Although oxidative stress is commonly associated with aging (1, 2), patients with Alzheimer disease (AD) 3 often exhibit increased oxidative damage (3-10) and subsequent neuronal loss in ␤-amyloid (A␤)-rich regions of the brain. The molecular mechanisms by which A␤ contributes to oxidative damage remain unclear (11)(12)(13)(14)(15)(16)(17)(18)(19). Understanding these mechanisms, however, is critical for developing effective methods to manage the disease. One mechanism for A␤-induced cellular oxidative stress proposes that A␤ peptides interact directly with cellular enzymes responsible for maintaining low physiological levels of reactive oxygen species (ROS) (20 -23). Two potential outcomes from such pathological protein-amyloid interactions are: 1) increased production of ROS, or 2) reduced degradation of ROS.The major ROS in cells are superoxide and the more reactive hydrogen peroxide (H 2 O 2 )-derived hydroxyl radical (24, 25). Both superoxide and H 2 O 2 are primarily produced in the mitochondria (26 -28). A␤ peptides have been shown to accumulate in the mitochondria (29, 30), and, therefore, could exert their detrimental effects through interaction with mitochondrial proteins (23, 31-34). Superoxide is produced by several enzymecatalyzed reactions in the mitochondria (25). Behl et al. (11) however, showed that a broad range of inhibitors of several of these enzymes had no effect on 〈␤ toxicity in clonal and primary neuronal cell cultures. Furthermore, Zhang et al. (35) reported that superoxide levels in cells were not substantially elevated upon exposure to 〈␤. These findings suggest that superoxide is not a dominant contributor to 〈␤ toxicity.On the other hand, 〈␤-induced cellular increase in H 2 O 2 or its metabolites is strongly correlated with 〈␤ toxicity (11, 13, 36). H 2 O 2 can be generated by several mitochondrial enzymes including monoamine oxidases, superoxide dismutase, and xanthine oxidase (25). Behl et al. (11) showed that inhibitors of monoamine oxidases and xanthine oxidase had no effect on 〈␤-induced H 2 O 2 accumulation or 〈␤ toxicity. Gsell et al. (37) also found that the activity of superoxide dismutase was unaltered in the brains of AD patients. Additionally, Rensink et al. (38) reported that the Dutch mutation of 〈␤ peptides (HCHWA-D 〈␤) did not bind directly to superoxide dismutase and Kaminsky et al. (39) reported only a relatively small effect of 〈␤ on superoxide dismutase activity and H 2 O 2 production upon chronic exposure of rat brains to 〈␤. These results suggest that 〈␤ peptides do not significantly affect production of H 2 O 2 in cells. Consequently, these findings imply that 〈␤-induced oxidative stress may arise from reduced degradation of H 2 O 2 in 〈␤-challenged cells.Degradation of H 2 O 2 in cells is primarily achieved by the enzymes catalase and glutathione peroxidase (GPx), both inside and outside of the mitochondria (25). Sagara et al. (40) found that cells resistant to 〈␤ toxicity had elevated levels of catalase and GPx. The activity of both enzymes was redu...

The tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG 4 , is a novel amyloid-binding small molecule that can penetrate the blood-brain barrier and protect cells from A␤-induced toxicity. However, the effects of A␤-targeting molecules on other cellular processes, including those that modulate synaptic plasticity, remain unknown. We report here that BTA-EG 4 decreases A␤ levels, alters cell surface expression of amyloid precursor protein (APP), and improves memory in wild-type mice. Interestingly, the BTA-EG 4 -mediated behavioral improvement is not correlated with LTP, but with increased spinogenesis. The higher dendritic spine density reflects an increase in the number of functional synapses as determined by increased miniature EPSC (mEPSC) frequency without changes in presynaptic parameters or postsynaptic mEPSC amplitude. Additionally, BTA-EG 4 requires APP to regulate dendritic spine density through a Ras signaling-dependent mechanism. Thus, BTA-EG 4 may provide broad therapeutic benefits for improving neuronal and cognitive function, and may have implications in neurodegenerative disease therapy.

A novel class of 2,4-disubstituted pyrimidines (7a-u, 8a-f, 9a-e) that possess substituents with varying steric and electronic properties at the C-2 and C-4 positions, were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. In vitro screening identified N-(naphth-1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine (9a) as the most potent AChE inhibitor (IC 50 = 5.5 μM). Among this class of compounds, 2-(4-methylpiperidin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine (9e) was identified as the most potent and selective BuChE inhibitor (IC 50 = 2.2 μM, Selectivity Index = 11.7) and was about 5.7-fold more potent compared to the commercial, approved reference drug galanthamine (BuChE IC 50 = 12.6 μM). In addition, the selective AChE inhibitor N-benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (7d), exhibited good inhibition of hAChE-induced aggregation of Aβ 1-40 fibrils (59% inhibition). Furthermore, molecular modeling studies indicate that a central pyrimidine ring serves as a suitable template to develop dual inhibitors of cholinesterase and AChE-induced Aβ aggregation thereby targeting multiple pathological routes in AD. KeywordsCholinesterase inhibitors ChEIs; Acetylcholinesterase AChE; Butyrylcholinesterase BuChE; Human acetylcholinesterase hAChE; Structure-activity relationship SAR; Amyloid-β Aβ; 5,5'-dithiobis(2-nitrobenzoic acid), DTNB; Thioflavin T ThT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide MTT

Our recent study demonstrated that an amyloid-β binding molecule, BTA-EG4, increases dendritic spine number via Ras-mediated signaling. To potentially optimize the potency of the BTA compounds, we synthesized and evaluated an amyloid-β binding analog of BTA-EG4 with increased solubility in aqueous solution, BTA-EG6. We initially examined the effects of BTA-EG6 on dendritic spine formation and found that BTA-EG6-treated primary hippocampal neurons had significantly increased dendritic spine number compared to control treatment. In addition, BTA-EG6 significantly increased the surface level of AMPA receptors. Upon investigation into the molecular mechanism by which BTA-EG6 promotes dendritic spine formation, we found that BTA-EG6 may exert its effects on spinogenesis via RasGRF1-ERK signaling, with potential involvement of other spinogenesis-related proteins such as Cdc42 and CDK5. Taken together, our data suggest that BTA-EG6 boosts spine and synapse number, which may have a beneficial effect of enhancing neuronal and synaptic function in the normal healthy brain.