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Biomedical subjects

M S Wolfe

Publications and source records attributed to M S Wolfe.

At least 19 recordsLinked to original sources
PubMed2005

Benefits of organic farming to biodiversity vary among taxa.

Habitat and biodiversity differences between matched pairs of organic and non-organic farms containing cereal crops in lowland England were assessed by a large-scale study of plants, invertebrates, birds and bats. Habitat extent, composition and management on organic farms was likely to favour higher levels of biodiversity and indeed organic farms tended to support higher numbers of species and overall abundance across most taxa. However, the magnitude of the response varied; plants showed larger and more consistent responses than other taxa. Variation in response across taxa may be partly a consequence of the small size and isolated context of many organic farms. Extension of organic farming could contribute to the restoration of biodiversity in agricultural landscapes.

Agriculture↗
PubMed2005

Notch-Delta signaling is required for spatial patterning and Müller glia differentiation in the zebrafish retina.

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Müller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis.

Animals↗
PubMed2001

A portrait of Alzheimer secretases--new features and familiar faces.

The amyloid beta-peptide (Abeta) is a principal component of the cerebral plaques found in the brains of patients with Alzeheimer's disease (AD). This insoluble 40- to 42-amino acid peptide is formed by the cleavage of the Abeta precursor protein (APP). The three proteases that cleave APP, alpha-, beta-, and gamma-secretases, have been implicated in the etiology of AD. beta-Secretase is a membrane-anchored protein with clear homology to soluble aspartyl proteases, and alpha-secretase displays characteristics of certain membrane-tethered metalloproteases. gamma-Secretase is apparently an oligomeric complex that includes the presenilins, which may be the catalytic component of this protease. Identification of the alpha-, beta-, and gamma-secretases provides potential targets for designing new drugs to treat AD.

ADAM Proteins↗
PubMed2001

Gamma -secretase inhibitors repress thymocyte development.

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is gamma-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Abeta42 peptides, precipitates of which are thought to cause the disease. Gamma-secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of gamma-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of gamma-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4-/CD8- state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological gamma-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of gamma-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

Amyloid Precursor Protein Secretases↗
PubMed2001

Effect of PS1 deficiency and an APP gamma-secretase inhibitor on Notch1 signaling in primary mammalian neurons.

Presenilin1 (PS1) has been implicated in normal Notch1 processing and signaling in several experimental systems. In the present study, the relationship between PS1 and Notch1 in mammalian neurons is studied by analyzing Notch1 cleavage and C-terminal nuclear translocation as well as Notch1 signaling via the transactivation of a CBF1-luciferase reporter construct. We show that full-length Notch1 [N1(FL)] transfected into wild type (WT) primary neurons is cleaved in the presence of its biological ligand Delta (Dl) and translocated to the nucleus within 1--3 min of ligand addition. PS1 deficient neurons show normal Notch1 insertion into the cellular membrane, yet lack Notch1 activation resulting in markedly inhibited nuclear translocation of the C-terminal Notch fragment (NICD). PS1 deficient neurons also have impaired Notch1 signaling which can be restored fully or partially to levels seen in WT littermates by transfection with WT or familial Alzheimer's disease-associated M146L mutant PS1, respectively. We also show that pharmacological inhibition of PS1-associated gamma-secretase activity parallels the effects of genetic PS1 deficiency in these assays. These results support the hypothesis that PS1 deficiency blocks neuronal Notch1 processing and signaling.

Alzheimer Disease↗
PubMed2001

gamma-Secretase inhibitors as molecular probes of presenilin function.

Mutations in the presenilins cause Alzheimer's disease (AD) and alter gamma-secretase activity to increase the production of the 42-residue amyloid-beta peptide (Abeta) found disproportionally in the cerebral plaques that characterize the disease. The serpentine presenilins are required for transmembrane cleavage of both the amyloid-beta precursor protein (APP) and the Notch receptor by y-secretase, and presenilins are biochemically associated with the protease. Inhibitors of gamma-secretase have provided critical clues to the function of presenilins. Pharmacological profiling suggested that gamma-secretase is an aspartyl protease, leading to the identification of two conserved aspartates important to presenilin's role in proteolysis. Conversion of transition-state analogue inhibitors of gamma-secretase to affinity reagents resulted in specific tagging of the heterodimeric form of presenilins, strongly suggesting that the active site of gamma-secretase lies at the interface of the presenilin heterodimer. Heterodimeric presenilin appears to be the catalytic portion of a multi-protein gamma-secretase complex.

Alzheimer Disease↗
PubMed2000

Difluoro ketone peptidomimetics suggest a large S1 pocket for Alzheimer's gamma-secretase: implications for inhibitor design.

The final step in the generation of the amyloid-beta protein (Abeta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit Abeta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked Abeta production (IC(50) approximately 5 microM). This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total Abeta and Abeta(42) production with the same rank order of potency and increased Abeta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.

Alzheimer Disease↗
PubMed2000

Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid beta-protein generation.

An unusual intramembranous cleavage of the beta-amyloid precursor protein (APP) by gamma-secretase is the final step in the generation of amyloid beta-peptide (Abeta). Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Abeta production by gamma-secretase. Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of gamma-secretase, can be coimmunoprecipitated with both PS1 and PS2. PS/C83 complexes were detected in cells expressing endogenous levels of PS. The complexes accumulate when gamma-secretase is inactivated either pharmacologically or by mutating the PS aspartates. PS1/C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions. In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of gamma-secretase, occurred earlier in endoplasmic reticulum-rich vesicles. The major portion of intracellular Abeta at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Abeta levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine. Furthermore, de novo generation of Abeta in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate. Thus, PSs are complexed with the gamma-secretase substrates C83 and C99 in the subcellular locations where Abeta is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.

Amyloid Precursor Protein Secretases↗
PubMed2000

The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation.

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid beta-protein (Abeta) identified the presenilins as important mediators of the gamma-secretase cleavage of beta-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Abeta production, providing evidence that PS1 either functions as a required diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for gamma-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp --> Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived gamma-secretase substrates, C83 and C99. The production of Abeta is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp --> Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Abeta. These results explain the residual Abeta production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Abeta generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Abeta therapeutically to prevent Alzheimer's disease.

Amyloid Precursor Protein Secretases↗
PubMed2000

FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a gamma-secretase inhibitor: evidence for direct involvement of PS1 in the gamma-secretase cleavage complex.

To investigate the mechanism of regulation of Ass production by familial Alzheimer's disease (FAD)-linked presenilin 1 (PS1), we used a cell-free system that allows de novo Ass generation to examine whether PS1 participates directly in the gamma-secretase reaction. Optimal Ass generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstatin A, consistent with the suggestion that gamma-secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Ass production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the gamma-secretase cleavage event.

Alzheimer Disease↗
PubMed2000

Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1.

The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.

Affinity Labels↗
PubMed2000

Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma-secretase processing of APP to reduce Abeta production.

Amino Acid Substitution↗