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1.
EXCLI J ; 16: 236-244, 2017.
Article in English | MEDLINE | ID: mdl-28507469

ABSTRACT

The purpose of the present investigation was to study the aerosolization, lungs deposition and pharmacokinetic study of inhalable submicron particles of budesonide in male Wistar rats. Submicron particles were prepared by antisolvent nanoprecipitation method and freeze-dried to obtain free flowing powder. The freeze-drying process yielded dry powder with desirable aerodynamic properties for inhalation therapy. An in-house model inhaler was designed to deliver medicine to lungs, optimized at dose level of 10 mg for 30 sec of fluidization. The in vitro aerosolization study demonstrates that submicron particles dissolve faster with improved aerosolization effect as compared to micronized budesonide. Both submicron and micron particles were compared for in vivo lungs deposition. The results showed that relatively high quantity of submicron particles reaches deep into the lungs as compared to micron particles. Most pronounced effect observed with submicron particles from pharmacokinetic parameters was the enhancement in peak plasma concentration (Cmax) by 28.85 %, and increase in area under concentration curve (AUC0-8h) by 30.33 % compared to micron sized particles. The results suggested that developed submicronized formulation of budesonide can be used for pulmonary drug delivery for high deposition to deep lungs tissues.

2.
Indian J Pharmacol ; 43(5): 582-5, 2011 Sep.
Article in English | MEDLINE | ID: mdl-22022005

ABSTRACT

OBJECTIVE: To establish the effect of Cinnamomum tamala leaves extract on diabetes and diabetes induced dyslipidemia in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Diabetes was induced by a single intravenous injection of streptozotocin (50 mg/kg body weight). Group I and II were kept as control and diabetic control respectively. And group III was further treated with ethanolic leaf extract of C. tamala (200 mg/kg body weight, orally) for a period of 40 days. Oral glucose tolerance test was performed before starting the experiment and blood glucose level was estimated. Statistical analysis was performed using one-way Analysis of Variance (using Statistical Package for the Social Sciences [SPSS] version 10.0) and student's 't'- test (Sigma Plot version 8.0). The values of P < 0.05 were considered as statistically significant. RESULTS: Treatment of diabetic animals with Cinnamomum tamala extract significantly lowered the blood glucose level, and maintained body weight and lipid-profile parameters towards near normal range. CONCLUSION: The extract exhibited antidiabetic and antidyslipidemic effect. Further, chemical and pharmacological investigations are required to elucidate the exact mechanism of action of this extract and to isolate the active principles responsible for these effects.

3.
Indian J Pharmacol ; 41(1): 23-7, 2009 Feb.
Article in English | MEDLINE | ID: mdl-20177577

ABSTRACT

OBJECTIVE: To estimate the hepatoprotective effects of the methanolic seed extract of Eugenia jambolana Lam. (Myrtaceae), in Wistar albino rats treated with carbon tetrachloride (CCl(4)). MATERIALS AND METHODS: Liver damage in rats treated with CCl(4) (1ml/kg/Bw, administered subcutaneously, on alternate days for one week) was studied by assessing parameters such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), acid phosphatase (ACP) and bilirubin (total and direct). The effect of co-administration of Eugenia jambolana Lam. (doses 100, 200 and 400 mg/kg p. o.) on the above parameters was investigated. These biochemical observations were supplemented by weight and histological examination of liver sections. Liv.52((R)) was used as positive control. Data were analyzed by one way ANOVA, followed by Scheff's/Dunnett's test. RESULTS: Administration of Eugenia jambolana Lam. (doses 100, 200 and 400 mg/kg p. o.) significantly prevented carbon tetrachloride induced elevation of serum SGOT, SGPT, ALP, ACP and bilirubin (total and direct) level. Histological examination of the liver section revealed hepatic regeneration, after administration of various doses of Eugenia jambolana Lam. The results were comparable to that of Liv.52((R)). CONCLUSION: The study suggests preventive action of Eugenia jambolana Lam. in carbon tetrachloride induced liver toxicity. Hepatic cell regeneration process was dose dependent.

