Abstract
A synergy between β-amyloid (Aβ) and tau appears to occur in Alzheimer disease (AD), but the mechanisms of interaction, and potential locations, are little understood. This study ...investigates the possibility of such interactions within the cortical synaptic compartments of APP/PS1 mice. We used label-free quantitative mass spectrometry to study the phosphoproteome of synaptosomes, covering 2400 phosphopeptides and providing an unbiased survey of phosphorylation changes associated with amyloid pathology. Hyperphosphorylation was detected on 36 synaptic proteins, many of which are associated with the cytoskeleton. Importantly, tau is one of the most hyperphosphorylated proteins at the synapse, upregulated at both proline-directed kinase (PDK) sites (S199/S202, S396/S404) and nonPDK sites (S400). These PDK sites correspond to well-known pathological tau epitopes in AD patients, recognized by AT8 and PHF-1 antibodies, respectively. Hyperphosphorylation at S199/S202, a rarely examined combination, was further validated in patient-derived human synaptosomes by immunoblotting. Global surveys of upregulated phosphosites revealed 2 potential kinase motifs, which resemble those of cyclin-dependent kinase 5 (CDK5, a PDK) and casein kinase II (CK2, a nonPDK). Our data demonstrate that, within synaptic compartments, amyloid pathology is associated with tau hyperphosphorylation at disease-relevant epitopes. This provides a plausible mechanism by which Aβ promotes the spreading of tauopathy.
Apolipoprotein E (ApoE) is a secreted apolipoprotein with three isoforms, E2, E3, and E4, that binds to lipids and facilitates their transport in the extracellular environment of the brain and the ...periphery. The E4 allele is a major genetic risk factor for the sporadic form of Alzheimer's disease (AD), and studies of human brain and mouse models have revealed that E4 significantly exacerbates the deposition of amyloid beta (Aβ). It has been suggested that this deposition could be attributed to the formation of soluble ApoE isoform–specific ApoE–Aβ complexes. However, previous studies have reported conflicting results regarding the directionality and strength of those interactions. In this study, using a series of flow cytometry assays that maintain the physiological integrity of ApoE–Aβ complexes, we systematically assessed the association of Aβ with ApoE2, E3, or E4. We used ApoE secreted from HEK cells or astrocytes overexpressing ApoE fused with a GFP tag. As a source of soluble Aβ peptide, we used synthetic Aβ40 or Aβ42 or physiological Aβ secreted from CHO cell lines overexpressing WT or V717F variant amyloid precursor protein (APP). We observed significant interactions between the different ApoE isoforms and Aβ, with E4 interacting with Aβ more strongly than the E2 and E3 isoforms. We also found subtle differences depending on the Aβ type and the ApoE-producing cell type. In conclusion, these results indicate that the strength of the ApoE–Aβ association depends on the source of Aβ or ApoE.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
There is a growing body of evidence that soluble oligomeric forms of amyloid β (Aβ) play a critical role in Alzheimer's disease (AD). Despite the importance of soluble Aβ oligomers as a therapeutic ...target for AD, the dynamic metabolism of these Aβ species in vivo has not been elucidated because of the difficulty in monitoring brain Aβ oligomers in living animals. Here, using a unique large pore‐sized membrane microdialysis, we characterized soluble Aβ oligomers in brain interstitial fluid (ISF) of awake, freely moving APP/PS1 transgenic and control WT mice. We could detect high‐molecular‐weight (HMW) and low‐molecular‐weight (LMW) Aβ oligomers in the brain ISF of living animals, which increased dramatically in an age‐dependent manner (5‐ to 8‐fold increase, 4 vs. 17‐18 mo). Notably, HMW Aβ decreased more slowly than other forms of Aβ after acute γ‐secretase inhibition % decrease from the baseline (HMW vs. LMW) was 36.9 vs. 74.1% (Aβ40, P<0.05) and 25.4 vs. 88.0% (Aβ42, P<0.01), suggesting that HMW Aβ oligomers clear more slowly than other forms from the brain. These data reveal the dynamic metabolism of neurotoxic Aβ oligomers in AD brain and could provide new insights into Aβ‐targeted therapies for AD.—Takeda, S., Hashimoto, T., Roe, A. D., Hori, Y., Spires‐Jones, T. L.and Hyman, B. T., Brain interstitial oligomeric amyloid β increases with age and is resistant to clearance from brain in a mouse model of Alzheimer's disease. FASEB J. 27, 3239–3248 (2013). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational ...differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Stress exposure and the corticotropin-releasing factor (CRF) system have been implicated as mechanistically involved in both Alzheimer's disease (AD) and associated rodent models. In particular, the ...major stress receptor, CRF receptor type 1 (CRFR1), modulates cellular activity in many AD-relevant brain areas, and has been demonstrated to impact both tau phosphorylation and amyloid-β (Aβ) pathways. The overarching goal of our laboratory is to develop and characterize agents that impact the CRF signaling system as disease-modifying treatments for AD. In the present study, we developed a novel transgenic mouse to determine whether partial or complete ablation of CRFR1 was feasible in an AD transgenic model and whether this type of treatment could impact Aβ pathology. Double transgenic AD mice (PSAPP) were crossed to mice null for CRFR1; resultant CRFR1 heterozygous (PSAPP-R1(+/-)) and homozygous (PSAPP-R1(-/-)) female offspring were used at 12 months of age to examine the impact of CRFR1 disruption on the severity of AD Aβ levels and pathology. We found that both PSAPP-R1(+/-) and PSAPP-R1(-/-) had significantly reduced Aβ burden in the hippocampus, insular, rhinal, and retrosplenial cortices. Accordingly, we observed dramatic reductions in Aβ peptides and AβPP-CTFs, providing support for a direct relationship between CRFR1 and Aβ production pathways. In summary, our results suggest that interference of CRFR1 in an AD model is tolerable and is efficacious in impacting Aβ neuropathology.
