Teresa Chu

Phenolic anti-inflammatory antioxidant reversal of Aβ-induced cognitive deficits and neuropathology

Both oxidative damage and inflammation have been implicated in age-related neurodegenerative diseases including Alzheimers Disease (AD). The yellow curry spice, curcumin, has both antioxidant and anti-inflammatory activities which confer significant protection against neurotoxic and genotoxic agents. We used 22 month Sprague-Dawley (SD) rats to compare the effects of the conventional NSAID, ibuprofen, and curcumin for their ability to protect against amyloid beta-protein (Abeta)-induced damage. Lipoprotein carrier-mediated, intracerebroventricular infusion of Abeta peptides induced oxidative damage, synaptophysin loss, a microglial response and widespread Abeta deposits. Dietary curcumin (2000 ppm), but not ibuprofen, suppressed oxidative damage (isoprostane levels) and synaptophysin loss. Both ibuprofen and curcumin reduced microgliosis in cortical layers, but curcumin increased microglial labeling within and adjacent to Abeta-ir deposits. In a second group of middle-aged female SD rats, 500 ppm dietary curcumin prevented Abeta-infusion induced spatial memory deficits in the Morris Water Maze and post-synaptic density (PSD)-95 loss and reduced Abeta deposits. Because of its low side-effect profile and long history of safe use, curcumin may find clinical application for AD prevention.

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.

Mapping biochemistry to metabolism : FDG-PET and amyloid burden in Alzheimer's disease

We evaluated the relationship between amyloid-beta protein (A beta) concentration and the metabolic abnormality in an Alzheimers disease (AD) patient as measured by [18F]fluorodeoxyglucose positron emission tomography (FDG-PET). Across most regions there were significant inverse correlations among FDG-PET intensity values and both insoluble. The temporal lobe samples showed no significant correlation between FDG-PET values and A beta deposition. Findings support A beta as contributing to the hypometabolism in regions of the AD brain that are still relatively viable metabolically; those regions with chronic pathologic damage, such as temporal cortex, may have other factors that contribute to metabolic deficits.

Estrogen (E2) and glucocorticoid (Gc) effects on microglia and Aβ clearance in vitro and in vivo

The accumulation of fibrillar aggregates of beta Amyloid (A beta) in Alzheimers Disease (AD) brain is associated with chronic brain inflammation. Although activated microglia (mu glia) can potentially clear toxic amyloid, chronic activation may lead to excessive production of neurotoxins. Recent epidemiological and clinical data have raised questions about the use of anti-inflammatory steroids (glucocorticoids, Gcs) and estrogens for treatment or prevention of AD. Since very little is known about steroid effects on mu glial interactions with amyloid, we investigated the effects of the synthetic Gc dexamethasone (DXM) and 17-beta estradiol (E2) in vitro in a murine mu glial-like N9 cell line on toxin production and intracellular A beta accumulation. To determine whether the steroid alterations of A beta uptake in vitro had relevance in vivo, we examined the effects of these steroids on A beta accumulation and mu glial responses to A beta infused into rat brain. Our in vitro data demonstrate for the first time that Gc dose-dependently enhanced mu glial A beta accumulation and support previous work showing that E2 enhances A beta uptake. Despite both steroids enhancing uptake, degradation was impeded, particularly with Gcs. Distinct differences between the two steroids were observed in their effect on toxin production and cell viability. Gc dose-dependently increased toxicity and potentiated A beta induction of nitric oxide, while E2 promoted cell viability and inhibited A beta induction of nitric oxide. The steroid enhancement of mu glial uptake and impedence of degradation observed in vitro were consistent with observations from in vivo studies. In the brains of A beta-infused rats, the mu glial staining in entorhinal cortex layer 3, not associated with A beta deposits was increased in response to A beta infusion and this effect was blocked by feeding rats prednisolone. In contrast, E2 enhanced mu glial staining in A beta-infused rats. A beta-immunoreactive (ir) deposits were quantitatively smaller, appeared denser, and were associated with robust mu glial responses. Despite the fact that steroid produced a smaller more focal deposit, total extracted A beta in cortical homogenate was elevated. Together, the in vivo and in vitro data support a role for steroids in plaque compaction. Our data are also consistent with the hypothesis that although E2 is less potent than Gc in impeding A beta degradation, long term exposure to both steroids could reduce A beta clearance and clinical utility. These data showing Gc potentiation of A beta-induced mu glial toxins may help explain the lack of epidemiological correlation for AD. The failure of both steroids to accelerate A beta degradation may explain their lack of efficacy for treatment of AD.

Intracellular Aβ is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.

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.

Methods for Evaluating in Vivo Rodent Models for Alzheimer’s Disease

In general, aged rodents do not show classical Alzheimer’s disease (AD) pathology. Although aged rats do not normally contain s-amyloid protein (As)-rich plaques nor neurofibrillary tangles (NFT), in rare instances, they do have amyloid plaques of unknown composition that contain dystrophic neurites (Vaughan and Peters 1981). Recent studies with mice transgenic for the s-amyloid precursor protein (APP) containing familial AD mutations have demonstrated that mice which produce high levels of As readily develop diffuse and neuritic s-amyloid deposits. As in AD, reactive astrocytes, activated microglia and dystrophic neurites cluster around and within the deposits. These dystrophic neurites contain abnormally phosphorylated tau protein between 6 and 12 months of age (Games et al. 1995) and other pathological cytoskeletal alterations (Masliah et al. 1996). Although similar to NFT, the dystrophic neurites in the transgenic mice do not meet all the criteria for NFT because paired helical filaments have not been found, even at 18 months. Furthermore, although age-dependent selective synapse loss occurs (Cole et al. 1999), there has not yet been demonstrated any quantifiable neuron loss. Collectively, these studies show that rodents producing large amounts of As can develop neuritic plaques closely resembling those found in AD and are therefore an excellent model of studying plaque pathogenesis.