4.
J Herb Pharmacother ; 6(1): 13-20, 2006.
Article in English | MEDLINE | ID: mdl-17135157

ABSTRACT

OBJECTIVE: To study the antisecretory and antiulcer activity of Asparagus racemosus Willd. (methanolic extract) and its action against indomethacin (a non-steroidal anti-inflammatory drug) plus pyloric ligation (PL)-induced gastric ulcers in rats. METHOD: Indomethacin plus PL-induced gastric ulceration model was used in the study. RESULTS: Treatment with Asparagus racemosus (Shatavari) crude extract (100 mg/kg/day orally) for fifteen days significantly reduced ulcer index when compared with control group. The reduction in gastric lesions was comparable to a standard antiulcer drug Ranitidine (30 mg/kg/ day orally). Crude extract also significantly reduced volume of gastric secretion, free acidity and total acidity. A significant increase in total carbohydrate (TC) and TC/total protein (TP) ratio of gastric juice was also observed. No significant change in the total protein was noted. CONCLUSION: Asparagus racemosus was found to be an effective antiulcerogenic agent, whose activity can well be compared with that of ranitidine hydrochloride. The results of this study suggest that Asparagus racemosus causes an inhibitory effect on release of gastric hydrochloric acid and protects gastric mucosal damage.


Subject(s)
Anti-Ulcer Agents/pharmacology , Asparagus Plant , Gastric Mucosa/drug effects , Pylorus/drug effects , Stomach Ulcer/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Ulcer Agents/administration & dosage , Dose-Response Relationship, Drug , Gastric Juice/drug effects , Indomethacin , Phytotherapy , Plant Roots , Pylorus/pathology , Ranitidine/administration & dosage , Rats , Rats, Wistar , Stomach/drug effects , Stomach Ulcer/etiology
5.
Mol Psychiatry ; 8(10): 863-78, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14515137

ABSTRACT

Presenilin 1 (PS1) plays a critical role in the nervous system development and PS1 mutations have been associated with familial Alzheimer's disease. PS1-deficient mice exhibit alterations in neural and vascular development and die in late embryogenesis. The present study was aimed at uncovering transcript networks that depend on intact PS1 function in the developing brain. To achieve this, we analyzed the brains of PS1-deficient and control animals at embryonic ages E12.5 and E14.5 using MG_U74Av2 oligonucleotide microarrays by Affymetrix. Based on the microarray data, overall molecular brain development appeared to be comparable between the E12.5 and E14.5 PS1-deficient and control embryos. However, in brains of PS1-deficient mice, we observed significant differences in the expression of genes encoding molecules that are associated with neural differentiation, extracellular matrix, vascular development, Notch-related signaling and lipid metabolism. Many of the expression differences between wild-type and PS1-deficient animals were present at both E12.5 and E14.5, whereas other transcript alterations were characteristic of only one developmental stage. The results suggest that the role of PS1 in development includes influences on a highly co-regulated transcript network; some of the genes participating in this expression network may contribute to the pathophysiology of Alzheimer's disease.


Subject(s)
Alzheimer Disease/genetics , Brain Chemistry/genetics , Brain/embryology , Brain/physiology , Membrane Proteins/genetics , Oligonucleotide Array Sequence Analysis , Alzheimer Disease/physiopathology , Animals , Female , Gene Expression Regulation, Developmental , In Situ Hybridization , Male , Mice , Mice, Mutant Strains , Pregnancy , Presenilin-1 , Transcription, Genetic
6.
Neuron ; 32(5): 911-26, 2001 Dec 06.
Article in English | MEDLINE | ID: mdl-11738035

ABSTRACT

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.


Subject(s)
Amyloid beta-Protein Precursor/analogs & derivatives , Hippocampus/growth & development , Membrane Proteins/deficiency , Membrane Proteins/genetics , Memory/physiology , Prosencephalon/growth & development , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Brain Chemistry/genetics , Electrophysiology , Hippocampus/pathology , Memory Disorders/genetics , Memory Disorders/pathology , Memory Disorders/physiopathology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Neurons/pathology , Presenilin-1 , Prosencephalon/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism
7.
J Biol Chem ; 276(47): 43756-60, 2001 Nov 23.
Article in English | MEDLINE | ID: mdl-11583985

ABSTRACT

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Endopeptidases/metabolism , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Cell Line , Molecular Sequence Data , Peptide Fragments/chemistry , Presenilin-1 , Presenilin-2 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
J Biol Chem ; 276(46): 43343-50, 2001 Nov 16.
Article in English | MEDLINE | ID: mdl-11564743

ABSTRACT

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid/chemistry , Membrane Proteins/chemistry , Membrane Proteins/genetics , Alanine/chemistry , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Biotinylation , Blotting, Western , Cell Line , Cell Membrane/metabolism , Codon , DNA, Complementary/metabolism , Endopeptidases/chemistry , Mice , Microscopy, Confocal , Microscopy, Electron , Microscopy, Fluorescence , Mutation , Peptides/chemistry , Precipitin Tests , Presenilin-1 , Protein Binding , Protein Structure, Tertiary , Protein Transport , Time Factors , Transfection , Tumor Cells, Cultured
9.
Trends Neurosci ; 24(11 Suppl): S2-6, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11881741

ABSTRACT

It is widely believed that the pathogenesis of Alzheimer's disease (AD) is intimately, if not causatively, associated with the deposition of approximately 4 kDa beta-amyloid (A beta) peptides in the cerebral cortex and hippocampus of affected individuals. A beta peptides are liberated from transmembrane proteins, termed beta-amyloid precursor proteins (APP), by the concerted action of beta- and gamma-secretase(s). Whereas the identity of beta-secretase is no longer in question, the identity of gamma-secretase, which is responsible for the intramembranous processing of APP, has never been more enigmatic. Considerable evidence has accrued to impugn the presenilins (PS) as the executioners of intramembranous processing of APP. Here, we summarize these observations and review recent evidence that argues against the prevailing hypothesis that PS function as gamma-secretases.


Subject(s)
Alzheimer Disease/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Receptors, Cell Surface , Transcription Factors , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Receptor, Notch1
11.
Neurobiol Dis ; 7(2): 99-117, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10783294

ABSTRACT

We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.


Subject(s)
Alzheimer Disease/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Subcellular Fractions/metabolism , Alzheimer Disease/pathology , Animals , Cell Culture Techniques , Cell Membrane/ultrastructure , Fluorescent Antibody Technique , Mice , Microscopy, Confocal , Microscopy, Electron , Presenilin-1 , Presenilin-2 , Subcellular Fractions/ultrastructure
12.
Neurobiol Dis ; 7(1): 54-63, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10671322

ABSTRACT

Mutations in presenilin 1 (PS1) are the most common causes of familial Alzheimer's disease (FAD). We examined synaptic physiology in hippocampal brain slices of transgenic mice expressing the FAD-linked PS1 deletion of exon 9 variant. Basal excitatory transmission and paired-pulse facilitation in PS1 mutant mice were unchanged. Short- and long-term potentiation of excitatory transmission following high-frequency stimulation were greater in transgenic mice expressing mutant PS1. Mutants had enhanced synaptic inhibition, which may be a compensatory change offsetting an abnormally sensitized plasticity of excitatory transmission. Increasing inhibitory transmission in mutant animals even more with a benzodiazepine reverted synaptic potentiation to the levels of controls. These results support the potential use of benzodiazepines in the treatment of familial Alzheimer's disease.


Subject(s)
Alzheimer Disease/genetics , Anti-Anxiety Agents/pharmacology , Excitatory Postsynaptic Potentials/physiology , Flunitrazepam/pharmacology , Hippocampus/physiology , Membrane Proteins/genetics , Synapses/physiology , Animals , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , Exons , GABA-A Receptor Antagonists , Genetic Variation , Hippocampus/drug effects , Hippocampus/physiopathology , Humans , In Vitro Techniques , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-1 , Sequence Deletion , Synapses/drug effects
13.
Nat Cell Biol ; 2(12): 863-70, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11146649

ABSTRACT

Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.


Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , Carrier Proteins/genetics , Heat-Shock Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Chaperones/genetics , Transcription Factors/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Cell Line , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , Genetic Variation , Humans , Mice , Mice, Knockout , Mice, Transgenic , Presenilin-1 , Presenilin-2 , Protein Denaturation , Protein Folding , Proteins/chemistry , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factor CHOP , Up-Regulation
14.
Neuron ; 28(2): 461-73, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11144356

ABSTRACT

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease.


Subject(s)
Alzheimer Disease/enzymology , Amyloid beta-Protein Precursor/analogs & derivatives , Amyloid beta-Protein Precursor/metabolism , Heme Oxygenase (Decyclizing)/antagonists & inhibitors , Heme Oxygenase (Decyclizing)/metabolism , Neurons/enzymology , Alzheimer Disease/etiology , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/toxicity , Animals , Bilirubin/metabolism , Binding, Competitive/genetics , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Dose-Response Relationship, Drug , Heme Oxygenase-1 , Hemin/toxicity , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Membrane Proteins , Mice , Mice, Transgenic , Mutation , Neurons/drug effects , Neurons/pathology , Oxidative Stress/genetics , Protein Structure, Tertiary/genetics , Transfection , Two-Hybrid System Techniques
15.
Ann N Y Acad Sci ; 920: 179-91, 2000.
Article in English | MEDLINE | ID: mdl-11193148

ABSTRACT

Transgenic animal models are useful in studying the features of APP- and PS1-linked FAD and SOD1-linked FALS. These models help to investigate the nature of the cellular/biochemical/molecular alterations in neural tissue; the character and evolution of neuronal and/or glial abnormalities; the ways mutant proteins cause damage to neurons; and the biochemical pathways associated with cell death. New technologies will help to define changes in a variety of genes/gene products and the events and conformational changes in mutant proteins that are implicated in pathogenic cascades. It is hoped such study will result in novel treatments for testing in transgenic models that can then be translated into new treatments for human neurodegenerative diseases.


Subject(s)
Neurodegenerative Diseases/genetics , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/deficiency , Amyloid beta-Protein Precursor/genetics , Amyloidosis/genetics , Animals , Disease Models, Animal , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Motor Neuron Disease/genetics , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/physiopathology , Presenilin-1 , Presenilin-2 , Superoxide Dismutase/genetics
16.
Ann N Y Acad Sci ; 920: 93-9, 2000.
Article in English | MEDLINE | ID: mdl-11193182

ABSTRACT

Vidal et al. (1999. Nature 399: 776-778) discovered that the underlying genetic lesion in familial British dementia (FBD) is a T-A transversion at the termination codon of a membrane protein, termed BRI. The mutation creates an arginine codon; translational read-through generates a novel protein, termed BRI-L, that is extended by 11 amino acids at the carboxyl-terminus. BRI-L is the precursor of the ABri peptide, a component of amyloid deposits in FBD brain. We demonstrate that both BRI and its mutant counterpart are constitutively processed by furin, resulting in the secretion of carboxyl-terminal peptide derivatives that correspond to all, or part of, ABri. Notably, elevated levels of peptides are generated from the mutant BRI precursor, suggesting that subtle conformational alterations at the carboxyl-terminus may influence furin-mediated processing. We have examined BRI/BRI-L processing by other members of the prohormone convertase (PC) family (PACE4, LPC, PC 5/6) and found that these enzymes also process BRI, albeit inefficiently. Moreover, BRI-L processing by the other PC members is severely compromised. Finally, our electron microscopic studies reveal that synthetic ABri peptides assemble into insoluble beta-pleated fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates amyloidogenic peptides that initiate the pathogenesis of FBD.


Subject(s)
Amyloid/genetics , Amyloid/metabolism , Brain/metabolism , Dementia/genetics , Peptide Fragments/genetics , Adaptor Proteins, Signal Transducing , Adult , Amyloid/chemistry , Animals , Cells, Cultured , Codon, Terminator , Furin , Humans , Mammals , Membrane Glycoproteins , Membrane Proteins , Mutation, Missense , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Subtilisins/metabolism , Transfection , United Kingdom
17.
J Mol Neurosci ; 15(3): 189-204, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11303783

