Walter Beech

β-Amyloid Oligomers Induce Phosphorylation of Tau and Inactivation of Insulin Receptor Substrate via c-Jun N-Terminal Kinase Signaling: Suppression by Omega-3 Fatty Acids and Curcumin

Both insulin resistance (type II diabetes) and β-amyloid (Aβ) oligomers are implicated in Alzheimers disease (AD). Here, we investigate the role of Aβ oligomer-induced c-Jun N-terminal kinase (JNK) activation leading to phosphorylation and degradation of the adaptor protein insulin receptor substrate-1 (IRS-1). IRS-1 couples insulin and other trophic factor receptors to downstream kinases and neuroprotective signaling. Increased phospho-IRS-1 is found in AD brain and insulin-resistant tissues from diabetics. Here, we report Aβ oligomers significantly increased active JNK and phosphorylation of IRS-1 (Ser616) and tau (Ser422) in cultured hippocampal neurons, whereas JNK inhibition blocked these responses. The omega-3 fatty acid docosahexaenoic acid (DHA) similarly inhibited JNK and the phosphorylation of IRS-1 and tau in cultured hippocampal neurons. Feeding 3xTg-AD transgenic mice a diet high in saturated and omega-6 fat increased active JNK and phosphorylated IRS-1 and tau. Treatment of the 3xTg-AD mice on high-fat diet with fish oil or curcumin or a combination of both for 4 months reduced phosphorylated JNK, IRS-1, and tau and prevented the degradation of total IRS-1. This was accompanied by improvement in Y-maze performance. Mice fed with fish oil and curcumin for 1 month had more significant effects on Y-maze, and the combination showed more significant inhibition of JNK, IRS-1, and tau phosphorylation. These data indicate JNK mediates Aβ oligomer inactivation of IRS-1 and phospho-tau pathology and that dietary treatment with fish oil/DHA, curcumin, or a combination of both has the potential to improve insulin/trophic signaling and cognitive deficits in AD.

Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice

We previously showed the non-steroidal anti-inflammatory drug (NSAID) ibuprofen suppresses inflammation and amyloid in the APPsw (Tg2576) Tg2576 transgenic mouse. The mechanism for these effects and the impact on behavior are unknown. We now show ibuprofens effects were not mediated by alterations in amyloid precursor protein (APP) expression or oxidative damage (carbonyls). Six months ibuprofen treatment in Tg+ females caused a decrease in open field behavior (p < 0.05), restoring values similar to Tg- mice. Reduced caspase activation per plaque provided further evidence for a neuroprotective action of ibuprofen. The impact of a shorter 3 month duration ibuprofen trial, beginning at a later age (from 14 to 17 months), was also investigated. Repeated measures ANOVA of Abeta levels (soluble and insoluble) demonstrated a significant ibuprofen treatment effect (p < 0.05). Post-hoc analysis showed that ibuprofen-dependent reductions of both soluble Abeta and Abeta42 were most marked in entorhinal cortex (p < 0.05). Although interleukin-1beta and insoluble Abeta were more effectively reduced with longer treatment, the magnitude of the effect on soluble Abeta was not dependent on treatment duration.

Selective inhibition of Aβ42 production by NSAID R-enantiomers

Non‐steroidal anti‐inflammatory drugs (NSAIDs) have been associated with reduced risk for Alzheimers disease (AD) and selected NSAIDs racemates suppress β‐amyloid (Aβ) accumulation in vivo and Aβ42 production in vitro. Clinical use of NSAIDs for preventing or treating AD has been hampered by dose‐limiting toxicity believed to be due to cyclooxygenase (COX)‐inhibition that is reportedly not essential for selective Aβ42 reduction. Profens have racemates and R‐enantiomers were supposed to be inactive forms. Here we demonstrate that R‐ibuprofen and R‐flurbiprofen, with poor COX‐inhibiting activity, reduce Aβ42 production by human cells. Although these R‐enantiomers inhibit nuclear factor‐κB (NF‐κB) activation and NF‐κB can selectively regulate Aβ42, Aβ42 reduction is not mediated by inhibition of NF‐κB activation. Because of its efficacy at lowering Aβ42 production and low toxicity profile, R‐flurbiprofen is a strong candidate for clinical development.

GSK3 inhibitors show benefits in an Alzheimer's disease (AD) model of neurodegeneration but adverse effects in control animals

The dysregulation of glycogen synthase kinase-3 (GSK3) has been implicated in Alzheimer disease (AD) pathogenesis and in Abeta-induced neurotoxicity, leading us to investigate it as a therapeutic target in an intracerebroventricular Abeta infusion model. Infusion of a specific GSK3 inhibitor SB216763 (SB) reduced a downstream target, phospho-glycogen synthase 39%, and increased glycogen levels 44%, suggesting effective inhibition of enzyme activity. Compared to vehicle, Abeta increased GSK3 activity, and was associated with elevations in levels of ptau, caspase-3, the tau kinase phospho-c-jun N-terminal kinase (pJNK), neuronal DNA fragmentation, and gliosis. Co-infusion of SB corrected all responses to Abeta infusion except the induction of gliosis and behavioral deficits in the Morris water maze. Nevertheless, SB alone was associated with induction of neurodegenerative markers and behavioral deficits. These data support a role for GSK3 hyperactivation in AD pathogenesis, but emphasize the importance of developing inhibitors that do not suppress constitutive activity.

p21-activated Kinase-aberrant Activation and Translocation in Alzheimer Disease Pathogenesis

Defects in dendritic spines and synapses contribute to cognitive deficits in mental retardation syndromes and, potentially, Alzheimer disease. p21-activated kinases (PAKs) regulate actin filaments and morphogenesis of dendritic spines regulated by the Rho family GTPases Rac and Cdc42. We previously reported that active PAK was markedly reduced in Alzheimer disease cytosol, accompanied by downstream loss of the spine actin-regulatory protein Drebrin. β-Amyloid (Aβ) oligomer was implicated in PAK defects. Here we demonstrate that PAK is aberrantly activated and translocated from cytosol to membrane in Alzheimer disease brain and in 22-month-old Tg2576 transgenic mice with Alzheimer disease. This active PAK coimmunoprecipitated with the small GTPase Rac and both translocated to granules. Aβ42 oligomer treatment of cultured hippocampal neurons induced similar effects, accompanied by reduction of dendrites that were protected by kinase-active but not kinase-dead PAK. Aβ42 oligomer treatment also significantly reduced N-methyl-d-aspartic acid receptor subunit NR2B phosphotyrosine labeling. The Src family tyrosine kinase inhibitor PP2 significantly blocked the PAK/Rac translocation but not the loss of p-NR2B in Aβ42 oligomer-treated neurons. Src family kinases are known to phosphorylate the Rac activator Tiam1, which has recently been shown to be Aβ-responsive. In addition, anti-oligomer curcumin comparatively suppressed PAK translocation in aged Tg2576 transgenic mice with Alzheimer amyloid pathology and in Aβ42 oligomer-treated cultured hippocampal neurons. Our results implicate aberrant PAK in Aβ oligomer-induced signaling and synaptic deficits in Alzheimer disease.

Evidence of Aβ-and transgene-dependent defects in ERK-CREB signaling in Alzheimer's models

Extracellular‐signal regulated kinase (ERK) signaling is critical for memory and tightly regulated by acute environmental stimuli. In Alzheimer disease transgenic models, active ERK is shown to first be increased, then later reduced, but whether these baseline changes reflect disruptions in ERK signaling is less clear. We investigated the influence of the familial Alzheimer’s disease transgene APPsw and β‐amyloid peptide (Aβ) immunoneutralization on cannulation injury‐associated (i.c.v. infusion) ERK activation. At both 12 and 22 months of age, the trauma‐associated activation of ERK observed in Tg− mice was dramatically attenuated in Tg+. In cortices of 22‐month‐old non‐infused mice, a reduction in ERK activation was observed in Tg+, relative to Tg− mice. Intracerebroventricular (i.c.v.) anti‐Aβ infusion significantly increased phosphorylated ERK, its substrate cAMP‐response element‐binding protein (CREB) and a downstream target, the NMDA receptor subunit. We also demonstrated that Aβ oligomer decreased active ERK and subsequently active CREB in human neuroblastoma cells, which could be prevented by oligomer immunoneutralization. Aβ oligomers also inhibited active ERK and CREB in primary neurons, in addition to reducing the downstream post‐synaptic protein NMDA receptor subunit. These effects were reversed by anti‐oligomer. Our data strongly support the existence of an APPsw transgene‐dependent and Aβ oligomer‐mediated defect in regulation of ERK activation.

Lipoprotein effects on aβ accumulation and degradation by microglia in vitro

An inflammatory response involving activated microglia in neuritic β‐amyloid plaques is found in Alzheimers disease (AD) brain. Because HDL lipoproteins have been shown to carry the β‐amyloid peptide (Aβ) in plasma and CSF, we have investigated the influence of plasma high‐density lipoprotein (HDL) and lipidated ApoE and ApoJ particles on the interaction of cultured rat microglia with Aβ1–42. Microglia degraded Aβ via a pathway sensitive to cytochalasin D and the scavenger receptor inhibitor, fucoidan. HDL increased the degradation of Aβ and the ratio of multimeric/monomeric Aβ in a dose‐dependent manner. In contrast, lipidated ApoJ and ApoE decreased the degradation of Aβ, and the effects were ApoE isoform‐dependent. Immuno‐electron microscopy revealed internalized Aβ in endosomes and lysosomes as well as cell‐associated Aβ in deep invaginations, which may be related to caveolae and surface‐connected compartments. These data suggest that lipoprotein‐dependent Aβ trafficking to microglia could be relevant to plaque pathogenesis in AD. J. Neurosci. Res. 57:504–520, 1999.

Effect of chloroquine and leupeptin on intracellular accumulation of amyloid‐beta (Aβ) 1–42 peptide in a murine N9 microglial cell line

Murine N9 microglia accumulated Aβ from media containing 0.67 μM Aβ within 6 h. In N9 and in primary rat microglia, chloroquine, which disrupts lysosomal pH, increased Aβ‐induced accumulation of Aβ, particularly Aβ1–42. Leupeptin similarly enhanced Aβ accumulation. The scavenger receptor antagonist fucoidan did not affect acute chloroquine‐dependent Aβ1–42 accumulation, demonstrating uptake of non‐aggregated Aβ. After prolonged incubations, chloroquine enhanced Aβ multimer (8–12 kDa) accumulation, an effect inhibited by fucoidan. Disruptions of the lysosomal system enhance Aβ and its multimer formation. Despite negligible effects of fucoidan on initial Aβ uptake, chronic exposure inhibits multimer accumulation, demonstrating a role for scavenger receptor in multimer accumulation.

Use of Copper and Insulin-Resistance to Accelerate Cognitive Deficits and Synaptic Protein Loss in a Rat Aβ-Infusion Alzheimer’s Disease Model

The rat amyloid-beta (Abeta) intracerebroventricular infusion can model aspects of Alzheimers disease (AD) and has predicted efficacy of therapies such as ibuprofen and curcumin in transgenic mouse models. High density lipoprotein (HDL), a normal plasma carrier of Abeta, is used to attenuate Abeta aggregation within the pump, causing Abeta-dependent toxicity and cognitive deficits within 3 months. Our goal was to identify factors that might accelerate onset of Abeta-dependent deficits to improve efficiency and cost-effectiveness of model. We focused on: 1) optimizing HDL-Abeta preparation for maximal toxicity; 2) evaluating the role of copper, a factor typically in water that can impact oligomer stability; and 3) determining impact of insulin resistance (type II diabetes), a risk factor for AD. In vitro studies were performed to determine doses of copper and methods of Abeta-HDL preparation that maximized toxicity. These preparations when infused resulted in earlier onset of cognitive deficits within 6 weeks post-infusion. Induction of insulin resistance did not exacerbate Abeta-dependent cognitive deficits, but did exacerbate synaptic protein loss. In summary, the newly described in vivo infusion model may be useful cost-effective method for screening for new therapeutic drugs for AD.

Protection of excitatory synapses by DHA through preserving function of NMDA-Tiam1-PAK signal pathway in Alzheimer models

Background: Epigenetic mechanisms such as post-translational histone modifications are increasingly recognized for their contribution to gene activation and silencing in the brain. Histone acetylation in particular has been shown to be important both in hippocampal long-term potentiation (LTP) and memory formation in mice. The involvement of the epigenetic modulation of memory formation has also been proposed in neuropathological models, although up to now no clear-cut connection has been demonstrated between histone modifications and Alzheimer’s disease (AD) etiology. Thus, we have undertaken preclinical studies in the APP/PS1 mouse model of AD to determine whether there are differences in histone acetylation levels during associative memory formation. Methods: Westen blot, LTP and contextual fear conditioning Results: After fear conditioning training, levels of hippocampal acetylated histone 4 (H4) in APP/PS1 mice were about 50% lower than in wild-type littermates. Interestingly, acute treatment with a histone deacetylase inhibitor, Trichostatin A (TSA), rescued the defect in CA3-CA1 LTP in slices from APP/PS1 mice. Moreover, TSA administration prior to training rescued both the defect in acetylated H4 levels and contextual freezing to wildtype values. This effect was likley due to amyloid-beta elevation as TSA rescued also the amyloid-beta induced defect in contextual fear memory. Conclusions: Based on this evidence, we propose the hypothesis that epigenetic mechanisms are involved in the altered synaptic function and memory associated with AD. In this respect, histone deacetylase inhibitors represent a new therapeutic target to effectively counteract disease progression.