Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.     

Docosahexaenoic acid-induced amelioration on impairment of memory learning in amyloid beta-infused rats relates to the decreases of amyloid beta and cholesterol levels in detergent-insoluble membrane fractions

We investigated the effects of dietary administration of docosahexaenoic acid (DHA; C22:6n-3) on the levels of amyloid beta (A beta) peptide (1-40) and cholesterol in the nonionic detergent Triton 100 x-insoluble membrane fractions (DIFs) of the cerebral cortex and, also, on learning-related memory in an animal model of Alzheimer's disease (AD) rats infused with A beta peptide (1-40) into the cerebral ventricle. The infusion increased the levels of A beta peptide and cholesterol in the DIFs concurrently with a significant increase in reference memory errors (measured by eight-arm radial-maze tasks) compared with those of vehicle rats. Conversely, the dietary administration of DHA to AD-model rats decreased the levels of A beta peptide and cholesterol in the DIFs, with the decrease being more prominent in the DHA-administered rats. Regression analysis revealed a significant positive correlation between A beta peptide and each of cholesterol, palmitic acid and stearic acid, and between the number of reference memory errors and each of cholesterol, palmitic, stearic and oleic acid; moreover, a significant negative correlation was observed between the number of reference memory errors and the molar ratio of DHA to palmitic plus stearic acid. These results suggest that DHA-induced protection of memory deficits in AD-model rats is related to the interactions of cholesterol, palmitic acid or stearic acid with A beta peptides in DIFs where DHA ameliorates these interactions.     

Neurochemical and behavioral effects of the intrahippocampal co-injection of beta-amyloid protein1-40 and ibotenic acid in rats

The present study was designed to investigate the effects of the intrahippocampal co-injection of beta-amyloid protein1-40 (Abeta(1-40)) with ibotenic acid (Ibo) on learning and memory in normal and aging model rats, and to explore the mechanism underlying the effects of the co-injection. The normal and aging rats were bilaterally injected Abeta(1-40) (4 microg for each side) with Ibo (2 microg for each side) into the hippocampus. Two weeks after the intrahippocampal injection, the exploratory behavior and learning-memory ability of the rats were tested by using open field, Y-maze and passive avoidance task. And the changes of membrane fluidity in hippocampal mitochondria, the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in hippocampus were also examined. The co-injection of Abeta(1-40) with Ibo induced tested rats a remarkable decrease in the explorative behaviors and a significant decline in learning-memory ability (P < 0.01). The neurochemical changes induced by the co-injection included a significant decrease in membrane fluidity of hippocampal mitochondria (P < 0.01), a significant decrease in the activity of SOD (P < 0.01), as well as a remarkable increase in the content of MDA (P < 0.01). The results suggest that co-injection of Abeta(1-40) with Ibo may induce an increase of hippocampal damage by peroxidation and a serious deficit in the learning and memory of the rats. The results also suggest that the co-injection of Abeta(1-40) with Ibo may provide a useful animal model for the Alzheimer's disease (AD) research.     

Synaptic plasma membrane-bound acetylcholinesterase activity is not affected by docosahexaenoic acid-induced decrease in membrane order

We investigated the effect of administration of docosahexaenoic acid (C22:6, n-3; 300 mg/kg.day, for 12 weeks) on the degree of membrane order and membrane-bound acetylcholinesterase activity of the cerebral cortex synaptic plasma membrane in male Wistar rats. Docosahexaenoic acid levels in the synaptic plasma membrane increased significantly by 16% over levels in control rats concomitant with an increase in the molar ratio of docosahexaenoic acid to arachidonic acid. Synaptic plasma membrane order, assessed by 1,6-diphenyl-1,3,5-hexatriene, which measures order of the bulk internal hydrophobic lipid core, decreased significantly in the docosahexaenoic acid-fed rats. Lateral mobility of both global and annular lipids measured by pyrene also increased. Acetylcholinesterase activity of the synaptic plasma membrane was unaffected, and synaptic plasma membrane phospholipid contents increased in the docosahexaenoic acid-fed rats, with a concomitant decrease in the cholesterol/phospholipid molar ratio. Lipid peroxide and reactive oxygen species, indicators of tissue oxidative stress, decreased in both the cerebral cortex synaptosome and homogenate of the docosahexaenoic acid-fed rats. Arrhenius plot showed a break point in acetylcholinesterase activity at 22 degrees C and 24 degrees C in plasma membranes from docosahexaenoic acid-fed and control rats, respectively. The present experiment indicates that chronic administration of docosahexaenoic acid does not affect synaptic acetylcholinesterase activity and evoke oxidative stress, although it increases the disorder of the global and annular lipids of rat synaptic plasma membranes.     

Green tea catechins prevent cognitive deficits caused by Abeta1-40 in rats

Amyloid beta peptide (Abeta)-induced oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). In contrast, green tea catechins confer potent antioxidative defense to brain neurons. Therefore, we examined whether long-term administration of green tea catechins [Polyphenon E (PE): 63% of epigallocatechin-3-gallate, 11% of epicatechin, 6% of (-)-epigallocatechin and 6% of (-)-epicatechin-gallate] prevents cognitive impairment in an animal model of AD, rats infused with Abeta(1-40) into the cerebral ventricle. Five-week-old male Wistar rats fed with an MF diet were randomly divided into two groups: 0.0% PE (rats administered with water only) and 0.5% PE (rats administered with 5 g/L of PE). Twenty weeks after the PE administration, the 0.0% PE group was divided into the Vehicle group (rats infused with the solvent used for dissolving Abeta) and the Abeta(1-40)-infused rat group (Abeta group), whereas the 0.5% PE group was divided into the PE+Vehicle group (PE-preadministered vehicle-infused rats) and the PE+Abeta group (PE-preadministered Abeta-infused rats). Abeta(1-40) or vehicle was infused into the cerebral ventricle using a mini osmotic pump. Behavioral changes in the rats were assessed by an eight-arm radial maze. PE administration for 26 weeks significantly decreased the Abeta-induced increase in the number of reference and working memory errors, with a concomitant reduction of hippocampal lipid peroxide (LPO; 40%) and cortico-hippocampal reactive oxygen species (ROS; 42% and 50%, respectively). Significantly reduced levels of LPO in the plasma (24%) and hippocampus (25%) as well as those of ROS in the hippocampus (23%) and cortex (41%) were found in the PE+Vehicle group as compared with the Vehicle group. Furthermore, rats with preadministered PE had higher ferric-reducing antioxidation power of plasma as compared with the Vehicle group. Our results suggest that long-term administration of green tea catechins provides effective prophylactic benefits against Abeta-induced cognitive impairment by increasing antioxidative defenses.     

Mechanisms of n-3 fatty acid-mediated development and maintenance of learning memory performance

Docosahexaenoic acid (DHA, 22:6n-3) is specifically enriched in the brain and mainly anchored in the neuronal membrane, where it is involved in the maintenance of normal neurological function. Most DHA accumulation in the brain takes place during brain development in the perinatal period. However, hippocampal DHA levels decrease with age and in the brain disorder Alzheimer's disease (AD), and this decrease is associated with reduced hippocampal-dependent spatial learning memory ability. A potential mechanism is proposed by which the n-3 fatty acids DHA and eicosapentaenoic acid (20:5n-3) aid the development and maintenance of spatial learning memory performance. The developing brain or hippocampal neurons can synthesize and take up DHA and incorporate it into membrane phospholipids, especially phosphatidylethanolamine, resulting in enhanced neurite outgrowth, synaptogenesis and neurogenesis. Exposure to n-3 fatty acids enhances synaptic plasticity by increasing long-term potentiation and synaptic protein expression to increase the dendritic spine density, number of c-Fos-positive neurons and neurogenesis in the hippocampus for learning memory processing. In aged rats, n-3 fatty acid supplementation reverses age-related changes and maintains learning memory performance. n-3 fatty acids have anti-oxidative stress, anti-inflammation, and anti-apoptosis effects, leading to neuron protection in the aged, damaged, and AD brain. Retinoid signaling may be involved in the effects of DHA on learning memory performance. Estrogen has similar effects to n-3 fatty acids on hippocampal function. It would be interesting to know if there is any interaction between DHA and estrogen so as to provide a better strategy for the development and maintenance of learning memory.     

Deficient Liver Biosynthesis of Docosahexaenoic Acid Correlates with Cognitive Impairment in Alzheimer's Disease

Reduced brain levels of docosahexaenoic acid (C22:6n-3), a neurotrophic and neuroprotective fatty acid, may contribute to cognitive decline in Alzheimer''s disease. Here, we investigated whether the liver enzyme system that provides docosahexaenoic acid to the brain is dysfunctional in this disease. Docosahexaenoic acid levels were reduced in temporal cortex, mid-frontal cortex and cerebellum of subjects with Alzheimer''s disease, compared to control subjects (P = 0.007). Mini Mental State Examination (MMSE) scores positively correlated with docosahexaenoic/α-linolenic ratios in temporal cortex (P = 0.005) and mid-frontal cortex (P = 0.018), but not cerebellum. Similarly, liver docosahexaenoic acid content was lower in Alzheimer''s disease patients than control subjects (P = 0.011). Liver docosahexaenoic/α-linolenic ratios correlated positively with MMSE scores (r = 0.78; P<0.0001), and negatively with global deterioration scale grades (P = 0.013). Docosahexaenoic acid precursors, including tetracosahexaenoic acid (C24:6n-3), were elevated in liver of Alzheimer''s disease patients (P = 0.041), whereas expression of peroxisomal d-bifunctional protein, which catalyzes the conversion of tetracosahexaenoic acid into docosahexaenoic acid, was reduced (P = 0.048). Other genes involved in docosahexaenoic acid metabolism were not affected. The results indicate that a deficit in d-bifunctional protein activity impairs docosahexaenoic acid biosynthesis in liver of Alzheimer''s disease patients, lessening the flux of this neuroprotective fatty acid to the brain.     

The protective effect of dietary eicosapentaenoic acid against impairment of spatial cognition learning ability in rats infused with amyloid beta(1-40)

BackgroundAmyloid β (Aβ) peptide (1–40) can cause cognitive impairment.Experimental designWe investigated whether dietary preadministration of eicosapentaenoic acid (EPA) is conducive to cognition learning ability and whether it protects against the impairment of learning ability in rats infused with Aβ peptide (1–40) into the cerebral ventricle.ResultsDietary EPA administered to rats for 12 weeks before the infusion of Aβ into the rat brain significantly decreased the number of reference memory errors (RMEs) and working memory errors (WMEs), suggesting that chronic administration of EPA improves cognition learning ability in rats. EPA preadministered to the Aβ-infused rats significantly reduced the increase in the number of RMEs and WMEs, with concurrent proportional increases in the levels of corticohippocampal EPA and docosahexaenoic acid (DHA) and in the DHA/arachidonic acid molar ratio. Decrease in oxidative stress in these tissues was evaluated by determining the reactive oxygen species and lipid peroxide levels. cDNA microarray analysis revealed that altered genes included those that control synaptic signal transduction, cell communication, membrane-related vesicular transport functions, and enzymes and several other proteins.ConclusionThe present study suggests that EPA, by acting as a precursor for DHA, ameliorates learning deficits associated with Alzheimer's disease and that these effects are modulated by the expression of proteins involved in neuronal plasticity.     

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo

Amyloid-beta (Abeta) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Abeta peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Abeta(25-35) and Abeta(1-40) for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Abeta(1-40) led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Abeta peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.     

Alzheimer's Abeta fused to green fluorescent protein induces growth stress and a heat shock response

The 42 amino acid Alzheimer's Abeta peptide is involved in the progression of Alzheimer's disease. Here we describe the effects of intracellular Abeta, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Abeta exhibited a lower growth yield and a heat shock response, showing that Abeta fusions promote stress in cells and supporting the notion that intracellular Abeta is a toxic molecule. These studies have relevance in understanding the role of Abeta in the death of neuronal cells, and indicate that yeast may be a new tractable model system for the screening for inhibitors of the stress caused by Abeta.     

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.     

Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimer's disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-beta peptide (Abeta), the aggregating peptide in Alzheimer's disease, is natively unfolded. The deposits found in Alzheimer's disease are composed of Abeta fibrillar aggregates rich in beta-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Abeta peptide was studied by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce alpha-helix structure in Abeta, but their hydrogenated analogous lead to beta-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Abeta fibrillogenesis.     