ABSTRACT

Presenilin 1 (PS1), a polytopic membrane protein, is required for endoproteolytic processing at gamma-secretase site within the transmembrane domain of amyloid precursor proteins (APP). In addition, PS1 and its orthologues facilitate signaling of Notch family members, cell-surface receptors that specify cell fates during development. To clarify the mechanism(s) by which PS facilitates Notch signaling, we examined human Jagged-2-dependent metabolism and activity of a chimeric full-length Notchl-GFP molecule expressed in fibroblasts with heterozygous, or homozygous deletions of PS1. We demonstrate that PS1 is required for facilitating Jagged 2-mediated proteolysis and that translocation and accumulation of NICD in the nucleus correlates with signaling activity. Moreover, in a ligand-independent, Ca2+-depletion paradigm, we demonstrate that PS1 facilitates endoproteolysis of a plasma-membrane-associated, Notch1-GFP derivative. Finally, we report that NICD production is inhibited by L-685,458, a potent and selective inhibitor that blocks solubilized gamma-secretase activity and Abeta production in cultured cells. These findings strongly suggest that intramembranous processing of APP and Notch 1 are mediated by similar, if not identical, proteases that require PS1 for their activation.


Subject(s)
Alzheimer Disease/genetics , Carrier Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Peptide Hydrolases/metabolism , Receptors, Cell Surface , Signal Transduction/genetics , Transcription Factors , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases , Carbamates , Carrier Proteins/genetics , Cell Line, Transformed/cytology , Cell Line, Transformed/metabolism , Cell Membrane/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Dipeptides , Endopeptidases/drug effects , Endopeptidases/metabolism , Fetus , Fibroblasts/cytology , Fibroblasts/metabolism , Green Fluorescent Proteins , Indicators and Reagents/metabolism , Intercellular Signaling Peptides and Proteins , Jagged-2 Protein , Luminescent Proteins/metabolism , Membrane Proteins/genetics , Mice , Mice, Knockout , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Hydrolases/genetics , Presenilin-1 , Protease Inhibitors/pharmacology , Receptor, Notch1 , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Translocation, Genetic/genetics
18.
Curr Biol ; 9(24): 1493-6, 1999.
Article in English | MEDLINE | ID: mdl-10607593

ABSTRACT

Studies in invertebrates have indicated a functional requirement for presenilin (PS) genes in the Notch pathway [1-5]. One model of Notch signal transduction suggests that proteolysis releases an activated Notch fragment that migrates to the nucleus and regulates gene transcription in concert with CBF1/Su(H)/lag1 (CSL) proteins [6-9]. Recent studies suggest that PS genes control the proteolysis and nuclear access of the Notch intracellular domain [3,4,10,11], offering a basis for the functional interaction of PS and Notch genes [12]. Here, we report that Notch1 signaling elicited by the ligand Delta1 was quantitatively unchanged in PS1-deficient primary embryonic fibroblasts (PEFs). Notch1 signals were measured by both the activation of the hairy/enhancer of split (HES1) promoter and by the antagonism of MyoD-induced muscle creatine kinase (MCK) promoter activity. A membrane-tethered ligand-independent Notch1 construct also showed full efficacy in both assays, despite its presumed requirement for cleavage. Although signaling through Notch1 persisted in PS1-deficient cells, we found a marked reduction in the appearance of a complex of a cleaved, intracellular Notch fragment (NICD) and a CSL protein, as previously reported [6] [10]. These studies reveal that PS1 is not required for ligand-dependent Notch signaling, and that PS1 and PS2 may be redundant. Our data also suggest that the identified NICD fragment may not be necessary for Notch signal transduction [9].


Subject(s)
Membrane Proteins/physiology , Receptors, Cell Surface , Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Binding Sites , Cells, Cultured , Gene Expression Regulation , Homeodomain Proteins/physiology , Intracellular Signaling Peptides and Proteins , Ligands , Membrane Proteins/chemistry , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , MyoD Protein/physiology , Presenilin-1 , Presenilin-2 , Promoter Regions, Genetic , Receptor, Notch1 , Signal Transduction , Transcription Factor HES-1
20.
Nat Neurosci ; 2(11): 984-8, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10526337

ABSTRACT

The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.


Subject(s)
Amyloid/biosynthesis , Dementia/genetics , Heredodegenerative Disorders, Nervous System/genetics , Neurofibrils/metabolism , Peptide Fragments/biosynthesis , Subtilisins/physiology , Adaptor Proteins, Signal Transducing , Animals , Furin , Heredodegenerative Disorders, Nervous System/metabolism , Membrane Glycoproteins , Membrane Proteins , Mice , Microscopy, Electron , Tumor Cells, Cultured , United Kingdom
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