/-)·5XFAD mice relative to BACE1(+/+)·5XFAD controls. Meanwhile, APP and sAPPα levels in BACE1(+/-)·5XFAD mice were higher than those of 5XFAD controls regardless of sex. Based on these observations, we next combined male and female data to examine the effects of BACE1 haploinsufficiency in 5XFAD mice at 12-14 months of age, as compared with those in 6-7-month-old 5XFAD mice. Oligomeric Aβ and C99 levels were dramatically elevated in older 5XFAD mice. Although the β-metabolites of APP were significantly reduced by BACE1 haploinsufficiency in both age groups, high levels of these toxic amyloidogenic fragments remained in 12-14-month-old BACE1(+/-)·5XFAD mice. The present findings are consistent with our previous behavioral data showing that BACE1 haploinsufficiency rescues memory deficits in 5XFAD mice irrespective of sex but only in the younger age group.

Soluble forms of amyloid-beta (Abeta) have been considered responsible for cognitive dysfunction prior to senile plaque formation in Alzheimer's disease (AD). As its mechanism is not well understood, we examined the effects of repeated i.c.v. infusion of soluble Alphabeta(25-35) on peptidergic system and glial cells in the pathogenesis of AD. The present study aims to investigate the protective effects of memantine on Abeta(25-35)-induced changes in peptidergic and glial systems. Infusion of Alphabeta(25-35) decreased the level of immunoreactive somatostatin (SS) and substance P (SP) in the hippocampus prior to neuronal loss or caspase activation, which is correlated with the loss of spine density and activation of inducible nitric-oxide synthase (iNOS). Biochemical experiment with peptide-degrading enzymes, prolyl oligopeptidase (POP) and endopeptidase 24.15 (EP 24.15) activities demonstrated a concomitant increase with the activation of glial marker proteins, glial fibrillary acidic protein (GFAP) and CD11b in the Abeta-treated hippocampus. Double immunostaining experiments of EP 24.15 and GFAP/CD11b antibodies clearly demonstrated the co-localization of neuro peptidases with astrocytes and microglia. Treatment with memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist significantly attenuated Abeta(25-35)-induced changes of neuropeptides, their metabolizing enzymes, glial marker proteins, and activation of iNOS. Taken together, the data implies that memantine exerts its protective effects by modulating the neuropeptide system as a consequence of suppressing the glial cells and oxidative stress in AD model rat brain regions.

RanBP9 is a multi-domain scaffolding protein known to integrate extracellular signaling with intracellular targets. We previously demonstrated that RanBP9 enhances Aβ generation and amyloid plaque burden which results in loss of specific pre- and postsynaptic proteins in vivo in a transgenic mouse model. Additionally, we showed that the levels of spinophilin, a marker of dendritic spines were inversely proportional to the RanBP9 protein levels within the synaptosomes isolated from AD brains. In the present study, we found reduced dendritic intersections within the layer 6 pyramidal neurons of the cortex as well as the hippocampus of RanBP9 transgenic mice compared to age-matched wild-type (WT) controls at 12 months of age but not at 6months. Similarly, the dendritic spine numbers were reduced in the cortex at only 12 months of age by 30% (p<0.01), but not at 6months. In the hippocampus also the spine densities were reduced at 12 months of age (38%, p<0.01) in the RanBP9 transgenic mice. Interestingly, the levels of phosphorylated form of cofilin, an actin binding protein that plays crucial role in the regulation of spine numbers were significantly decreased in the cortical synaptosomes at only 12months of age by 26% (p<0.01). In the hippocampal synaptosomes, the decrease in cofilin levels were 36% (p<0.01) at 12 months of age. Thus dendritic arbor and spine density were directly correlated to the levels of phosphorylated form of cofilin in the RanBP9 transgenic mice. Similarly, cortical synaptosomes showed a 20% (p<0.01) reduction in the levels of spinophilin in the RanBP9 transgenic mice. These results provided the physical basis for the loss of synaptic proteins by RanBP9 and most importantly it also explains the impaired spatial learning and memory skills previously observed in the RanBP9 transgenic mice.

Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.