Neurotoxic Abeta peptides increase oxidative stress in vivo through NMDA-receptor and nitric-oxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro

Beta amyloid (Abeta) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Abeta to increase oxygen radical production in vivo. The 1-40 Abeta fragment increased 2,3-dehydroxybenzoic acid efflux more than the 1-28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Abeta peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Abeta(25-35) and Abeta(35-25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N-ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Abeta aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Abeta(25-35) but not by Abeta(35-25). Neurotoxic Abeta peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Abeta levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed-forward hypothesis of Abeta neurotoxicity.     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Abeta(25-35) in comparison to Abeta(1-42)

Abeta(1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Abeta aggregation and amyloid plaque formation. Abeta(25-35), a fragment from the C-terminal end of Abeta(1-42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Abeta(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Abeta(1-42). When added after vesicle formation, Abeta(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Abeta(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Abeta(25-35) retained the same beta-sheet structure irrespective of the mode of addition, the longer Abeta(1-42) appeared to have an increase in beta-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Abeta(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

Hydroperoxide metabolism in trout gill tissue: effect of glutathione on lipoxygenase products generated from arachidonic acid and docosahexaenoic acid

The generation of oxygenated products from arachidonic acid and docosahexaenoic acid by the n-9 lipoxygenase of trout gill was monitored as a function of substrate concentration and added glutathione. In the absence of added glutathione up to 50% of the substrate consumed by the lipoxygenase was ultimately converted non-enzymatically to trihydroxy derivatives of the initial n-9 hydroperoxide enzyme product. The presence of added glutathione progressively increased conversion of the respective fatty acid hydroperoxides to the n-9 monohydroxy derivatives of arachidonic and docosahexaenoic acids while concomitantly decreasing the yield of trihydroxy derivatives, consistent with its role as a cosubstrate in the peroxidase reaction. The stability and net turnover of the lipoxygenase were also significantly improved by the addition of glutathione. The relative distribution of monohydroxy and trihydroxy products from either arachidonic acid or docosahexaenoic acid were similarly affected and equally sensitive to the glutathione concentration. These data suggest that in animals, the hydroperoxides of n-6 and n-3 polyunsaturated fatty acids generated by lipoxygenases are equally metabolized by the peroxide scavenging capabilities of the tissue.     

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimer's disease. It is characterized by the accumulation of amyloid beta peptide (Abeta) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. alpha-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of alphaA-crystallin may inhibit Abeta fibrillogenesis and toxicity. The peptide chaperone (mini-alphaA-crystallin), having an Abeta interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-alphaA-crystallin arrests the fibril formation of Abeta peptides. Mini-alphaA-crystallin also suppresses the toxic action of Abeta on rat pheochromocytoma (PC 12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-alphaA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimer's disease.     

Chronic treatment with trans resveratrol prevents intracerebroventricular streptozotocin induced cognitive impairment and oxidative stress in rats

We have recently shown free radical generation is associated with cognitive impairment in intracerebroventricular (ICV) streptozotocin (STZ) model of sporadic dementia of Alzheimer's type in rats. Trans resveratrol is a polyphenolic compound and is known to have antioxidant activity. In the present study, the effect of trans resveratrol was investigated on ICV STZ induced cognitive impairment and oxidative stress in rats. Adult male Wistar rats were injected with ICV STZ bilaterally, on day 1 and day 3. The learning and memory behavior was assessed using passive avoidance paradigms, elevated plus maze and the closed field activity test while the parameters of oxidative stress assessed were malondialdehyde [MDA] and glutathione. The rats were treated with trans resveratrol chronically at doses of 10 and 20 mg/kg,i.p. for 21 days starting from day 1 of STZ injection. Trans resveratrol treatment significantly prevented ICV STZ induced cognitive impairment. There was a rise in brain glutathione and an insignificant increase in brain MDA in trans resveratrol treated ICV STZ rats as compared to significantly elevated brain MDA levels in the vehicle treated ICV STZ animals. The study demonstrates the effectiveness of trans resveratrol in preventing the cognitive deficits as well as the oxidative stress caused by ICV STZ in rats and it's potential in the treatment of neurodegenerative diseases such as Alzheimer's disease.     

Docosahexaenoic acid (22:6, n-3) is metabolized to lipoxygenase reaction products in the retina

Docosahexaenoic acid (22:6, n-3), a major component of retinal phospholipids, is a substrate for active lipoxygenation in intact canine retinas incubated in vitro with [U-14C]docosahexaenoic acid. The major lipoxygenase reaction product was identified by high performance liquid chromatography and gas chromatography-mass spectrometry as 11-hydroxy-4,7,9-(trans)13,16,19 docosahexaenoic acid. Other mono- and di-hydroxy derivatives also were detected. The synthesis of these compounds was inhibited by the antioxidant and lipoxygenase inhibitor, nordihydroguaiaretic acid, but was not inhibited by indomethacin or esculetin.     

Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.