Clinical studies suggest that exposure to stress can increase risk for Alzheimer’s disease (AD). Although the precise links between stress and vulnerability to develop AD remain uncertain, recent ...animal work suggests that stress may promote susceptibility to AD pathology by activating tau kinases and inducing tau phosphorylation (tau‐P). Our previous findings indicate the differential involvement of corticotropin‐releasing factor receptor (CRFR) types 1 and 2 in regulating tau‐P in the hippocampus induced by acute restraint, an emotional stressor. To assess the generality of CRFR involvement in stress‐induced tau‐P and tau kinase activity, the present study extends our investigation to a well‐characterized physiological stressor, i.e. immune challenge induced by bacterial lipopolysaccharide (LPS). Acute systemic administration of LPS (100 μg/kg) robustly increased hippocampal (but not isocortical or cerebellar) tau‐P, peaking at 40–120 min postinjection and abating thereafter. Assessments of the genotype dependence of this effect yielded results that were distinct from the restraint model. Treatment with LPS increased phosphorylation in wild‐type, single and double CRFR knockouts with only subtle variation, which included a reliable exaggeration of tau‐P responses in CRFR1‐deficient mice. Parallel analyses implicated glycogen synthase kinase‐3 and cyclin‐dependent kinase‐5 as likely cellular mediators of LPS‐induced tau‐P. Conversely, our data suggest that temperature‐dependent fluctuations in tau protein phosphatase 2A (PP2A) may not play a role in this context. Thus, neither the strict CRFR1 dependence of restraint‐induced tau‐P nor the exaggeration of these responses in CRFR2 null mice generalize to the LPS model. CRFR mediation of stress‐induced hippocampal tau‐P may be limited to emotional stressors.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Neurofibrillary tangles (NFTs), a marker of neuronal alterations in Alzheimer's disease (AD) and other tauopathies, are comprised of aggregates of hyperphosphorylated tau protein. We recently studied ...the formation of NFTs in the entorhinal cortex (EC) and their subsequent propagation through neural circuits in the rTgTauEC mouse model (de Calignon et al., 2012). We now examine the consequences of suppressing transgene expression with doxycycline on the NFT-associated pathological features of neuronal system deafferentation, NFT progression and propagation, and neuronal loss. At 21 months of age we observe that EC axonal lesions are associated with an abnormal sprouting response of acetylcholinesterase (AChE)-positive fibers, a phenotype reminiscent of human AD. At 24 months, NFTs progress, tau inclusions propagate to the dentate gyrus, and neuronal loss is evident. Suppression of the transgene expression from 18 to 24 months led to reversal of AChE sprouting, resolution of Gallyas-positive and Alz50-positive NFTs, and abrogation of progressive neuronal loss. These data suggest that propagation of NFTs, as well as some of the neural system consequences of NFTs, can be reversed in an animal model of NFT-associated toxicity, providing proof in principle that these lesions can be halted, even in established disease.
Abstract Introduction Little is known about the utility of plasma amyloid beta (Aβ) in clinical trials of Alzheimer's disease (AD). Methods We analyzed longitudinal plasma samples from two large ...multicenter clinical trials: (1) donezepil and vitamin E in mild cognitive impairment (n = 405, 24 months) and (2) simvastatin in mild to moderate AD (n = 225, 18 months). Results Baseline plasma Aβ was not related to cognitive or clinical progression. We observed a decrease in plasma Aβ40 and 42 among apolipoprotein E epsilon 4 ( APOE ε4) carriers relative to noncarriers in the mild cognitive impairment trial. Patients treated with simvastatin showed a significant increase in Aβ compared with placebo. We found significant storage time effects and considerable plate-to-plate variation. Discussion We found no support for the utility of plasma Aβ as a prognostic factor or correlate of cognitive change. Analysis of stored specimens requires careful standardization and experimental design, but plasma Aβ may prove useful in pharmacodynamic studies of antiamyloid drugs.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Apolipoprotein E (APOE) effects on brain function remain controversial. Removal of APOE not only impairs cognitive functions but also reduces neuritic amyloid plaques in mouse models of Alzheimer's ...disease (AD). Can APOE simultaneously protect and impair neural circuits? Here, we dissociated the role of APOE in AD versus aging to determine its effects on neuronal function and synaptic integrity. Using two-photon calcium imaging in awake mice to record visually evoked responses, we found that genetic removal of APOE improved neuronal responses in adult APP/PSEN1 mice (8-10 mo). These animals also exhibited fewer neuritic plaques with less surrounding synapse loss, fewer neuritic dystrophies, and reactive glia. Surprisingly, the lack of APOE in aged mice (18-20 mo), even in the absence of amyloid, disrupted visually evoked responses. These results suggest a dissociation in APOE's role in AD versus aging: APOE may be neurotoxic during early stages of amyloid deposition, although being neuroprotective in latter stages of aging.
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