ABSTRACT
The Parkinson's disease (PD)-causative leucine-rich repeat kinase 2 (LRRK2) belongs to the Roco family of G-proteins comprising a Ras-of-complex (Roc) domain followed by a C-terminal of Roc (COR) domain in tandem (called Roc-COR domain). Two prokaryotic Roc-COR domains have been characterized as 'G proteins activated by guanine nucleotide-dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high-level expression of human LRRK2 Roc-COR domain (LRRK2 Roc-COR). Biochemical analyses of LRRK2 Roc-COR reveal that it forms homodimers, with the C-terminal portion of COR mediating its dimerization. Furthermore, it co-purifies and binds Mg2+ GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km and kcat of 22 nM and 4.70 × 10-4 min-1 , respectively. Thus, even though LRRK2 Roc-COR forms GAD-like homodimers, it exhibits conventional Ras-like GTPase properties, with high-affinity binding of Mg2+ -GTP/GDP and low intrinsic catalytic activity. The PD-causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full-length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc-COR. As this mutation does not directly affect the GTPase activity of the isolated Roc-COR tandem, it is possible that the effects of this mutation on full-length LRRK2 occur via other functional domains. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
Subject(s)
GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Genes, ras/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Animals , Dimerization , Escherichia coli , Gene Expression Regulation, Enzymologic/genetics , Guanine Nucleotides/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Magnesium/metabolism , Mice , Mutation/genetics , Neuropeptides/biosynthesis , Neuropeptides/genetics , Protein Multimerization , Protein Structure, Secondary/genetics , Recombinant Proteins , rac1 GTP-Binding Protein/biosynthesis , rac1 GTP-Binding Protein/geneticsABSTRACT
The abnormal accumulation of alpha-synuclein (α-syn) has been linked to a number of neurodegenerative disorders, the most noteworthy of which is Parkinson's disease. Alpha-synuclein itself is not toxic and fulfills various physiological roles in the central nervous system. However, specific types of aggregates have been shown to be toxic, and metals have been linked to the assembly of these toxic aggregates. In this paper, we have characterized a transgenic mouse that overexpresses the A53T mutation of human α-syn, specifically assessing cognition, motor performance, and subtle anatomical markers that have all been observed in synucleinopathies in humans. We hypothesized that treatment with the moderate-affinity metal chelator, clioquinol (CQ), would reduce the interaction between metals and α-syn to subsequently improve the phenotype of the A53T animal model. We showed that CQ prevents an iron-synuclein interaction, the formation of urea-soluble α-syn aggregates, α-syn-related substantia nigra pars compacta cell loss, reduction in dendritic spine density of hippocampal and caudate putamen medium spiny neurons, and the decline in motor and cognitive function. In conclusion, our data suggests that CQ is capable of mitigating the pathological metal/α-syn interactions, suggesting that the modulation of metal ions warrants further study as a therapeutic approach for the synucleinopathies.
Subject(s)
Brain/pathology , Clioquinol/therapeutic use , Cognition Disorders , Movement Disorders , Mutation/genetics , alpha-Synuclein/genetics , Animals , Brain/metabolism , Clioquinol/pharmacology , Cognition Disorders/drug therapy , Cognition Disorders/genetics , Cognition Disorders/pathology , Disease Models, Animal , Exploratory Behavior/drug effects , Humans , Maze Learning/drug effects , Mice , Mice, Transgenic , Movement Disorders/drug therapy , Movement Disorders/genetics , Movement Disorders/pathology , Protein Aggregation, Pathological/drug therapy , Protein Aggregation, Pathological/genetics , Recognition, Psychology/drug effects , Silver Staining , Spatial Learning/drug effects , alpha-Synuclein/metabolismABSTRACT
Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.
Subject(s)
Bacterial Proteins/chemistry , Parkinson Disease/enzymology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protozoan Proteins/chemistry , Animals , Binding Sites , Conserved Sequence , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/genetics , Protein Structure, SecondaryABSTRACT
Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.
Subject(s)
Mutation , Parkinson Disease/genetics , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Computational Biology , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Protein Serine-Threonine Kinases/genetics , Sequence Homology, Amino AcidABSTRACT
Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1.
Subject(s)
Protein Kinases/genetics , Protein Structure, Tertiary , Animals , Catalytic Domain , Humans , Mitochondria/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Phosphorylation , Protein Kinases/metabolism , Protein Transport , Substrate SpecificityABSTRACT
Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.
Subject(s)
Cognition/drug effects , Motor Activity/drug effects , Neuroprotective Agents/therapeutic use , Organometallic Compounds/therapeutic use , Parkinson Disease/drug therapy , Radiopharmaceuticals/therapeutic use , Thiosemicarbazones/therapeutic use , Animals , Cell Line, Tumor , Coordination Complexes , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Neuroprotective Agents/pharmacology , Organometallic Compounds/pharmacology , Parkinson Disease/psychology , Positron-Emission Tomography , Radiopharmaceuticals/pharmacology , Rats , Rats, Sprague-Dawley , Thiosemicarbazones/pharmacology , alpha-Synuclein/chemistryABSTRACT
We have previously identified presenilin-1 (PS1), the active component of the γ-secretase complex, as an interacting protein of the amyloid-associated enzyme acetylcholinesterase (AChE). In this study, we have explored the consequences of AChE-PS1 interactions. Treatment of SH-SY5Y cells with the AChE-inhibitor tacrine decreased PS1 levels, in parallel with increase in the secretion of amyloid precursor protein APPα, whereas the cholinergic agonist carbachol had no effect on PS1. AChE knockdown with siRNA also decreased PS1 levels, while AChE overexpression exerted opposing effect. AChE-deficient also had decreased PS1. Mice administered with tacrine or donepezil displayed lower levels of brain PS1. However, sustained AChE inhibition failed to exert long-term effect on PS1. This limited duration of response may be due to AChE upregulation caused by chronic inhibition. Finally, we exposed SH-SY5Y cells to ß-amyloid (Aß)42 which triggered elevation of both AChE and PS1 levels. The Aß42-induced PS1 increase was abolished by siRNA AChE pretreatment, suggesting that AChE may participate in the pathological feedback loop between PS1 and Aß. Our results provide insight into AChE-amyloid interrelationships.
Subject(s)
Acetylcholinesterase/biosynthesis , Acetylcholinesterase/genetics , Presenilin-1/antagonists & inhibitors , Presenilin-1/metabolism , Acetylcholinesterase/metabolism , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/toxicity , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Line, Tumor , Down-Regulation/genetics , Down-Regulation/physiology , Feedback, Physiological/physiology , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/biosynthesis , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Knockdown Techniques , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred ICR , Mice, Knockout , Neuroblastoma/genetics , Neuroblastoma/metabolism , Peptide Fragments/toxicity , Presenilin-1/deficiency , RNA, Small Interfering/genetics , Up-Regulation/geneticsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Organometallic Compounds/chemistry , Peroxynitrous Acid/metabolism , Superoxide Dismutase/genetics , Thiosemicarbazones/chemistry , Animals , Antioxidants/chemistry , Astrocytes/cytology , Coordination Complexes , Copper/chemistry , DNA-Binding Proteins/pharmacology , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/cytology , Neurodegenerative Diseases/embryology , Neurons/metabolism , Oxidative Stress , Oxygen/chemistry , Spinal Cord/pathology , Superoxide Dismutase-1 , TransgenesABSTRACT
C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed ß-synuclein as a potential CHK substrate, and Y127 in ß-synuclein as the preferential phosphorylation site. Using peptides derived from ß-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides ß-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Structural Homology, Protein , beta-Synuclein/chemistry , src Homology Domains , Amino Acid Motifs , Amino Acid Sequence , Animals , CSK Tyrosine-Protein Kinase , Catalytic Domain , Cytosol/enzymology , Cytosol/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Peptide Library , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Rats , Substrate Specificity , beta-Synuclein/metabolism , src-Family KinasesABSTRACT
Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.
Subject(s)
Bacterial Proteins/metabolism , Mitochondria/metabolism , Animals , Bacterial Proteins/chemistry , Base Sequence , DNA Primers , Mice , Polymerase Chain Reaction , Signal TransductionABSTRACT
Parkinson's disease (PD) is a severe neurodegenerative disorder characterised by loss of dopaminergic neurons of the substantia nigra. The pathological hallmarks are cytoplasmic inclusions termed Lewy bodies consisting primarily of aggregated alpha-synuclein (alphaSN). Different lines of transgenic mice have been developed to model PD but have failed to recapitulate the hallmarks of this disease. Since treatment of rodents with the pesticide rotenone can reproduce nigrostriatal cell loss and other features of PD, we aimed to test chronic oral administration of rotenone to transgenic mice over-expressing human alphaSN with the A53T mutation. Initial assessment of this transgenic line for compensatory molecular changes indicated decreased brain beta-synuclein expression and significantly increased levels of the PD-associated oxidative stress response protein, DJ-1, and the E3 ubiquitin ligase enzyme, Parkin. Rotenone treatment of 30 mg/kg for 25 doses over a 35-day period was tolerated in the transgenic mice and resulted in decreased spontaneous locomotor movement and increased cytoplasmic alphaSN expression. The mitochondrial Parkinson's-associated PTEN-induced kinase 1 protein levels were also increased in transgenic mouse brain after rotenone treatment; there was no change in brain dopamine levels or nigrostriatal cell loss. These hA53T alphaSN transgenic mice provide a useful model for presymptomatic Parkinson's features and are valuable for study of associated compensatory changes in early Parkinson's disease stages.
Subject(s)
Oncogene Proteins/genetics , Parkinsonian Disorders/genetics , Protein Kinases/genetics , Rotenone/toxicity , Ubiquitin-Protein Ligases/genetics , Uncoupling Agents/toxicity , Up-Regulation/drug effects , alpha-Synuclein/genetics , Animals , Disease Models, Animal , Humans , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Oncogene Proteins/biosynthesis , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/physiopathology , Peroxiredoxins , Protein Deglycase DJ-1 , Protein Kinases/biosynthesis , Ubiquitin-Protein Ligases/biosynthesis , alpha-Synuclein/biosynthesisABSTRACT
Gamma-secretase is involved in the production of Aß amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aß and the APP intracellular domain (AICD). Metal ions play an important role in Aß aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg(2+) stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca(2+) and Mg(2+) stabilize γ-secretase and enhance its activity.
ABSTRACT
1. The Src-family protein tyrosine kinases (SFKs) are multidomain oncogenic protein tyrosine kinases. Their overactivation contributes to cancer formation and progression. Thus, synthetic inhibitors of SFKs are being developed as therapeutics for cancer treatment. Understanding the regulatory and catalytic mechanisms of SFKs is necessary for the development of therapeutic SFK inhibitors. 2. Although many upstream regulators and protein substrates of SFKs have been identified, both the mechanisms of activation and catalysis of SFKs are not fully understood. In particular, it is still unclear how the inactive SFKs undergo conformational transition during activation. The mechanism governing the binding of substrates and the release of products during catalysis is another area that requires investigation. 3. Several recent publications indicate the presence of a 'hydrophobic spine' formed by four conserved interacting hydrophobic residues in the kinase domain of SFKs. In the present review, we discuss how the assembly and disassembly of the hydrophobic spine residues may govern conformational transition of SFKs during activation. In addition to regulation of kinase activity, the hydrophobic spine is implicated to be involved in catalysis. It has been postulated recently that perturbation of the hydrophobic spine residues is a key step in catalysis. 4. Further investigations to decipher the roles of the hydrophobic spine residues in regulation and catalysis of SFKs will benefit the development of therapeutic SFK inhibitors for cancer treatment.
