Brain Perivascular Macrophages Initiate the Neurovascular Dysfunction of Alzheimer Aß Peptides.

RATIONALE: Increasing evidence indicates that alterations of the cerebral microcirculation may play a role in Alzheimer disease, the leading cause of late-life dementia. The amyloid-ß peptide (Aß), a key pathogenic factor in Alzheimer disease, induces profound alterations in neurovascular regulation through the innate immunity receptor CD36 (cluster of differentiation 36), which, in turn, activates a Nox2-containing NADPH oxidase, leading to cerebrovascular oxidative stress. Brain perivascular macrophages (PVM) located in the perivascular space, a major site of brain Aß collection and clearance, are juxtaposed to the wall of intracerebral resistance vessels and are a powerful source of reactive oxygen species. OBJECTIVE: We tested the hypothesis that PVM are the main source of reactive oxygen species responsible for the cerebrovascular actions of Aß and that CD36 and Nox2 in PVM are the molecular substrates of the effect. METHODS AND RESULTS: Selective depletion of PVM using intracerebroventricular injection of clodronate abrogates the reactive oxygen species production and cerebrovascular dysfunction induced by Aß applied directly to the cerebral cortex, administered intravascularly, or overproduced in the brain of transgenic mice expressing mutated forms of the amyloid precursor protein (Tg2576 mice). In addition, using bone marrow chimeras, we demonstrate that PVM are the cells expressing CD36 and Nox2 responsible for the dysfunction. Thus, deletion of CD36 or Nox2 from PVM abrogates the deleterious vascular effects of Aß, whereas wild-type PVM reconstitute the vascular dysfunction in CD36-null mice. CONCLUSIONS: The data identify PVM as a previously unrecognized effector of the damaging neurovascular actions of Aß and unveil a new mechanism by which brain-resident innate immune cells and their receptors may contribute to the pathobiology of Alzheimer disease.

Stabilizing the Hsp70-Tau Complex Promotes Turnover in Models of Tauopathy.

Heat shock protein 70 (Hsp70) is a chaperone that normally scans the proteome and initiates the turnover of some proteins (termed clients) by linking them to the degradation pathways. This activity is critical to normal protein homeostasis, yet it appears to fail in diseases associated with abnormal protein accumulation. It is not clear why Hsp70 promotes client degradation under some conditions, while sparing that protein under others. Here, we used a combination of chemical biology and genetic strategies to systematically perturb the affinity of Hsp70 for the model client, tau. This approach revealed that tight complexes between Hsp70 and tau were associated with enhanced turnover while transient interactions favored tau retention. These results suggest that client affinity is one important parameter governing Hsp70-mediated quality control.

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis.

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

Disrupted SOX10 function causes spongiform neurodegeneration in gray tremor mice.

Mice homozygous for the gray tremor (gt) mutation have a pleiotropic phenotype that includes pigmentation defects, megacolon, whole body tremors, sporadic seizures, hypo- and dys-myelination of the central nervous system (CNS) and peripheral nervous system, vacuolation of the CNS, and early death. Vacuolation similar to that caused by prions was originally reported to be transmissible, but subsequent studies showed the inherited disease was not infectious. The gt mutation mapped to distal mouse chromosome 15, to the same region as Sox10, which encodes a transcription factor with essential roles in neural crest survival and differentiation. As dominant mutations in mouse or human SOX10 cause white spotting and intestinal aganglionosis, we screened the Sox10 coding region for mutations in gt/gt DNA. An adenosine to guanine transversion was identified in exon 2 that changes a highly conserved glutamic acid residue in the SOX10 DNA binding domain to glycine. This mutant allele was not seen in wildtype mice, including the related GT/Le strain, and failed to complement a Sox10 null allele. Gene expression analysis revealed significant down-regulation of genes involved in myelin lipid biosynthesis pathways in gt/gt brains. Knockout mice for some of these genes develop CNS vacuolation and/or myelination defects, suggesting that their down-regulation may contribute to these phenotypes in gt mutants and could underlie the neurological phenotypes associated with peripheral demyelinating neuropathy-central dysmyelinating leukodystrophy-Waardenburg syndrome-Hirschsprung disease, caused by mutations in human SOX10.

Age-dependent neurovascular dysfunction and damage in a mouse model of cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Accumulation of amyloid-ß in cerebral blood vessels occurs in familial and sporadic forms of cerebral amyloid angiopathy and is a prominent feature of Alzheimer disease. However, the functional correlates of the vascular pathology induced by cerebral amyloid angiopathy and the mechanisms involved have not been fully established. METHODS: We used male transgenic mice expressing the Swedish, Iowa, and Dutch mutations of the amyloid precursor protein (Tg-SwDI) to examine the effect of cerebral amyloid angiopathy on cerebrovascular structure and function. Somatosensory cortex cerebral blood flow was monitored by laser-Doppler flowmetry in anesthetized Tg-SwDI mice and wild-type littermates equipped with a cranial window. RESULTS: Tg-SwDI mice exhibited reductions in cerebral blood flow responses to whisker stimulation, endothelium-dependent vasodilators, or hypercapnia at 3 months when compared with wild-type mice, whereas the response to adenosine was not attenuated. However, at 18 and 24 months, all cerebrovascular responses were markedly reduced. At this time, there was evidence of cerebrovascular amyloid deposition, smooth muscle fragmentation, and pericyte loss. Neocortical superfusion with the free radical scavenger manganic(I-II)meso-tetrakis(4-benzoic acid) porphyrin rescued endothelium-dependent responses and functional hyperemia completely at 3 months but only partially at 18 months. CONCLUSIONS: Tg-SwDI mice exhibit a profound age-dependent cerebrovascular dysfunction, involving multiple regulatory mechanisms. Early in the disease process, oxidative stress is responsible for most of the vascular dysfunction, but with advancing disease structural alterations of the vasomotor apparatus also play a role. Early therapeutic interventions are likely to have the best chance to counteract the deleterious vascular effects of cerebral amyloid angiopathy.

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs.

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into ß-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.

LIV-1 ZIP ectodomain shedding in prion-infected mice resembles cellular response to transition metal starvation.

We recently documented the co-purification of members of the LIV-1 subfamily of ZIP (Zrt-, Irt-like Protein) zinc transporters (LZTs) with the cellular prion protein (PrP(C)) and, subsequently, established that the prion gene family descended from an ancestral LZT gene. Here, we begin to address whether the study of LZTs can shed light on the biology of prion proteins in health and disease. Starting from an observation of an abnormal LZT immunoreactive band in prion-infected mice, subsequent cell biological analyses uncovered a surprisingly coordinated biology of ZIP10 (an LZT member) and prion proteins that involves alterations to N-glycosylation and endoproteolysis in response to manipulations to the extracellular divalent cation milieu. Starving cells of manganese or zinc, but not copper, causes shedding of the N1 fragment of PrP(C) and of the ectodomain of ZIP10. For ZIP10, this posttranslational biology is influenced by an interaction between its PrP-like ectodomain and a conserved metal coordination site within its C-terminal multi-spanning transmembrane domain. The transition metal starvation-induced cleavage of ZIP10 can be differentiated by an immature N-glycosylation signature from a constitutive cleavage targeting the same site. Data from this work provide a first glimpse into a hitherto neglected molecular biology that ties PrP to its LZT cousins and suggest that manganese or zinc starvation may contribute to the etiology of prion disease in mice.

Disruption of Fnip1 reveals a metabolic checkpoint controlling B lymphocyte development.

The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.

The effect of HIV protease inhibitors on amyloid-ß peptide degradation and synthesis in human cells and Alzheimer's disease animal model.

Combined antiretroviral therapy (ART) tremendously improved the lifespan and symptoms associated with AIDS-defining illness in affected individuals. However, chronic ART-treated patients frequently develop age-dependent complications, including dementia, diabetes, and hyperlipidemia: all risk factors of Alzheimer's disease. Importantly, the effect of ART compounds on amyloid generation and clearance has never been systematically examined. Nine prescribed HIV protease inhibitors were tested for their effect on amyloid-ß peptide (Aß) clearance in primary cultured human monocyte-derived macrophages. Atazanavir, ritonavir, and saquinavir modestly inhibited of Aß degradation, while lopinavir, nelfinavir, and ritonavir enhanced secretion of undigested Aß after phagocytosis. Lopinavir, nelfinavir, ritonavir, and saquinavir inhibited endogenous Aß40 production from primary cultured human cortical neurons, which were associated with reduction in Beta-site APP Converting Enzyme 1 (BACE1) and γ-secretase enzyme activities. However, ART compounds showed little inhibition of purified BACE1 activity in vitro, suggesting the indirect effect of ART compounds on BACE1 activity in neurons. Finally, nefinavir or lopinavir/ritonavir (Kaletra) were orally administered for 30 days into APP SCID mice expressing a double mutant form of APP 695 (KM670/671NL + V717F) in homozygosity for the scid allele of Prkdc. There was no difference in beta-amyloidosis by ART drug administration as determined by both immunohistochemistry and ELISA measurements although the therapeutic doses of the ART compounds was present in the brain. These data demonstrated that ART drugs can inhibit Aß clearance in macrophages and Aß production in neurons, but these effects did not significantly alter Aß accumulation in the mouse brain.

Caspase activation precedes and leads to tangles.

Studies of post-mortem tissue have shown that the location of fibrillar tau deposits, called neurofibrillary tangles (NFT), matches closely with regions of massive neuronal death, severe cytological abnormalities, and markers of caspase activation and apoptosis, leading to the idea that tangles cause neurodegeneration in Alzheimer's disease and tau-related frontotemporal dementia. However, using in vivo multiphoton imaging to observe tangles and activation of executioner caspases in living tau transgenic mice (Tg4510 strain), we find the opposite: caspase activation occurs first, and precedes tangle formation by hours to days. New tangles form within a day. After a new tangle forms, the neuron remains alive and caspase activity seems to be suppressed. Similarly, introduction of wild-type 4-repeat tau (tau-4R) into wild-type animals triggered caspase activation, tau truncation and tau aggregation. Adeno-associated virus-mediated expression of a construct mimicking caspase-cleaved tau into wild-type mice led to the appearance of intracellular aggregates, tangle-related conformational- and phospho-epitopes, and the recruitment of full-length endogenous tau to the aggregates. On the basis of these data, we propose a new model in which caspase activation cleaves tau to initiate tangle formation, then truncated tau recruits normal tau to misfold and form tangles. Because tangle-bearing neurons are long-lived, we suggest that tangles are 'off pathway' to acute neuronal death. Soluble tau species, rather than fibrillar tau, may be the critical toxic moiety underlying neurodegeneration.

Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C) interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP(C) paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C) and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C) with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc). A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C) organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

New variants in the Enpp1 and Ptpn6 genes cause low BMD, crystal-related arthropathy, and vascular calcification.

A large genome-wide, recessive, N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW-H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G(3) hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight-adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z-score >2.8. Genome-wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately -30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.

Identification of three loci affecting HDL-cholesterol levels in a screen for chemically induced recessive mutations in mice.

We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.

The Prion Disease Database: a comprehensive transcriptome resource for systems biology research in prion diseases.

Prion diseases reflect conformational conversion of benign isoforms of prion protein (PrP(C)) to malignant PrP(Sc) isoforms. Networks perturbed by PrP(Sc) accumulation and their ties to pathological events are poorly understood. Time-course transcriptomic and phenotypic data in animal models are critical for understanding prion-perturbed networks in systems biology studies. Here, we present the Prion Disease Database (PDDB), the most comprehensive data resource on mouse prion diseases to date. The PDDB contains: (i) time-course mRNA measurements spanning the interval from prion inoculation through appearance of clinical signs in eight mouse strain-prion strain combinations and (ii) histoblots showing temporal PrP(Sc) accumulation patterns in brains from each mouse-prion combination. To facilitate prion research, the PDDB also provides a suite of analytical tools for reconstructing dynamic networks via integration of temporal mRNA and interaction data and for analyzing these networks to generate hypotheses.Database URL:http://prion.systemsbiology.net.

A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity.

Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4(-)CD8(-) (double negative) to CD4(+)CD8(+) (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor kappaB (NF-kappaB)-dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-kappaB-dependent transcription proceeds independently of Hem1 and F-actin polymerization.

Cholesterol transporter ATP-binding cassette A1 (ABCA1) is elevated in prion disease and affects PrPC and PrPSc concentrations in cultured cells.

Prion diseases are transmissible neurodegenerative disorders of prion protein (PrP) conformation. Prion replication by conversion of benign PrPC isoforms into disease-specific PrPSc isoforms is intimately involved in prion disease pathogenesis and may be initiated in cholesterol-rich caveolae-like domains (CLD). Concentrations of the cholesterol transporter ATP-binding cassette A1 protein (ABCA1) are elevated in pre-clinical scrapie prion-infected mice and in prion-infected cells in vitro. Elevation of ABCA1 in prion-infected brain is not a direct consequence of local PrPSc accumulation, indeed levels of ABCA1 are comparable in brain regions that differ dramatically in the amount of PrPSc. Similarly, ABCA1 concentrations are identical in normal mice, transgenic mice overexpressing PrP and PrP knockout mice. In contrast, PrPC and PrPSc levels, but not Prnp mRNA, were increased by overexpression of ABCA1 in N2a neuroblastoma cells and scrapie prion-infected N2a cells (ScN2a). Conversely, RNAi-mediated knock down of Abca1 expression decreased the concentrations of PrPC in N2a cells and of PrPSc in ScN2a cells. These results suggest that ABCA1's effects on PrPC levels are post-translational and may reflect an increase in of PrPC stability, mediated either indirectly by increasing membrane cholesterol and CLD formation or by other functions of ABCA1. The increased supply of PrPC available for conversion would lead to increased PrPSc formation.

Nox2-derived radicals contribute to neurovascular and behavioral dysfunction in mice overexpressing the amyloid precursor protein.

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-beta peptide (Abeta) 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.

The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections.

The cellular prion protein, PrP(C), is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('DeltaPrP'). This paradigm has facilitated mapping of activity determinants in PrP(C) and implicated a cryptic PrP(C)-like protein, 'pi'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrP(C), but is low in cerebellar granular neurons (CGNs) containing PrP(C) and high in PrP(C)-deficient dendritic processes. In Prnp(0/0) CGNs, Sho transgenes were PrP(C)-like in their ability to counteract neurotoxic effects of either Doppel or DeltaPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for pi, and since it engenders a PrP(C)-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

NADPH-oxidase-derived reactive oxygen species mediate the cerebrovascular dysfunction induced by the amyloid beta peptide.

Overproduction of the amyloid beta (Abeta) peptide is a key factor in the pathogenesis of Alzheimer's disease (AD), but the mechanisms of its pathogenic effects have not been defined. Patients with AD have cerebrovascular alterations attributable to the deleterious effects of Abeta on cerebral blood vessels. We report here that NADPH oxidase, the major source of free radicals in blood vessels, is responsible for the cerebrovascular dysregulation induced by Abeta. Thus, the free-radical production and the associated alterations in vasoregulation induced by Abeta are abrogated by the NADPH oxidase peptide inhibitor gp91ds-tat and are not observed in mice lacking the catalytic subunit of NADPH oxidase (gp91phox). Furthermore, oxidative stress and cerebrovascular dysfunction do not occur in transgenic mice overexpressing the amyloid precursor protein but lacking gp91phox. The mechanisms by which NADPH oxidase-derived radicals mediate the cerebrovascular dysfunction involve reduced bioavailability of nitric oxide. Thus, a gp91phox-containing NADPH oxidase is the critical link between Abeta and cerebrovascular dysfunction, which may underlie the alteration in cerebral blood flow regulation observed in AD patients.

In vivo reduction of amyloid-beta by a mutant copper transporter.

Cu ions have been suggested to enhance the assembly and pathogenic potential of the Alzheimer's disease amyloid-beta (Abeta) peptide. To explore this relationship in vivo, toxic-milk (txJ) mice with a mutant ATPase7b transporter favoring elevated Cu levels were analyzed in combination with the transgenic (Tg) CRND8 amyloid precursor protein mice exhibiting robust Abeta deposition. Unexpectedly, TgCRND8 mice homozygous for the recessive txJ mutation examined at 6 months of age exhibited a reduced number of amyloid plaques and diminished plasma Abeta levels. In addition, homozygosity for txJ increased survival of young TgCRND8 mice and lowered endogenous CNS Abeta at times before detectable increases in Cu in the CNS. These data suggest that the beneficial effect of the txJ mutation on CNS Abeta burden may proceed by a previously undescribed mechanism, likely involving increased clearance of peripheral pools of Abeta peptide.