Summary Alzheimer's disease (AD) is a neurodegenerative disorder in which vascular pathology plays an important role. Since the β-amyloid peptide (Aβ) is a critical factor in this disease, we examined its relationship to fibrin clot formation in AD. In vitro and in vivo experiments showed that fibrin clots formed in the presence of Aβ are structurally abnormal and resistant to degradation. Fibrin(ogen) was observed in blood vessels positive for amyloid in mouse and human AD samples, and intravital brain imaging of clot formation and dissolution revealed abnormal thrombosis and fibrinolysis in AD mice. Moreover, depletion of fibrinogen lessened cerebral amyloid angiopathy pathology and reduced cognitive impairment in AD mice. These experiments suggest that one important contribution of Aβ to AD is via its effects on fibrin clots, implicating fibrin(ogen) as a potential critical factor in this disease.

Alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of Alzheimer's disease (AD), but their role in the amyloid deposition and cognitive impairment associated with AD remains unclear. We used mice overexpressing the Swedish mutation of the amyloid precursor protein (Tg2576) as a model of AD to examine the role of reactive oxygen species produced by NADPH oxidase in the cerebrovascular alterations, amyloid deposition, and behavioral deficits observed in these mice. We found that 12-to 15-month-old Tg2576 mice lacking the catalytic subunit Nox2 of NADPH oxidase do not develop oxidative stress, cerebrovascular dysfunction, or behavioral deficits. These improvements occurred without reductions in brain amyloid-␤ peptide (A␤) levels or amyloid plaques. The findings unveil a previously unrecognized role of Nox2-derived radicals in the behavioral deficits of Tg2576 mice and provide a link between the neurovascular dysfunction and cognitive decline associated with amyloid pathology.Alzheimer's disease ͉ cerebral blood flow ͉ tg2576 T he amyloid-␤ peptide (A␤) is central to the pathogenesis of Alzheimer's disease (AD), the most common form of dementia in the elderly (1). A␤ peptides are cleaved from the amyloid precursor protein (APP) by two aspartyl proteases, termed ␤-and ␥-secretases, and form deposits in the brain parenchyma (amyloid plaques) and around blood vessels (amyloid angiopathy) (2). The mechanisms by which A␤ leads to cognitive impairment have not been completely elucidated, although recent evidence suggests that small aggregates of A␤ may be key pathogenic factors by disrupting synaptic function and inducing neuronal death (2).However, A␤ also exerts powerful effects on cerebral blood vessels (3). In vitro and in vivo studies have demonstrated that A␤ enhances vasoconstriction, impairs responses to vasodilators, and reduces cerebral blood flow (CBF) (4, 5). In addition, transgenic mice overexpressing APP and A␤ have major alterations in resting CBF and in key cerebrovascular control mechanisms (5-9). For example, the increase in CBF induced by neural activity (functional hyperemia), a response that matches the brain's energy demands with its blood supply, and the ability of cerebral endothelial cells to regulate CBF are profoundly impaired in mice overexpressing APP (7, 10). The vasoconstriction induced by A␤ may underlie the marked reductions in CBF observed in the early stages of AD (11). The harmful cerebrovascular effects of A␤, in concert with epidemiological and pathological findings linking AD with cerebrovascular diseases (12-16), have suggested that A␤ has deleterious actions both on neurons and cerebral blood vessels, which may act synergistically to induce brain dysfunction in AD (3,17).The cerebrovascular alterations observed in mice overexpressing APP are associated with vascular oxidative stress and are counteracted by free radical scavengers (6,18,19), implicating reactive oxygen species (ROS) in the dysfunction. A major source...

