Rationale:The exact etiology of sporadic Alzheimer disease (AD) is unclear, but it is interesting that several cardiovascular risk factors are associated with higher incidence of AD. The link between these risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of nitric oxide (NO).Objective: To determine the relationship between endothelial derived NO and the expression and processing of amyloid precursor protein (APP). Key Words: endothelium Ⅲ amyloid precursor protein Ⅲ Alzheimer's disease Ⅲ cerebrovascular biology Ⅲ ␤ amyloid A lzheimer's disease (AD) is a chronic neurodegenerative disease affecting more than 5 million persons in the United States and more than 20 million worldwide. 1 AD is characterized by progressive loss of neurons, cognitive decline, and 2 defining histopathologies: extracellular amyloid plaques and intracellular tangles composed primarily of amyloid ␤ (A␤) peptide and hyperphosphorylated tau, respectively. 2 Furthermore, AD is often accompanied by cerebrovascular dysfunction, as well as amyloid deposition within the cerebral vessels, termed cerebral amyloid angiopathy. 3 A␤ is generated from sequential cleavages of its parent molecule, the amyloid precursor protein (APP), by the activities of ␤-site APP-cleaving enzyme (BACE)1 and ␥ secretase. 4,5 Importantly, A␤ has been shown to exert a plethora of effects on endothelial phenotype, including angiogenesis, proliferation, adhesion, and responsiveness to vasoactive molecules. 6,7 Moreover, it has been suggested that APP has functional roles in coagulation, adhesion, and inflammation. 8 The exact etiology of sporadic AD is unclear, but it is interesting that cardiovascular risk factors including hypertension, hypercholesterolemia, diabetes mellitus, aging, and sedentary lifestyle are associated with higher incidence of AD. 9 The link between cardiovascular risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of nitric oxide (NO). 10 In the cerebral circulation, endothelial NO is generated by endothelial nitric oxide synthase (eNOS), which, under basal conditions, is expressed exclusively in endothelial cells. 11 NO is an extremely important signaling molecule responsible for maintaining vascular homeostasis by promoting vasodilatation, inhibiting platelet aggregation and leukocyte adhesion. 12 Taken together, these data suggest that NO availability may be a common link between cardiovascular risk factors and the development of AD; therefore, we examine here the role of endothelial-derived NO in modulating brain and microvascular APP expression and processing and generation of the amyloidogenic fragment A␤. Methods and Results:

Endothelial nitric oxide (NO) is generated by constitutively active endothelial nitric oxide synthase (eNOS), an essential enzyme responsible for cardiovascular homeostasis. Historically, endothelial NO was first recognized as a major vasodilator involved in control of vasomotor function and local blood flow. In this review, our attention is focused on the emerging role of endothelial NO in linking cerebrovascular function with cognition. We will discuss the recognized ability of endothelial NO to modulate processing of amyloid precursor protein (APP), influence functional status of microglia, and affect cognitive function. Existing evidence suggests that the loss of NO in cultured human cerebrovascular endothelium causes increased expression of APP and β-site APP-cleaving enzyme 1 (BACE1) thereby resulting in increased secretion of amyloid β peptides (Aβ1-40 and Aβ1-42). Furthermore, increased expression of APP and BACE1 as well as increased production of Aβ peptides was detected in the cerebral microvasculature and brain tissue of eNOS-deficient mice. Since Aβ peptides are considered major cytotoxic molecules responsible for the pathogenesis of Alzheimer's disease, these observations support the concept that a loss of endothelial NO might significantly contribute to the initiation and progression of cognitive decline. In addition, genetic inactivation of eNOS causes activation of microglia and promotes a pro-inflammatory phenotype in the brain. Behavioural analysis revealed that eNOS-deficient mice exhibit impaired cognitive performance thereby indicating that selective loss of endothelial NO has a detrimental effect on the function of neuronal cells. Together with findings from prior studies demonstrating the ability of endothelial NO to affect synaptic plasticity, mitochondrial biogenesis, and function of neuronal progenitor cells, it is becoming apparent that the role of endothelial NO in the control of central nervous system function is very complex. We propose that endothelial NO represents the key molecule linking cerebrovascular and neuronal function.

Recent Parkinson's disease research has focused on understanding the function of the cytosolic protein, ␣-synuclein, and its contribution to disease mechanisms. Within neurons, ␣-synuclein is hypothesized to have a role in regulating synaptic plasticity, vesicle release, and trafficking. In contrast, glial-expressed ␣-synuclein remains poorly described. Here, we examine the consequence of a loss of ␣-synuclein expression on microglial activation. Using a postnatal brain-derived culture system, we defined the phenotype of microglia from wildtype and knock-out ␣-synuclein mice (Scna Ϫ/Ϫ ). Scna Ϫ / Ϫ microglia displayed a basally increased reactive phenotype compared with the wild-type cells and an exacerbated reactive phenotype after stimulation. They also exhibited dramatic morphologic differences compared with wild-type, presenting as large, ramified cells filled with vacuole-like structures. This corresponded with increased protein levels of activation markers, CD68 and ␤1 integrin, in the Scna Ϫ / Ϫ cells. More importantly, Scna Ϫ / Ϫ microglia, after stimulation, secreted elevated levels of proinflammatory cytokines, TNF␣ (tumor necrosis factor ␣) and IL-6 (interleukin-6), compared with wild type. However, despite the reactive phenotype, Scna Ϫ / Ϫ cells had impaired phagocytic ability. We demonstrate for the first time that ␣-synuclein plays a critical role in modulating microglial activation state. We suggest that altered microglial ␣-synuclein expression will affect their phenotype as has already been demonstrated in neurons. This has direct ramifications for the contribution of microglia to the pathophysiology of disease, particularly in familial cases linked to altered ␣-synuclein expression.

Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer’s disease (AD). A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14–15 month) endothelial nitric oxide synthase deficient (eNOS−/−) mice. APP, β-site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS−/− mice as compared to LMA wild type controls. APP and Aβ1-40 were increased in hippocampal tissue of eNOS−/− mice as compared to wild type mice. LMA eNOS−/− mice displayed an increased inflammatory phenotype as compared to LMA wild type mice. Importantly, LMA eNOS−/− mice performed worse in a radial arm maze test of spatial learning and memory as compared to LMA wild type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline.

Alpha-synuclein (Snca) is an abundant small cytosolic protein (140 amino acids) that is expressed in the brain, although its physiological role is poorly defined. Consistent with its ubiquitous distribution in the brain, we and others have established a role for Snca in brain lipid metabolism and downstream events such as neuroinflammation. In astrocytes, Snca is important for fatty acid uptake and trafficking, where its deletion decreases 16:0 and 20:4n-6 uptake and alters targeting to specific lipid pools. Although Snca has no impact on 22:6n-3 uptake into astrocytes, it is important for its targeting to lipid pools. Similar results for fatty acid uptake from the plasma are seen in studies using whole mice coupled with steady-state kinetic modeling. We demonstrate in gene-ablated mice a significant reduction in the incorporation rate of 20:4n-6 into brain phospholipid pools due to reduced recycling of 20:4n-6 through the ER-localized long-chain acyl-CoA synthetases (Acsl). This reduction results in a compensatory increase in the incorporation rate of 22:6n-3 into brain phospholipids. Snca is also important for brain and astrocyte cholesterol metabolism, where its deletion results in an elevation of cholesterol and cholesteryl esters. This increase may be due to the interaction of Snca with membrane-bound enzymes involved in lipid metabolism such as Acsl. Snca is critical in modulating brain prostanoid formation and microglial activities. In the absence of Snca, microglia are basally activated and demonstrate increased proinflammatory cytokine secretion. Thus, Snca, through its modulation of brain lipid metabolism, has a critical role in brain inflammatory responses.