ABSTRACT
Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer's disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.
Subject(s)
Adenosine/analogs & derivatives , Drug Design , Hydrolases/antagonists & inhibitors , S-Adenosylhomocysteine , Adenosine/chemistry , Adenosine/pharmacology , Animals , Brain Chemistry , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Homocysteine/blood , Hydrogen Bonding , Inhibitory Concentration 50 , Models, Molecular , Rats , S-Adenosylhomocysteine/chemistry , Substrate SpecificityABSTRACT
The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinson's disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via beta-catenin promotes the differentiation of Nurr1(+) dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that beta-catenin and Nurr1 functionally interact at multiple levels. In the absence of beta-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. Beta-catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and beta-catenin. Since KCNIP4 interacts with presenilins, the Alzheimer's disease-associated proteins that promote beta-catenin degradation, we tested the possibility that KCNIP4 induction regulates beta-catenin signaling. KCNIP4 induction limited beta-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of beta-catenin activity was absent in PS1(-/-) cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and beta-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Gene Regulatory Networks , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Wnt Proteins/metabolism , Animals , Base Sequence , Cell Line , Cell Nucleus/metabolism , Kv Channel-Interacting Proteins/metabolism , Lymphoid Enhancer-Binding Factor 1/metabolism , Mice , Models, Biological , Molecular Sequence Data , Nuclear Receptor Subfamily 4, Group A, Member 2 , Presenilin-1/metabolism , Protein Binding , Protein Processing, Post-Translational , Rats , Repressor Proteins/metabolism , beta Catenin/metabolismABSTRACT
Androgens increase muscle mass, decrease fat mass, and reduce high-density lipoprotein cholesterol (HDL), but the relationship between body composition, lipoprotein metabolism, and androgens has not been explained. Here we treated ovariectomized cynomolgus monkeys with 5alpha-dihydrotestosterone (DHT) or vehicle for 14 d and measured lipoprotein and triglycerides. Nuclear magnetic resonance analysis revealed that DHT dose-dependently reduced the cholesterol content of large HDL particles and decreased mean HDL particle size (P < 0.01) and also tended to lower low-density lipoprotein cholesterol without altering other lipoprotein particles. Liver and visceral fat biopsies taken before and after DHT treatment for 1 or 14 d were analyzed by genome-wide microarrays. In liver, DHT did not alter the expression of most genes involved in cholesterol synthesis or uptake but rapidly increased small heterodimer partner (SHP) RNA, along with concomitant repression of CYP7A1, a target of SHP transcriptional repression and the rate-limiting enzyme in bile acid synthesis. DHT regulation of SHP and CYP7A1 also occurs in rats, indicating a conserved mechanism. In adipose tissue, pathway analyses suggested coordinate regulation of adipogenesis, tissue remodeling, and lipid homeostasis. Genes encoding IGF-I and beta-catenin were induced, as were extracellular matrix, cell adhesion, and cytoskeletal components, whereas there was consistent down-regulation of genes involved in triacylglycerol metabolism. Interestingly, cholesterol ester transfer protein RNA was induced rapidly in monkey adipose tissue, whereas its inhibitor apolipoprotein CI was repressed. These data provide insight into the androgenic regulation of lipoprotein homeostasis and suggest that changes in adipose lipoprotein metabolism could contribute to HDL cholesterol reduction.
Subject(s)
Adipose Tissue/metabolism , Cholesterol, HDL/blood , Dihydrotestosterone/pharmacology , Animals , Body Composition , Cholesterol 7-alpha-Hydroxylase/genetics , Cholesterol 7-alpha-Hydroxylase/physiology , Cholesterol Ester Transfer Proteins/genetics , Cholesterol, LDL/blood , Dose-Response Relationship, Drug , Female , Liver/metabolism , Macaca fascicularis , Oligonucleotide Array Sequence Analysis , Particle Size , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/geneticsABSTRACT
Accumulation of small soluble assemblies of amyloid-ß (Aß)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.
Subject(s)
Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/biosynthesis , Immunoassay/methods , Peptide Fragments/analysis , Peptide Fragments/biosynthesis , Alzheimer Disease/immunology , Animals , Dimerization , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epitopes/genetics , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Light , Microscopy, Atomic Force , Neurons/metabolism , Protein Conformation , Rats , Reproducibility of Results , Scattering, RadiationABSTRACT
Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Genome/physiology , Ubiquitin-Protein Ligases/metabolism , Amyloid Precursor Protein Secretases/genetics , Aspartic Acid Endopeptidases/genetics , Cell Line , Cyclophilins/genetics , Cyclophilins/metabolism , Gene Expression/physiology , Humans , Luciferases/metabolism , Microarray Analysis/methods , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transfection/methods , Ubiquitin-Protein Ligases/geneticsABSTRACT
The androgen receptor (AR), when complexed with 5alpha-dihydrotestosterone (DHT), supports the survival and proliferation of prostate cells, a process critical for normal development, benign prostatic hypertrophy, and tumorigenesis. However, the androgen-responsive genetic pathways that control prostate cell division and differentiation are largely unknown. To identify such pathways, we examined gene expression in the ventral prostate 6 and 24 h after DHT administration to androgen-depleted rats. 234 transcripts were expressed significantly differently from controls (p < 0.05) at both time points and were subjected to extensive data mining. Functional clustering of the data reveals that the majority of these genes can be classified as participating in induction of secretory activity, metabolic activation, and intracellular signaling/signal transduction, indicating that AR rapidly modulates the expression of genes involved in proliferation and differentiation in the prostate. Notably AR represses the expression of several key cell cycle inhibitors, while modulating members of the wnt and notch signaling pathways, multiple growth factors, and peptide hormone signaling systems, and genes involved in MAP kinase and calcium signaling. Analysis of these data also suggested that p53 activity is negatively regulated by AR activation even though p53 RNA was unchanged. Experiments in LNCaP prostate cancer cells reveal that AR inhibits p53 protein accumulation in the nucleus, providing a post-transcriptional mechanism by which androgens control prostate cell growth and survival. In summary these data provide a comprehensive view of the earliest events in AR-mediated prostate cell proliferation in vivo, and suggest that nuclear exclusion of p53 is a critical step in prostate growth.