ABSTRACT
Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein to Abeta peptide. Moreover, cholesterol-rich apoE-containing lipoproteins may also promote Abeta clearance. Agonists of the liver X receptor (LXR) transcriptionally induce genes involved in intracellular lipid efflux and transport, including apoE. Thus, LXR agonists have the potential to both inhibit APP processing and promote Abeta clearance. Here we show that LXR agonist, TO901317, increased hippocampal ABCA1 and apoE and decreased Abeta42 levels in APP transgenic mice. TO901317 had no significant effects on levels of Abeta40, full length APP, or the APP processing products. Next, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the contextual memory deficit in these mice. These data demonstrate that LXR agonists do not directly inhibit APP processing but rather facilitate the clearance of Abeta42 and may represent a novel therapeutic approach to Alzheimer's disease.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , DNA-Binding Proteins/agonists , Hippocampus/metabolism , Memory/drug effects , Peptide Fragments/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Sulfonamides/pharmacology , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Alzheimer Disease/metabolism , Animals , Apolipoproteins E/metabolism , Hippocampus/drug effects , Humans , Hydrocarbons, Fluorinated , Liver X Receptors , Male , Memory/physiology , Mice , Mice, Transgenic , Orphan Nuclear Receptors , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.
Subject(s)
Apoptosis/genetics , Laser Scanning Cytometry/methods , Neurons/pathology , Serine Endopeptidases/genetics , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Caspase 3/metabolism , Caspase Inhibitors , Cells, Cultured , Cerebellum/cytology , Cerebellum/enzymology , Cerebellum/pathology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/genetics , Neurons/drug effects , Neurons/enzymology , Point Mutation , Proprotein Convertase 9 , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Serine , Serine Endopeptidases/metabolism , TransfectionABSTRACT
NAcht Leucine-rich-repeat Protein 1 (NALP1) contains a putative nucleotide binding site, a region of leucine-rich repeats, and death domain folds at both termini providing protein/protein association functions such as caspase recruitment. We report here that NALP1 gene expression was induced in primary cerebellar granule neurons (CGN) upon injury. Up-regulation of NALP1 was also observed in a model of transient focal ischemia induced by middle cerebral artery occlusion. We investigated the biological consequence of over-expression of NALP1 in both HeLa cells and in CGN. Expression of recombinant NALP1 stimulated cell death in both HeLa cells and CGN by an apoptotic mechanism, demonstrated by the induction of apoptotic nuclear morphology and activation of the apoptotic enzyme caspase-3. Also described here are studies on the mechanism of action studies including deletion analyses and investigations of nucleotide binding, which begin to elucidate a regulatory function for NALP1 in neuronal apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Cerebellum/cytology , Neurons/metabolism , Neurons/pathology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis Regulatory Proteins , Binding Sites , Caspase 3 , Caspases/metabolism , Cells, Cultured , Deoxyadenine Nucleotides/metabolism , Deoxyadenine Nucleotides/pharmacology , Enzyme Activation , HeLa Cells , Humans , Mutagenesis , NLR Proteins , Protein Binding , RNA, Messenger/metabolism , Rats , TransfectionABSTRACT
The NARC 1 gene encodes a novel proteinase K family proteinase. The domain structure of rat Narc 1 resembles that of the subtilisin-like proprotein convertases (SPCs), except that rNarc 1 lacks the canonical P-domain of SPCs, retaining only the RGD motif as part of what might be a cryptically functioning P-domain. Narc 1 undergoes autocatalytic intramolecular processing at the site LVFAQ/, resulting in the cleavage of its prosegment and the generation of an active proteinase with a broad alkaline pH optimum and no apparent calcium requirement for activity. Both primary and secondary structural determinants influence Narc 1 substrate recognition. Our functional characterization of Narc 1 reinforces the inference drawn from the analysis of its predicted structure that this enzyme is most closely related to representatives of the proteinase K family, but that it is also sufficiently different to warrant its possible classification in a separate sub-family.