Previous studies demonstrated that ␣-synuclein (␣-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346 -1349). Dopamine inhibition of ␣-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not ␣-syn oxidation, because mutagenesis of Met, His, and Tyr residues in ␣-syn did not abrogate this inhibition. However, truncation of ␣-syn at residue 125 restored the ability of ␣-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified ␣-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of ␣-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of ␣-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits ␣-syn fibrillization by inducing structural changes in ␣-syn that can occur through the interaction of dopaminochrome with the 125 YEMPS 129 motif of ␣-syn. These results suggest that the dopamine autoxidation can prevent ␣-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote ␣-syn aggregation in Parkinson's disease. Parkinson disease (PD)1 is the most common neurodegenerative movement disorder, as it affects over one million people in North America and four million worldwide (1). PD is clinically diagnosed by four characteristic features, bradykinesia, postural instability, motor rigidity, and resting tremor. Pathologically, there is a progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in a significant decrease in dopamine levels in the striatum followed by motor impairments in PD patients (1-3). In addition to neuron loss, intracellular proteinaceous lesions are found in different PD brain regions that are termed Lewy bodies (LBs) and Lewy neurites. LBs are found in the remaining dopaminergic neurons of the substantia nigra (4, 5), but they also occur in other brainstem neurons as well as in those of the thalamus, hypothalamus, cortex, olfactory bulb, and other brain regions (4, 6, 7). These inclusions are now known to be comprised of filamentous polymers of ␣-synuclein (␣-syn) protein (5, 8 -14).␣-Syn is a 140-amino acid heat-stable protein that is predominantly found in presynaptic terminals of cells of the central nervous system (15)(16)(17)(18). Studies have shown that pathological inclusions comprised of ␣-syn are found in neurodegenerative disorders other than PD, including the LB variant of Alzheimer's disease, dementia with LBs, multiple system atrophy, and related diseases collectiv...

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Increasing evidence supports a vascular contribution to Alzheimer's disease (AD), but a direct connection between AD and the circulatory system has not been established. Previous work has shown that blood clots formed in the presence of the β-amyloid peptide (Aβ), which has been implicated in AD, have an abnormal structure and are resistant to degradation in vitro and in vivo. In the present study, we show that Aβ specifically interacts with fibrinogen with a K d of 26.3 ± 6.7 nM, that the binding site is located near the C terminus of the fibrinogen β-chain, and that the binding causes fibrinogen to oligomerize. These results suggest that the interaction between Aβ and fibrinogen modifies fibrinogen's structure, which may then lead to abnormal fibrin clot formation. Overall, our study indicates that the interaction between Aβ and fibrinogen may be an important contributor to the vascular abnormalities found in AD. A lzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline and subsequent death. Effective long-term treatments and preventive measures are not available, and new therapeutic targets are needed. Substantial evidence indicates that the β-amyloid (Aβ) peptide, which is derived from the Aβ precursor protein (APP), is involved in AD (1-3). Aβ is soluble in its monomeric or oligomeric states, but can aggregate into fibrils and deposit as extracellular plaques in the brain parenchyma. However, the severity of dementia does not correlate well with the amount of extracellular amyloid plaques and the mechanism by which Aβ causes neurodegeneration is still unclear (4).Aβ can also accumulate in brain blood vessels, a condition known as cerebral amyloid angiopathy (CAA). CAA is characterized by deposition of Aβ within cerebral vessels, resulting in degenerative vascular changes (5-7). In mouse models of AD, endothelial cells in CAA vessels show early dysfunction, which reduces their response to vasodilators (8) and impairs the regulation of blood flow (9, 10). Many patients with AD present vascular symptoms, including altered cerebral blood flow, damaged cerebral vasculature, and abnormal hemostasis (11). Cerebral blood flow is reduced and many vascular defects are present in patients with AD (12). Vascular diseases such as atherosclerosis correlate in severity with dementia and other symptoms of sporadic AD (13-15). Vascular abnormalities could therefore play an important role in AD, but a direct connection remains unknown.Fibrinogen is the primary protein component of blood clots. It is 45 nm in length with identical globular domains at each end, which are connected by rod-like strands. It is composed of three pairs of polypeptide chains, designated Aα, Bβ, and γ, which are connected by disulfide bonds (16). When fibrinopeptides A and B of fibrinogen are cleaved by the serine protease thrombin, fibrinogen noncovalently polymerizes to form protofibrils, which then branch to form an insoluble fibrin clot. This clot network forms a mesh around platelets to impede blood fl...