benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Alzheimer-Disease

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Endopeptidases; Humans; Isoenzymes; Molecular Sequence Data

Topics: Acetylcholinesterase; Aluminum Chloride; Alzheimer Disease; Amyloid beta-Peptides; Animals; Butyrylcholinesterase; Cholinesterase Inhibitors; Donepezil; Dose-Response Relationship, Drug; Drug Development; Humans; Ligands; Molecular Structure; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Structure-Activity Relationship; Zebrafish

Reduced retrograde memory performance at the cognitive level and aggregation/deposition of amyloid beta (Aβ) in the brain at the cellular level are some of the hallmarks of Alzheimer's Disease (AD). A molecular system that participates in the removal of proteins with an altered conformation is the Ubiquitin-Proteasome System (UPS). Impairments of the UPS in wild-type (WT) mice lead to defective clearance of Aβ and prevent long-term plasticity of synaptic transmission. Here we show data whereby in contrast to WT mice, the inhibition of proteasome-mediated protein degradation in an animal model of AD by MG132 or lactacystin restores impaired activity-dependent synaptic plasticity and its associative interaction, synaptic tagging and capture (STC) in vitro, as well as associative long-term memory in vivo. This augmentation of synaptic plasticity and memory is mediated by the mTOR pathway and protein synthesis. Our data offer novel insights into the rebalancing of proteins relevant for synaptic plasticity which are regulated by UPS in AD-like animal models. In addition, the data provide evidence that proteasome inhibitors might be effective in reinstating synaptic plasticity and memory performance in AD, and therefore offer a new potential therapeutic option for AD treatment.

Topics: Alzheimer Disease; Animals; Behavior, Animal; Cysteine Proteinase Inhibitors; Disease Models, Animal; Leupeptins; Male; Memory Disorders; Memory, Long-Term; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuronal Plasticity; Proteasome Endopeptidase Complex

Cav1.2 is the pore-forming subunit of L-type voltage-gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen-mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β-estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin-proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29-deubiquitinating enzyme TRAF-binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane-permeable PEST peptides prevented ERα-mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα-induced Cav1.2 degradation involved K29-linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Calcium Channels, L-Type; Cell Line, Tumor; Cognitive Dysfunction; Disease Models, Animal; Estradiol; Estrogen Receptor alpha; Female; Gene Knockdown Techniques; Humans; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Oligopeptides; Phenols; Proteasome Inhibitors; Proteolysis; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Transfection; Ubiquitin; Ubiquitination

Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.

Topics: Alternative Splicing; Alzheimer Disease; Amyotrophic Lateral Sclerosis; Astrocytes; Autophagy; Brain; Cells, Cultured; Cellular Senescence; Coculture Techniques; Genetic Vectors; Humans; Interleukin-6; Leupeptins; Neurons; Neuroprotection; Protein Isoforms; RNA Interference; RNA, Small Interfering; Sequestosome-1 Protein; Serine-Arginine Splicing Factors; Tumor Suppressor Protein p53

Lithium is one of the most widely used mood-stabilizing agents for the treatment of bipolar disorder. Lithium is also a potent inhibitor of glycogen synthase kinase-3β (GSK3β) activity, which is linked to Alzheimer's disease (AD). In experiments with cultured HEK293T cells, we show here that GSK3β stabilizes synaptic acetylcholinesterase (AChE-S), a critical component of AD development. Cells treated with lithium exhibited rapid proteasomal degradation of AChE-S. Furthermore treatment of the cells with MG132, an inhibitor of the 26S proteasome, prevented the destabilizing effect of lithium on AChE-S. Taken together, these findings suggest that regulation of AChE-S protein stability may be an important biological target of lithium therapy.

Topics: Acetylcholinesterase; Alzheimer Disease; Amino Acid Substitution; Animals; Enzyme Inhibitors; Enzyme Stability; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; GPI-Linked Proteins; HEK293 Cells; Humans; Leupeptins; Lithium; Mutagenesis, Site-Directed; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Recombinant Proteins

Spatial control of RhoGTPase-inactivating GAP components remains largely enigmatic. We describe a brain-specific RhoGAP splice variant, BARGIN (BGIN), which comprises a combination of BAR, GAP, and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42-amino acid N-terminal stretch. The partial CIN domain is a poly-ubiquitin (poly-Ub)-binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as reactive oxygen species (ROS) generation by the Nox1 complex. Given that Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/Ub interactions are observed through colocalization in tangle aggregates in the Alzheimer's disease (AD) brain. Moreover, enhanced BGIN membrane distribution correlates with reduced Rac1 activity in AD brain tissue. Finally, BGIN contributes to Rac1 inhibition and ROS generation in an amyloid precursor protein (APP) proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relay poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Brain; cdc42 GTP-Binding Protein; Cell Membrane; Gene Knockdown Techniques; GTPase-Activating Proteins; HEK293 Cells; HeLa Cells; Humans; Leupeptins; Molecular Sequence Data; NADPH Oxidase 1; NADPH Oxidases; Phosphoric Monoester Hydrolases; Polyubiquitin; Proteasome Inhibitors; Protein Isoforms; Protein Structure, Tertiary; Protein Transport; rac1 GTP-Binding Protein; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