Subject(s)
Allosteric Site/genetics , Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism , CSK Tyrosine-Protein Kinase , Catalysis , Drug Delivery Systems/methods , Humans , Models, Biological , Mutation/physiology , Protein Conformation , Protein Kinases/genetics , Protein Kinases/metabolism , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins pp60(c-src)/physiology , Signal TransductionABSTRACT
OBJECTIVE: This study questions whether increased dopamine (DA) turnover in nigral neurons leads to formation of Lewy bodies (LBs), the characteristic alpha-synuclein-containing cytoplasmic inclusion of Parkinson disease (PD). METHODS: Mice with targeted deletion of the dopamine D(2) receptor gene (D(2)R[-/-]) have higher striatal and nigral dopamine turnover and elevated oxidative stress. These mice were examined for evidence of histological, biochemical, and gene expression changes consistent with a synucleinopathy. RESULTS: LB-like cytoplasmic inclusions containing alpha-synuclein and ubiquitin were present in substantia nigra pars compacta (SNpc) neurons of older D(2)R(-/-) mice, and were also occasionally seen in aged wild-type mice. These inclusions displaced the nucleus of affected cells and were eosinophilic. Diffuse cytosolic alpha-synuclein immunoreactivity in SNpc neurons increased with age in both wild-type and D(2)R(-/-) mice, most likely because of redistribution of alpha-synuclein from striatal terminals to SNpc cell bodies. Gene and protein expression studies indicated endoplasmic reticulum (ER) stress and changes in trafficking and autophagic pathways in D(2)R(-/-) SNpc. These changes were accompanied by a loss of DA terminals in the dorsal striatum, although there was no evidence of progressive cell death in the SNpc. INTERPRETATION: Increased sprouting and DA turnover, as observed in PD and D(2)R(-/-) mice, augments LB-like inclusions and axonal degeneration of dopaminergic neurons. These changes are associated with ER stress and autophagy.
Subject(s)
Parkinson Disease/genetics , Parkinson Disease/metabolism , Receptors, Dopamine D2/deficiency , Receptors, Dopamine D2/genetics , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/genetics , Parkinson Disease/physiopathology , Receptors, Dopamine D2/physiology , Substantia Nigra/metabolism , Substantia Nigra/pathology , Substantia Nigra/physiopathology , alpha-Synuclein/metabolismABSTRACT
BACKGROUND: Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. beta-cells depend on zinc for both insulin crystallization and regulation of cell mass. METHODOLOGY/PRINCIPAL FINDINGS: This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in beta-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a beta-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced beta-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals. CONCLUSION/SIGNIFICANCE: Zinc transporting proteins in beta-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.
Subject(s)
Carrier Proteins/metabolism , Glucose/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Membrane Proteins/metabolism , Stress, Physiological/drug effects , Zinc/pharmacology , Animals , Blood Glucose/drug effects , Carrier Proteins/genetics , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Death/drug effects , Cell Line , Chelating Agents/pharmacology , Dose-Response Relationship, Drug , Fasting/blood , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Glucose/metabolism , Hyperglycemia/metabolism , Insulin/genetics , Insulin Secretion , Insulin-Secreting Cells/cytology , Membrane Proteins/genetics , Membrane Transport Proteins , Mice , Mice, Knockout , Rats , Streptozocin , Zinc Transporter 8ABSTRACT
Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), beta-amyloid peptide (Abeta) aggregation, and amyloid formation. Abeta.copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the beta-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. beta- and gamma-secretases, and Abeta have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Abeta synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Abeta.copper complexes are more likely to form. This explains the paradoxical hypermetallation of Abeta with copper under tissue copper deficiency conditions in AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Copper/deficiency , Gene Expression Regulation , Membrane Microdomains/metabolism , Animals , Brain/metabolism , Cell Line, Tumor , Cholesterol/metabolism , Copper/metabolism , DNA, Complementary/metabolism , Endocytosis , Humans , Mice , Mice, Transgenic , Models, BiologicalABSTRACT
BACKGROUND: Retinal ganglion cell loss is considered to be a cause of visual impairment in Alzheimer;s patients. Alterations in amyloid precursor protein (APP) processing and amyloid-beta (Abeta) accumulation, key molecules associated with Alzheimer;s disease pathogenesis, may therefore contribute to retinal damage. We therefore investigated retinal APP processing and eye morphology in Alzheimer;s transgenic mouse models. METHODS: Eyes and brain samples of 2- to 18-month-old transgenic mice expressing human APP with the double Swedish mutation (APPswe) (APP K595N/M596L)(Tg2576) were compared with eyes and brain tissue from wild-type background C57BL6xSJL controls. In addition, 6- to 12-month-old double transgenic mice over-expressing human APPswe and mutant presenilin 1 with exon 9 deletion (APPswe/PS1-dE9) were compared with background controls of C57BL6xC3H strain. Tissue samples were fixed in formalin for immunohistochemistry, and dissected retinal and cerebellar extracts were frozen for Western blotting and enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies 1E8 and WO2 were used for immunohistochemical detection of APP and Abeta, whereas Abeta 42/40 levels were assayed by ELISA. APP and processed fragments were detected biochemically by Western blotting with domain-specific antibodies, using antibody WO2 (Abeta) and rabbit antibody 369 to the C-terminal domain of APP. RESULTS: Immunocytochemistry revealed strong cytoplasmic expression of APP and possibly Abeta in retinal ganglion cells and inner nuclear layer cells, and in lens and corneal epithelia for APP transgenic mice. Retinas from the APP transgenic mouse strains contained 18 to 70 kDa APP proteolytic products that were not detected in the cerebellum. We found a higher proportion of APP alpha-secretase generated C-terminal fragments in transgenic retinal tissues than beta-secretase-generated C-terminal fragments. Very low level Abeta was detected in transgenic retinas by ELISA; retinal Abeta 42 was 75 times less than for transgenic brain. Abeta was not detected in mouse retina by Western blotting in our study, indicating much less generation of Abeta in retina than brain tissue. CONCLUSIONS: Alzheimer's mouse model retinas present with different APP proteolytic products and have a significantly lower production of amyloidogenic Abeta than found in brain.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Disease Models, Animal , Retinal Diseases/metabolism , Retinal Ganglion Cells/metabolism , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases , Animals , Blotting, Western , Cerebral Cortex/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelium, Corneal/metabolism , Lens, Crystalline/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Retinal Diseases/pathology , Retinal Ganglion Cells/pathologyABSTRACT
Up to 40% of Parkinson's disease patients suffer from anxiety, but little is known about the mechanisms involved. We used the elevated plus maze and open field test to evaluate groups of young adult mice expressing different levels of alpha-synuclein, including mice transgenic for human alpha-synuclein with the A53T mutation. Compared to alpha-synuclein knock-out mice and wild-type controls, alpha-synuclein A53T transgenic mice exhibited reduced anxiety-like behaviour by spending markedly greater amounts of time on the maze open arms and by a higher proportion of entries to the open arms. In the open field, transgenic mice showed a trend towards reduced locomotor habituation and increased thigmotaxis. These results indicate a possible role for alpha-synuclein in anxiety-like behaviours.
Subject(s)
Anxiety/genetics , Anxiety/physiopathology , Mutation/physiology , alpha-Synuclein/genetics , Analysis of Variance , Animals , Behavior, Animal/physiology , Exploratory Behavior/physiology , Humans , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/geneticsABSTRACT
Presenilin 1 (PS1) plays a critical role in the gamma-secretase processing of the amyloid precursor protein to generate the beta-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships.
Subject(s)
Acetylcholinesterase/metabolism , Presenilin-1/metabolism , Acetylcholinesterase/genetics , Adult , Aged , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Animals , Brain/metabolism , Brain/pathology , COS Cells , Cells, Cultured , Chlorocebus aethiops , Embryo, Mammalian , Female , Glycosylation , Humans , Immunohistochemistry , Immunoprecipitation , Male , Mice , Mice, Transgenic , Middle Aged , Mutation , Presenilin-1/genetics , Protein Binding , Tissue Extracts/metabolismABSTRACT
Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis.