sirolimus and lactacystin

Topics: Acetylcysteine; Animals; Autophagosomes; Autophagy; C9orf72 Protein; Cell Line; Gene Expression Regulation; Gene Knockout Techniques; Macrolides; Mice; Neurons; Organ Specificity; Proteasome Endopeptidase Complex; Proteolysis; Sirolimus

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, autophagy, translation, and survival. Dysregulation of mTOR signaling is associated with cancer, diabetes, and autism. However, a role for mTOR signaling in neuronal death is not well delineated. Here we show that global ischemia triggers a transient increase in mTOR phosphorylation at S2448, whereas decreasing p-mTOR and functional activity in selectively vulnerable hippocampal CA1 neurons. The decrease in mTOR coincides with an increase in biochemical markers of autophagy, pS317-ULK-1, pS14-Beclin-1, and LC3-II, a decrease in the cargo adaptor p62, and an increase in autophagic flux, a functional readout of autophagy. This is significant in that autophagy, a catabolic process downstream of mTORC1, promotes the formation of autophagosomes that capture and target cytoplasmic components to lysosomes. Inhibitors of the lysosomal (but not proteasomal) pathway rescued the ischemia-induced decrease in mTOR, consistent with degradation of mTOR via the autophagy/lysosomal pathway. Administration of the mTORC1 inhibitor rapamycin or acute knockdown of mTOR promotes autophagy and attenuates ischemia-induced neuronal death, indicating an inverse causal relation between mTOR, autophagy, and neuronal death. Our findings identify a novel and previously unappreciated mechanism by which mTOR self-regulates its own levels in hippocampal neurons in a clinically relevant model of ischemic stroke.

Topics: Acetylcysteine; Adenine; AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Beclin-1; Cells, Cultured; Hippocampus; Ischemia; Leupeptins; Lysosomes; Male; Microtubule-Associated Proteins; Neurons; Phosphorylation; Rats; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2-related inhibitory peptide; or the blocker of L-voltage-dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.

Topics: Acetylcysteine; Animals; Anisomycin; Behavior, Animal; Calcium Channel Blockers; Conditioning, Classical; Excitatory Amino Acid Antagonists; Fear; Hippocampus; Proteasome Endopeptidase Complex; Protein Kinase Inhibitors; Rats; Sirolimus; Ubiquitin

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two most important components of cellular mechanisms for protein degradation. In the present study we investigated the functional relationship of the two systems and the interactional role of p53 in vitro. Our study showed that the proteasome inhibitor lactacystin induced an increase in p53 level and autophagy activity, whereas inhibition of p53 by pifithrin-alpha or small interference RNA (siRNA) of p53 attenuated the autophagy induction and increased protein aggregation. Furthermore, we found that pretreatment with the autophagy inhibitor 3-methyladenine or beclin 1 siRNA further activated p53 and its downstream apoptotic pathways, while the autophagy inducer rapamycin showed the opposite effects. Moreover, we demonstrated that rapamycin pretreatment increased tyrosine hydroxylase (TH) protein level in dopamine (DA) neurons, which was associated with its induction of autophagy to degrade aggregated proteins. Our results suggest that p53 can mediate proteasomal inhibition-induced autophagy enhancement which in turn can partially block p53 or its downstream mitochondria-dependent apoptotic pathways. Further autophagy induction with rapamycin protects DA neurons from lactacystin-mediated cell death by downregulating p53 and its related apoptotic pathways and by inducing autophagy to degrade aggregated proteins. Therefore, rapamycin may be a promising drug for protection against neuronal injury relevant to Parkinson disease (PD). Our studies thus provide a mechanistic insight into the functional link between the two protein degradation systems.

Topics: Acetylcysteine; Apoptosis; Autophagy; Cell Line, Tumor; Cytoprotection; Dopamine; Enzyme Inhibitors; Humans; Mitochondria; Nerve Degeneration; Neurons; Phagosomes; Phosphorylation; Proteasome Inhibitors; Protein Processing, Post-Translational; Sirolimus; Time Factors; Tumor Suppressor Protein p53

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two most important cellular mechanisms for protein degradation. To investigate the role of autophagy in reversing neuronal injury, the proteasome inhibitor lactacystin was used to cause UPS dysfunction in differentiated PC12 cells and in C57BL/6 mice and rapamycin was used as an autophagy enhancer. The results showed that rapamycin pre-treatment attenuated lactacystin-induced apoptosis and reduced lactacystin-induced ubiquitinated protein aggregation in differentiated PC12 cells. The observed protection was partially blocked by the autophagy inhibitor 3-methyladenine. Furthermore, post-treatment of mice with rapamycin significantly attenuated lactacystin-induced loss of nigral DA neurons and the reduction of striatal DA levels. The lactacystin-induced high molecular ubiquitinated proteins were also attenuated by rapamycin treatment in vivo. In addition, as a chemical compound, rapamycin caused an increase of bcl2 protein level and blocked the release of cytochrome c from mitochondria to cytosal. We concluded that the neuroprotective effect of rapamycin is partially mediated by autophagy enhancement through enhanced degradation of misfolded proteins and autophagy enhancement may be considered to be a promising strategy to prevent diseases associated with misfolded/aggregated proteins, such as Parkinson's disease.

Topics: Acetylcysteine; Animals; Autophagy; Cells, Cultured; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroprotective Agents; Parkinson Disease; PC12 Cells; Protein Folding; Rats; Sirolimus

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chromones; Epidermal Growth Factor; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney Tubules; Leupeptins; Lysosomes; Methylamines; Morpholines; PAX2 Transcription Factor; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; Adenoviridae; Amino Acids; Animals; Biological Transport; Carrier Proteins; Cell Cycle Proteins; Cell Line; Cysteine Endopeptidases; Cytosol; Deoxyglucose; Down-Regulation; Enzyme Inhibitors; Eukaryotic Initiation Factors; Glucose; Humans; Immunoblotting; Insulin; Insulin Receptor Substrate Proteins; Mice; Multienzyme Complexes; Phosphoproteins; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Serine; Signal Transduction; Sirolimus; Subcellular Fractions; Threonine; Time Factors; TOR Serine-Threonine Kinases; Tyrosine

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.

Topics: 3T3 Cells; Acetylcysteine; Adenoviridae; Adipocytes; Animals; Cell Line; Cysteine Endopeptidases; Deoxyglucose; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression; Humans; Insulin; Insulin Receptor Substrate Proteins; Kidney; Mice; Multienzyme Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proteasome Endopeptidase Complex; Signal Transduction; Sirolimus; Transfection