Incomplete lysosomal acidification in microglia inhibits the degradation of fibrillar forms of Alzheimer's amyloid β peptide (fAβ). Here we show that in primary microglia a chloride transporter, ClC-7, is not delivered efficiently to lysosomes, causing incomplete lysosomal acidification. ClC-7 protein is synthesized by microglia but it is mistargeted and appears to be degraded by an endoplasmic reticulum-associated degradation pathway. Activation of microglia with macrophage colony-stimulating factor induces trafficking of ClC-7 to lysosomes, leading to lysosomal acidification and increased fAβ degradation. ClC-7 associates with another protein, Ostm1, which plays an important role in its correct lysosomal targeting. Expression of both ClC-7 and Ostm1 is increased in activated microglia, which can account for the increased delivery of ClC-7 to lysosomes. Our findings suggest a novel mechanism of lysosomal pH regulation in activated microglia that is required for fAβ degradation.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cells, Cultured; Chloride Channels; Humans; Hydrogen-Ion Concentration; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Macrophage Colony-Stimulating Factor; Membrane Proteins; Mice; Mice, Transgenic; Microglia; Proteasome Inhibitors; Protein Transport; RNA Interference

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.

Topics: Aging; Alzheimer Disease; Animals; Astrocytes; Cells, Cultured; Cysteine Proteinase Inhibitors; Food, Formulated; Hydrophobic and Hydrophilic Interactions; Leupeptins; Male; Neurons; Oxidation-Reduction; Proteasome Endopeptidase Complex; Proteins; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Ubiquitination

Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad. In the present study, we assessed the SCA8 repeat size ranges in Taiwanese Parkinson's disease, Alzheimer's disease and atypical parkinsonism and investigated the genetic variation modulating ATXN8 expression. Thirteen large SCA8 alleles and a novel ATXN8 -62 G/A promoter SNP were found. There is a significant difference in the proportion of the individuals carrying SCA8 larger alleles in atypical parkinsonism (P = 0.044) as compared to that in the control subjects. In lymphoblastoid cells carrying SCA8 large alleles, treatment of MG-132 or staurosporine significantly increases the cell death or caspase 3 activity. Although expressed at low steady-state, ATXN8 expression level is significantly higher (P = 0.012) in cells with SCA8 large alleles than that of the control cells. The ATXN8 transcriptional activity was significantly higher in the luciferase reporter construct containing the -62G allele than that containing the -62A allele in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that ATXN8 gene -62 G/A polymorphism may be functional in modulating ATXN8 expression.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Base Sequence; Case-Control Studies; Cell Line; Central Nervous System Diseases; DNA Primers; Female; Gene Expression; Humans; Leupeptins; Lymphocytes; Male; Microfilament Proteins; Middle Aged; Nerve Tissue Proteins; Parkinson Disease; Parkinsonian Disorders; Polymorphism, Single Nucleotide; Pyrimidines; RNA, Long Noncoding; RNA, Untranslated; Spinocerebellar Ataxias; Staurosporine; Sulfonamides; Trinucleotide Repeat Expansion

G(q/11) protein-coupled muscarinic receptors are known to regulate the release of soluble amyloid precursor protein (sAPPalpha) produced by alpha-secretase processing; however, their signaling mechanisms remain to be elucidated. It has been reported that a muscarinic agonist activates nuclear factor (NF)-kappaB, a transcription factor that has been shown to play an important role in the Alzheimer disease brain, and that NF-kappaB activation is regulated by intracellular Ca2+ level. In the present study, we investigated whether NF-kappaB activation plays a role in muscarinic receptor-mediated sAPPalpha release enhancement and contributes to a changed capacitative Ca2+ entry (CCE), which was suggested to be involved in the muscarinic receptor-mediated stimulation of sAPPalpha release. Muscarinic receptor-mediated NF-kappaB activation was confirmed by observing the translocation of the active subunit (p65) of NF-kappaB to the nucleus by the muscarinic agonist, oxotremorine M (oxoM), in SH-SY5Y neuroblastoma cells expressing muscarinic receptors that are predominantly of the M3 subtype. NF-kappaB activation and sAPPalpha release enhancement induced by oxoM were inhibited by NF-kappaB inhibitors, such as an NF-kappaB peptide inhibitor (SN50), an IkappaB alpha kinase inhibitor (BAY11-7085), a proteasome inhibitor (MG132), the inhibitor of proteasome activity and IkappaB phosphorylation, pyrrolidine dithiocarbamate, the novel NF-kappaB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino) quinazoline), and by an intracellular Ca2+ chelator (TMB-8). Furthermore, both oxoM-induced NF-kappaB activation and sAPPalpha release were antagonized by CCE inhibitors (gadolinium or SKF96365) but not by voltage-gated Ca2+-channel blockers. On the other hand, treatment of cells with NF-kappaB inhibitors (SN50, BAY11-7085, MG132, or pyrrolidine dithiocarbamate) did not inhibit muscarinic receptor-mediated CCE. These findings provide evidence for the involvement of NF-kappaB regulated by CCE in muscarinic receptor-mediated sAPPalpha release enhancement.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Calcium; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Humans; Leupeptins; NF-kappa B; Nitriles; Peptides; Proteasome Endopeptidase Complex; Receptors, Muscarinic; Sulfones

Increasing evidence suggests that an inhibition of the proteasome, as demonstrated in Parkinson's disease, might be involved in Alzheimer's disease. In this disease and other Tauopathies, Tau proteins are hyperphosphorylated and aggregated within degenerating neurons. In this state, Tau is also ubiquitinated, suggesting that the proteasome might be involved in Tau proteolysis. Thus, to investigate if proteasome inhibition leads to accumulation, hyperphosphorylation and aggregation of Tau, we used neuroblastoma cells overexpressing Tau proteins. Surprisingly, we showed that the inhibition of the proteasome led to a bidirectional degradation of Tau. Following this result, the cellular mechanisms that may degrade Tau were investigated.

Topics: Alzheimer Disease; Antibodies, Phospho-Specific; Caspases; Cell Extracts; Cell Line, Tumor; Humans; Leupeptins; Neurons; Phosphorylation; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; tau Proteins

Epidemiologic studies indicated that non-steroidal anti-inflammatory drugs (NSAIDs) might prevent or delay the clinical features of Alzheimer disease (AD). The pharmacological activity of NSAIDs is generally attributed to inhibition of cyclooxygenase and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Based on the antineoplastic and apoptotic effects of PPARgamma activation in a number of cell types, we hypothesized that NSAIDs could protect neurons by controlling the regulation of cell cycle. Recent work suggests that uncoordinated expression of cell cycle molecules and perturbation of cell cycle checkpoints may be one of the mechanisms by which post-mitotic neurons die. Since cell cycle dysfunction is not restricted to neurons in AD, we found it interesting to study the role of PPARgamma activation on cell proliferation in immortalized lymphocytes from AD patients. We report here that 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), but not NSAIDs or thiazolidinediones inhibited the serum-mediated enhancement of cell proliferation in AD by blocking the events critical for G1/S transition. The cyclopentenone induced a partial inhibition of retinoblastoma protein phosphorylation and increased levels of the CDK inhibitor p27kip1.

Topics: Aged; Alzheimer Disease; Analysis of Variance; Apoptosis; Case-Control Studies; Cell Cycle; Cell Line; Cell Proliferation; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation; Humans; Leupeptins; Lymphocytes; Male; Peroxisome Proliferator-Activated Receptors; Phosphorylation; PPAR gamma; Prostaglandin D2; Retinoblastoma Protein; Time Factors

Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.

Topics: Alzheimer Disease; Animals; Boronic Acids; Cells, Cultured; Cysteine Endopeptidases; Fibroblasts; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Models, Animal; Multienzyme Complexes; Myocytes, Cardiac; Neurodegenerative Diseases; Neurons; Oligopeptides; Organ Specificity; Parkinson Disease; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tissue Distribution; Ubiquitin

Recent studies of gamma-secretase have pointed out that it may be comprised of a multisubunit complex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanism of this enzymatic activity will provide important information for developing gamma-secretase inhibitors in Alzheimer's disease therapy. Here we describe the biochemical characterization of gamma-secretase activities using a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressing C99, the substrate of gamma-secretase. Upon incubation at 37 degrees C, C99 is cleaved by the endogenous gamma-secretase, and Abeta peptides are liberated. Abeta40 and Abeta42 gamma-secretase activities are very similar in terms of their kinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both Abeta40 and Abeta42 production. Pepstatin A inhibited Abeta40 and Abeta42 gamma-secretase activities with similar potency. Peptide difluoroketone and peptide aldehyde inhibitors inhibited Abeta40 production in a dose-dependent fashion, enhanced Abeta42 production at low concentrations, and inhibited Abeta42 production at high concentrations. Although the selective increase of Abeta42 by low concentrations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a membrane-based assay system suggests that these compounds increase Abeta42 by a direct effect on gamma-secretase. The ability of these compounds to increase Abeta42 production may reflect allosteric modulation of the gamma-secretase complex by a mechanism related to that responsible for the increase of Abeta42 production by mutations in presenilins.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Brefeldin A; Cell Line; Cell Membrane; Cell-Free System; Endopeptidases; Enzyme Activation; Humans; Hydrolysis; Leupeptins; Membrane Proteins; Oligopeptides; Peptide Fragments; Protease Inhibitors; Protein Transport; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity