Amyloid-β (Aβ) accumulation in the brain extracellular space is a hallmark of Alzheimer's disease (AD). The factors regulating this process are only partly understood. Aβ aggregation is a concentration-dependent process that is likely to be dependent on changes in brain interstitial fluid (ISF) levels of Aβ. Using in vivo microdialysis, we found that ISF Aβ levels correlated with wakefulness. ISF Aβ levels also significantly increased during acute sleep deprivation and during orexin infusion, whereas they decreased with infusion of a dual orexin receptor antagonist. Importantly, chronic sleep restriction significantly increased and a dual orexin receptor antagonist decreased Aβ plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of AD.Alzheimer's disease (AD) is the most common cause of dementia. The accumulation of the amyloid-β (Aβ) peptide in the brain extracellular space is a critical event in the pathogenesis of AD. Aβ is produced by neurons and secreted into the brain interstitial fluid (ISF). An initiating factor in AD pathogenesis occurs when soluble, monomeric Aβ undergoes a conformational change and converts into forms such as oligomers, protofibrils, and fibrils. The accumulation of these forms of Aβ is concentration-dependent and confers toxicity (1). Elucidating factors that regulate soluble Aβ levels is important for understanding AD pathogenesis. Synaptic activity regulates the release of Aβ from neurons into the ISF (2,3). How ISF Aβ is regulated by normal physiology is poorly understood.To investigate ISF Aβ metabolism, we monitored hippocampal Aβ levels using in vivo microdialysis in both wild-type mice and human APP transgenic (Tg2576) mice, which express a mutated form of human amyloid precursor protein (APP) (4). ISF Aβ was assessed in Tg2576 mice at 3 months of age, several months earlier than Aβ deposition begins. We found diurnal variation of ISF Aβ levels. Aβ levels were significantly increased during the dark period compared to the light period (Fig. 1A). ISF Aβ levels fluctuated over a 24-hour period with mean levels during the light period being ~75% of mean Aβ levels during the dark period (Fig. 1B). ISF Aβ levels were significantly correlated with the amount of time spent awake (Fig. 1, C-D). Conversely, ISF Aβ levels were negatively correlated with the amount of time spent

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Aggregation of the amyloid-␤ (A␤) peptide in the extracellular space of the brain is critical in the pathogenesis of Alzheimer's disease. A␤ is produced by neurons and released into the brain interstitial fluid (ISF), a process regulated by synaptic activity. To determine whether behavioral stressors can regulate ISF A␤ levels, we assessed the effects of chronic and acute stress paradigms in amyloid precursor protein transgenic mice. Isolation stress over 3 months increased A␤ levels by 84%. Similarly, acute restraint stress increased A␤ levels over hours. Exogenous corticotropin-releasing factor (CRF) but not corticosterone mimicked the effects of acute restraint stress. Inhibition of endogenous CRF receptors or neuronal activity blocked the effects of acute stress on A␤. Thus, behavioral stressors can rapidly increase ISF A␤ through neuronal activity in a CRF-dependent manner, and the results suggest a mechanism by which behavioral stress may affect Alzheimer's disease pathogenesis.Alzheimer's disease ͉ synaptic activity ͉ environmental stress ͉ microdialysis ͉ transgenic E vidence indicates that the aggregation and accumulation of the amyloid-␤ (A␤) peptide in the brain extracellular space is a key initiating event in the pathogenesis of Alzheimer's disease (AD) (1). A number of studies demonstrate that aggregation of A␤ is concentration-dependent (2). Increasing the amount of A␤ produced by 50% or specifically increasing the more fibrillogenic A␤ 42 either by APP gene dose or mutations in amyloid precursor protein (APP), PS1, or PS2, accelerates the onset of A␤ deposition and AD (3). Conversely, decreasing A␤ by decreasing cleavage of APP or by enhancing clearance of A␤ delays the onset of A␤ deposition (4). Thus, determining factors that regulate the levels of A␤ in the brain extracellular space, where it likely changes conformation and aggregates, may provide insight into AD pathogenesis and treatment.A␤ is produced in the brain primarily by neurons after cleavage of APP by ␤-and ␥-secretase (1). A␤ levels in the extracellular space are then influenced by factors regulating its release from neurons as well as postsecretory events such as transport and clearance. Recent evidence (5, 6) has shown that A␤ release from neurons is regulated by neuronal and specifically synaptic activity over minutes to hours. However, whether behavioral manipulations regulate synaptic activity and interstitial fluid (ISF) A␤ levels has not been addressed.Evidence in both humans and animals suggests that environmental stressors may increase risk for AD or AD pathology. In humans, persons without dementia who are prone to psychological distress are more likely to develop AD (7,8). Also, plasma levels of the stress hormone, cortisol, are correlated with the rate of dementia progression in patients with AD (9). In mouse models of AD, animals subjected to isolation stress over months had decreased learning performance and accelerated A␤ deposition (10). To explore the potential mechanisms and links between behavioral stressors and A...

Twelve polyphenols (1-12) possessing tyrosinase inhibitory properties were isolated from the methanol (95%) extract of Morus lhou. The isolated compounds consisted of four flavanones (1-4), four flavones (5-8), and four phenylbenzofuranes (9-12). Moracin derivative 12 proved to be new a compound which was fully characterized. Compounds 1-12 were evaluated for both monophenolase and diphenolase (the two steps catalyzed by tyrosinase) inhibition to identify the structural characteristics required for mushroom tyrosinase inhibition. We observed that all parent compounds (1, 5, and 9) possessing an unsubstituted resorcinol group were highly effective inhibitors of monophenolase activity (IC(50) values of 1.3, 1.2, and 7.4 microM). The potency of the inhibitors diminished with alkyl substitution on either the aromatic ring or the hydroxyl functions. Interestingly, flavone 5 was shown to possess only monophenolase inhibitory activity, but flavanone 1 and phenylbenzofuran 9 inhibited diphenolase as well as monophenolase significantly. The inhibitory mode of these species was also dependent upon the skeleton: phenylbenzofuran 9 manifested a simple competitive inhibition mode for monophenolase and diphenolase; on the other hand flavanone 1 (monophenolase, k(3) = 0.1966 min(-1) microM(-1), k(4) = 0.0082 min(-1), and K(i)(app) = 0.0468 microM; diphenolase, k(3) = 0.0014 min(-1) microM(-1), k(4) = 0.0013 min(-1), and K(i)(app) = 0.8996 microM) and flavone 5 both showed time-dependent inhibition against monophenolase. Compound 1 operated according to the simple reversible slow binding model whereas compound 5 operated under the enzyme isomerization model.