73 results on '"R Jane, Rylett"'
Search Results
2. SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins
- Author
-
Trevor M. Morey, Mohammad Ali Esmaeili, Martin L. Duennwald, and R. Jane Rylett
- Subjects
protein stability and degradation ,protein half-life ,pulse-chase analysis ,click chemistry ,SPAAC ,mammalian cells ,Biology (General) ,QH301-705.5 - Abstract
Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.
- Published
- 2021
- Full Text
- View/download PDF
3. Into the Fourth Dimension: Dysregulation of Genome Architecture in Aging and Alzheimer’s Disease
- Author
-
Warren Winick-Ng and R. Jane Rylett
- Subjects
Alzheimer’s disease ,epigenetics ,gene regulation ,genome architecture ,protein mislocalization ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons. Dysregulation of epigenetic processes have been observed in the aging brain and in patients with AD, though there is not yet agreement on the impact of these changes on transcription. New evidence shows that proteins involved in genome organization have altered expression and localization in the AD brain, suggesting that the genomic landscape may play a critical role in the development of AD. This review discusses the role of the chromatin organizers and epigenetic modifiers in post-mitotic cells, the aging brain, and in the development and progression of AD. How these new insights can be used to help determine disease risk and inform treatment strategies will also be discussed.
- Published
- 2018
- Full Text
- View/download PDF
4. Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP
- Author
-
Trevor M. Morey, Warren Winick-Ng, Claudia Seah, and R. Jane Rylett
- Subjects
choline acetyltransferase ,BioID ,heat shock proteins ,ubiquitination ,CHIP/Stub1 ,proximity-ligation assay ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease.
- Published
- 2017
- Full Text
- View/download PDF
5. Amino-Terminal β-Amyloid Antibody Blocks β-Amyloid-Mediated Inhibition of the High-Affinity Choline Transporter CHT
- Author
-
Leah K. Cuddy, Claudia Seah, Stephen H. Pasternak, and R. Jane Rylett
- Subjects
protein trafficking ,cholinergic ,β-amyloid ,Alzheimer’s disease ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Alzheimer’s disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived β-amyloid peptides (Aβ) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aβ peptides released from neural cells. The Aβ-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA1). BafA1 also attenuated the Aβ-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aβ on CHT activity. Importantly, neutralizing Aβ using an anti-Aβ antibody directed at the N-terminal amino acids 1–16 of Aβ, but not by an antibody directed at the mid-region amino acids 22–35 of Aβ, attenuates the effect of Aβ on CHT activity and trafficking. This indicates that a specific N-terminal Aβ epitope, or specific conformation of soluble Aβ, may impair CHT activity. Therefore, Aβ immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.
- Published
- 2017
- Full Text
- View/download PDF
6. Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice
- Author
-
Shawn Albers, Fatima Inthathirath, Sandeep K. Gill, Warren Winick-Ng, Ewa Jaworski, Daisy Y.L. Wong, Robert Gros, and R. Jane Rylett
- Subjects
Alzheimer disease ,Amyloid ,Amyloidogenic ,BACE1 ,Cholinergic ,Choline acetyltransferase ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1–42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1–42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons.
- Published
- 2014
- Full Text
- View/download PDF
7. La COVID-19 et les priorités de recherche sur le vieillissement
- Author
-
Joanne Goldberg, Susan Rogers, Patricia Versteegh, Flamine Alary, and R. Jane Rylett
- Subjects
2019-20 coronavirus outbreak ,Health (social science) ,Coronavirus disease 2019 (COVID-19) ,adultes plus âgés ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Article ,Health(social science) ,03 medical and health sciences ,0302 clinical medicine ,Institut du vieillissement ,Political science ,Institute of Aging ,030212 general & internal medicine ,older adults ,research prioritization ,030304 developmental biology ,Community and Home Care ,0303 health sciences ,Instituts de recherche en santé du Canada ,aging ,Canadian Institutes of Health Research ,vieillissement ,Geriatrics and Gerontology ,Covid-19 ,Gerontology ,Humanities ,priorisation de la recherche - Abstract
RésuméCet article présente les domaines prioritaires de recherche sur les impacts de la pandémie de COVID-19 chez les personnes âgées telles qu’ils ont été identifiés par l’Institut du vieillissement des IRSC (IV-IRSC). Le processus utilisé par l’IV-IRSC a comporté plusieurs phases itératives qui ont permis d’identifier trois secteurs prioritaires parmi les besoins de la recherche relative à la COVID-19, et quatre axes thématiques transversaux. Les secteurs de recherche prioritaires sont : 1) la réponse des personnes âgées à la maladie, à la vaccination et aux traitements, 2) la santé mentale et l’isolement, et 3) les milieux de soins soutenants. Les quatre thèmes transversaux sont : a) l’Équité, la diversité et l’inclusion (EDI), b) les considérations éthiques et morales, c) les pratiques fondées sur les données probantes, et d) les technologies numériques de la santé. Les priorités décrites dans cet article guideront les réponses de l’IV-IRSC aux besoins de la recherche sur la COVID-19.
- Published
- 2020
8. SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins
- Author
-
Mohammad Ali Esmaeili, R. Jane Rylett, Martin L. Duennwald, and Trevor M. Morey
- Subjects
protein stability and degradation ,protein half-life ,QH301-705.5 ,Cell ,Computational biology ,yeast ,pulse-chase analysis ,Cell and Developmental Biology ,03 medical and health sciences ,0302 clinical medicine ,Methods ,medicine ,SPAAC ,mammalian cells ,Biology (General) ,Cytotoxicity ,Cellular proteins ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Measurement method ,Chemistry ,Cell Biology ,Yeast ,Amino acid ,medicine.anatomical_structure ,click chemistry ,Click chemistry ,Bioorthogonal chemistry ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.
- Published
- 2021
9. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.
- Author
-
Katherine J Fishwick and R Jane Rylett
- Subjects
Medicine ,Science - Abstract
Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.
- Published
- 2015
- Full Text
- View/download PDF
10. Infiltrating Hematogenous Macrophages Aggregate Around β-Amyloid Plaques in an Age- and Sex-Dependent Manner in a Mouse Model of Alzheimer Disease
- Author
-
Gregory A. Dekaban, Shawn Albers, R. Jane Rylett, Jennifer Yang, Natalie Kozyrev, Marco A. M. Prado, Vania F. Prado, and Gregory J. Fonseca
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Amyloid ,Inflammation ,Mice, Transgenic ,Plaque, Amyloid ,Biology ,Pathology and Forensic Medicine ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mice ,0302 clinical medicine ,Alzheimer Disease ,medicine ,Macrophage ,Animals ,Neuroinflammation ,030304 developmental biology ,0303 health sciences ,Amyloid beta-Peptides ,Microglia ,Macrophages ,Neurotoxicity ,Brain ,General Medicine ,medicine.disease ,Disease Models, Animal ,medicine.anatomical_structure ,Neurology ,Gliosis ,Female ,Neurology (clinical) ,medicine.symptom ,Alzheimer's disease ,030217 neurology & neurosurgery - Abstract
β-Amyloid (Aβ) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMϕ) from hematogenous macrophages (hMϕ), as they are morphologically and phenotypically similar when activated. To distinguish between mMϕ and hMϕ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMϕ, but not in mMϕ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMϕ from hMϕ. Mutant mice demonstrated robust increases in Aβ1–42, area of Aβ plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aβ accumulation, paralleled by the accumulation of hMϕ around Aβ plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aβ plaques was age- and sex-dependent and preceded cognitive impairment.
- Published
- 2020
11. Integrating sex and gender into neurodegeneration research: A six-component strategy
- Author
-
Howard Chertkow, R. Jane Rylett, Ashley F. Curtis, and Mary C. Tierney
- Subjects
Sex and gender guidelines ,Gerontology ,Best practice ,Gender ,Featured Article ,medicine.disease ,3. Good health ,03 medical and health sciences ,Psychiatry and Mental health ,0302 clinical medicine ,Gender research ,5. Gender equality ,CCNA ,Sex differences ,medicine ,Dementia ,Sex ,030212 general & internal medicine ,Neurology (clinical) ,Neurodegeneration ,10. No inequality ,Psychology ,Inclusion (education) ,030217 neurology & neurosurgery - Abstract
Introduction Despite important sex differences, there remains a paucity of studies examining sex and gender differences in neurodegeneration. The Canadian Consortium on Neurodegeneration in Aging (CCNA), a national network of researchers, provides an ideal platform to incorporate sex and gender. Methods CCNA's Women, Gender, Sex and Dementia program developed and implemented a six-component strategy involving executive oversight, training, research collaboration, progress report assessment, results dissemination, and ongoing manuscript review. The inclusion of sex and gender in current and planned CCNA projects was examined in two progress reporting periods in 2016. Results Sex and gender research productivity increased substantially for both preclinical (36%–45%) and human (56%–60%) cohorts. The main barrier was lack of funding. Discussion The Women, Gender, Sex and Dementia strategy resulted in a major increase of sex and gender into research on neurodegenerative disorders. This best practice model could be utilized by a wide variety of large multidisciplinary groups., Highlights • There is a paucity of research on sex and gender in neurodegenerative disorders. • A six-component strategy to integrate sex and gender into research was developed. • Strategy implemented in national network of researchers studying neurodegeneration. • Sex and gender research productivity increased for preclinical and human research. • Strategy could be adapted and utilized across various multidisciplinary groups.
- Published
- 2017
12. Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants
- Author
-
Shawn Albers, R. Jane Rylett, Brian H. Shilton, and Trevor M. Morey
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Immunoprecipitation ,education ,Protein degradation ,Biochemistry ,Catalysis ,Choline O-Acetyltransferase ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Ubiquitin ,mental disorders ,medicine ,Animals ,Humans ,Cells, Cultured ,health care economics and organizations ,biology ,Ubiquitination ,Choline acetyltransferase ,Molecular biology ,Cholinergic Neurons ,humanities ,030104 developmental biology ,nervous system ,Proteasome ,Mutation ,Proteolysis ,Proteasome inhibitor ,biology.protein ,Cholinergic ,030217 neurology & neurosurgery ,Acetylcholine ,medicine.drug - Abstract
Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.
- Published
- 2016
13. Editorial: Aging in Indigenous Populations
- Author
-
R. Jane Rylett
- Subjects
Community and Home Care ,Aging ,Canada ,Community-Based Participatory Research ,Health (social science) ,Academies and Institutes ,MEDLINE ,Library science ,Community-based participatory research ,Indigenous ,Humans ,Indigenous Canadians ,Sociology ,Geriatrics and Gerontology ,Gerontology ,Introductory Journal Article - Published
- 2019
14. Mobility and Cognition in Seniors. Report from the 2008 Institute of Aging (CIHR) Mobility and Cognition Workshop
- Author
-
Manuel Montero-Odasso, David Galinsky, Paul Stoole, Aleksandra Zecevic, Karen Gopaul, Mark Speechley, Jennie Wells, Richard Camicioli, Louis Bherer, Afua Oteng-Amoako, Susan W. Muir-Hunter, Jeffrey W. Jutai, Sarah Woolmore-Goodwin, Stephanie A. Studenski, R. Jane Rylett, and Timothy J. Doherty
- Subjects
Gerontology ,Physical Therapy ,business.industry ,Psychological intervention ,Reviews ,Poison control ,Human factors and ergonomics ,Standardized test ,Cognition ,dual-task gait ,Suicide prevention ,mobility ,MCI ,gait variability ,exercises ,Mood ,executive function ,falls ,Parkinson’s disease ,Medicine ,Geriatrics and Gerontology ,Cognitive decline ,business - Abstract
Background The annual Scientific Meeting of the Canadian Association on Gerontology was held on October 24 and 25, 2008 in London, Ontario. Prior to the annual meeting, mobility and cognition experts met on October 23, 2008 to engage in a pre-conference workshop. Methods Discussions during the workshop addressed novel areas of research and knowledge and research gaps pertaining to the interaction between mobility and cognition in seniors. Results Workshop presenters moved from the neuromuscular, biomechanics, and neurology of gait impairments, and falls through the role of cognition and mood on mobility regulation to the whole person in the environment. Research gaps were identified. Conclusions Despite a consensus that mobility and cognition are increasingly correlated as people age, several gaps in our understanding of mechanisms and how to assess the interaction were recognized. The gaps originally identified in 2008 are still pertinent today. Common and standardized assessments for “mobility and cognition” are still not in place in current practice. Interventions that target mobility and cognitive decline as a single entity are still lacking.
- Published
- 2015
15. P2‐333: Integration of Sex and Gender in the Research of the Canadian Consortium on Neurodegeneration in Aging (CCNA)
- Author
-
Mary C. Tierney, R. Jane Rylett, Nicolette Baines, Cara Tannenbaum, Lynn Posluns, and Howard Chertkow
- Subjects
Gerontology ,Psychiatry and Mental health ,Cellular and Molecular Neuroscience ,Developmental Neuroscience ,Epidemiology ,CCNA ,Health Policy ,Neurodegeneration ,medicine ,Neurology (clinical) ,Geriatrics and Gerontology ,medicine.disease ,Psychology - Published
- 2016
16. P1‐020: The Role of Neuroinflammation in Alzheimer's Disease
- Author
-
Shawn Albers, Vania F. Prado, Natalie Kozyrev, Shuxin Zhang, R. Jane Rylett, and Gregory A. Dekaban
- Subjects
Psychiatry and Mental health ,Cellular and Molecular Neuroscience ,Developmental Neuroscience ,Epidemiology ,business.industry ,Health Policy ,Medicine ,Neurology (clinical) ,Disease ,Geriatrics and Gerontology ,business ,Neuroscience ,Neuroinflammation - Published
- 2016
17. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions
- Author
-
Warren Winick-Ng, Bryan Heit, Trevor M. Morey, Jennifer Winick-Ng, R. Jane Rylett, and Fabiana A. Caetano
- Subjects
0301 basic medicine ,Chromatin Immunoprecipitation ,education ,Biology ,Article ,Cell Line ,Choline O-Acetyltransferase ,Epigenesis, Genetic ,03 medical and health sciences ,Amyloid beta-Protein Precursor ,0302 clinical medicine ,mental disorders ,medicine ,Amyloid precursor protein ,Humans ,Scaffold/matrix attachment region ,Promoter Regions, Genetic ,health care economics and organizations ,Genetics ,Cell Nucleus ,Neurons ,Messenger RNA ,Multidisciplinary ,Amyloid beta-Peptides ,Binding protein ,High-Throughput Nucleotide Sequencing ,Matrix Attachment Region Binding Proteins ,Matrix Attachment Regions ,Choline acetyltransferase ,humanities ,Introns ,Cell biology ,Chromatin ,Molecular Weight ,Cell nucleus ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,biology.protein ,Chromatin immunoprecipitation ,030217 neurology & neurosurgery - Abstract
The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure.
- Published
- 2016
18. Amyloid-beta oligomers increase the localization of prion protein at the cell surface
- Author
-
Marco A. M. Prado, Fabiana A. Caetano, Vilma R. Martins, Ana Paula Wasilewska-Sampaio, André Luiz Sena Guimarães, Flavio H. Beraldo, Vania F. Prado, Sergio T. Ferreira, Sofia Jürgensen, Pedro H. F. Hirata, R. Jane Rylett, Daisy Y.L. Wong, Glaucia N. M. Hajj, Cleiton F. Machado, Ivana A. Souza, and Fernanda G. De Felice
- Subjects
chemistry.chemical_classification ,Amyloid ,biology ,Amyloid beta ,animal diseases ,Cell ,Peptide ,Endocytosis ,Biochemistry ,nervous system diseases ,Cellular and Molecular Neuroscience ,medicine.anatomical_structure ,chemistry ,Cell culture ,Biotinylation ,mental disorders ,biology.protein ,medicine ,Biophysics ,Receptor ,Neuroscience - Abstract
J. Neurochem. (2011) 117, 538–553. Abstract In Alzheimer’s disease, the amyloid-β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrPC) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrPC-dependent manner, as might be expected if Aβ oligomers use PrPC as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrPC at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrPC. In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrPC, but we also found that after Aβ oligomer-treatment PrPC formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrPC-dependent way and that they can affect PrPC trafficking, increasing its localization at the cell surface.
- Published
- 2011
19. Regulated recycling and plasma membrane recruitment of the high-affinity choline transporter
- Author
-
Vania F. Prado, Stefanie A. G. Black, Metta Pinthong, R. Jane Rylett, Alexis C. Gordon, Marco A. M. Prado, Stephen S. G. Ferguson, and Fabiola M. Ribeiro
- Subjects
Synaptic cleft ,General Neuroscience ,Vesicle ,media_common.quotation_subject ,HEK 293 cells ,Depolarization ,Biology ,Cell biology ,Choline transporter ,chemistry.chemical_compound ,chemistry ,Choline ,Internalization ,Intracellular ,media_common - Abstract
The high-affinity choline transporter (CHT1) is responsible for uptake of choline from the synaptic cleft and supplying choline for acetylcholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K + -depolarization promotes Ca 2+ -dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K + -depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular transporters to the recycling pool.
- Published
- 2007
20. 82-kDa choline acetyltransferase is in nuclei of cholinergic neurons in human CNS and altered in aging and Alzheimer disease
- Author
-
Tomas Dobransky, Sandeep K. Gill, R. Jane Rylett, Margaret Ishak, Vahram Haroutunian, and Kenneth L. Davis
- Subjects
Adult ,Central Nervous System ,Male ,Aging ,medicine.medical_specialty ,Adolescent ,education ,Biology ,Choline O-Acetyltransferase ,chemistry.chemical_compound ,Alzheimer Disease ,Internal medicine ,mental disorders ,medicine ,Humans ,Immunoprecipitation ,Cholinergic neuron ,health care economics and organizations ,Aged ,Aged, 80 and over ,Cell Nucleus ,Neurons ,General Neuroscience ,Infant, Newborn ,Human brain ,Middle Aged ,medicine.disease ,Choline acetyltransferase ,Acetylcholinesterase ,humanities ,Molecular Weight ,Endocrinology ,Nerve growth factor ,medicine.anatomical_structure ,nervous system ,chemistry ,Cholinergic ,Female ,Neurology (clinical) ,Geriatrics and Gerontology ,Alzheimer's disease ,Neuroscience ,Acetylcholine ,Subcellular Fractions ,Developmental Biology ,medicine.drug - Abstract
Cholinergic neurons express choline acetyltransferase (ChAT) which synthesizes acetylcholine. We show here for the first time that primate-specific 82-kDa ChAT is expressed in nuclei of cholinergic neurons in human brain and spinal cord; isoform-specific antibodies were used to compare localization patterns and temporal expression of the more abundant 69-kDa ChAT and primate-specific 82-kDa ChAT in necropsy tissues. The 82-kDa ChAT co-localizes with 69-kDa ChAT in well-characterized cholinergic areas, but is also found in the claustrum which does not contain 69-kDa ChAT. Cholinergic neuron function changes with increasing age and are targeted in neurodegenerative diseases such as AD, thus we compared expression and subcellular localization of 69- and 82-kDa ChAT in necropsy brain samples from control subjects of varying ages and from Alzheimer disease (AD) subjects. The 82-kDa ChAT protein was expressed in cholinergic neurons in brain from birth until the eighth decade of life and in AD, but the subcellular staining pattern and proportion of neurons that were immunopositive changed with increasing age and in AD.
- Published
- 2007
21. The 'ins' and 'outs' of the high-affinity choline transporter CHT1
- Author
-
Stephen S. G. Ferguson, Marco A. M. Prado, R. Jane Rylett, Vania F. Prado, Fabiola M. Ribeiro, and Stefanie A. G. Black
- Subjects
Synaptic cleft ,Amino Acid Motifs ,Presynaptic Terminals ,Synaptic Membranes ,Biology ,Endocytosis ,Synaptic Transmission ,Biochemistry ,Synaptic vesicle ,Exocytosis ,Reuptake ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Neurotransmitter ,Cation Transport Proteins ,Acetylcholine ,Cell biology ,Choline transporter ,Protein Transport ,chemistry ,Neuroscience ,medicine.drug - Abstract
Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.
- Published
- 2006
22. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT
- Author
-
R. Jane Rylett and Katherine J. Fishwick
- Subjects
medicine.medical_specialty ,medicine.medical_treatment ,Morpholines ,lcsh:Medicine ,Biology ,Choline ,Membrane Potentials ,03 medical and health sciences ,chemistry.chemical_compound ,Phosphatidylinositol 3-Kinases ,0302 clinical medicine ,Insulin resistance ,Internal medicine ,Cell Line, Tumor ,medicine ,Humans ,Insulin ,lcsh:Science ,Protein kinase B ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,lcsh:R ,Cell Membrane ,Membrane Transport Proteins ,medicine.disease ,Endocytosis ,Choline transporter ,Enzyme Activation ,Insulin receptor ,Endocrinology ,chemistry ,Chromones ,biology.protein ,Cholinergic ,lcsh:Q ,Proto-Oncogene Proteins c-akt ,030217 neurology & neurosurgery ,Acetylcholine ,medicine.drug ,Signal Transduction ,Research Article - Abstract
Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.
- Published
- 2014
23. Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: Detection of NeuN
- Author
-
Margaret Ishak, R. Jane Rylett, and Sandeep K. Gill
- Subjects
Pathology ,medicine.medical_specialty ,Indoles ,Tissue Fixation ,Nerve Tissue Proteins ,Naphthalenes ,Immunofluorescence ,Epitope ,Fixatives ,Mice ,Necrosis ,chemistry.chemical_compound ,Formaldehyde ,Borates ,medicine ,Animals ,Humans ,Nuclear protein ,Coloring Agents ,Paraffin Embedding ,medicine.diagnostic_test ,biology ,General Neuroscience ,Antigens, Nuclear ,Hydrogen-Ion Concentration ,Immunohistochemistry ,Molecular biology ,Rats ,Staining ,Spinal Cord ,Antigen retrieval ,chemistry ,Phosphopyruvate Hydratase ,biology.protein ,NeuN ,Azo Compounds ,Immunostaining - Abstract
Immunohistochemical and immunofluorescence staining approaches are powerful tools for characterization of the endogenous protein expression and subcellular compartmentalization. However, several technical problems hamper identification of low-abundance nuclear proteins in archival formalin-fixed, paraffin-embedded human neural tissue. These include loss of protein antigenicity during tissue fixation and processing, and intrinsic auto-fluorescence associated with the tissue related to its fixation and the presence of lipofuscin. We evaluated several antigen retrieval methods to establish a strategy for detection of neuronal nuclear proteins in human spinal cord formalin-fixed, paraffin-embedded tissue. Thus, using immunostaining of the neuron-specific nuclear protein NeuN as the outcome measure, we found that heating tissue sections in an alkaline pH buffer unmasked protein epitopes most effectively. Moreover, staining by immunohistochemistry with diaminobenzidine tetrahydrochloride chromagen was superior to immunofluorescence labeling, likely due to the signal amplification steps included in the former approach. Auto-fluorescence in the tissue sections can be effectively reduced, but a sufficient fluorescence signal associated with specific antibody labeling could not be detected above this background for NeuN in the nucleus.
- Published
- 2005
24. Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase
- Author
-
R. Jane Rylett, C. Shaun Demeris, Margaret Ishak, and Bettina E. Kalisch
- Subjects
biology ,MAP kinase kinase kinase ,Cyclin-dependent kinase 2 ,Mitogen-activated protein kinase kinase ,Biochemistry ,MAP2K7 ,Cell biology ,Cellular and Molecular Neuroscience ,biology.protein ,Cyclin-dependent kinase 9 ,ASK1 ,c-Raf ,MAPK14 - Abstract
Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.
- Published
- 2003
25. Identification of a Novel Nuclear Localization Signal Common to 69- and 82-kDa Human Choline Acetyltransferase
- Author
-
Sandeep K. Gill, Moshmi Bhattacharya, Stephen S. G. Ferguson, and R. Jane Rylett
- Subjects
DNA, Complementary ,Heterogeneous nuclear ribonucleoprotein ,Molecular Sequence Data ,Nuclear Localization Signals ,Biology ,Biochemistry ,Cell Line ,Choline O-Acetyltransferase ,chemistry.chemical_compound ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Nuclear protein ,Nuclear export signal ,Molecular Biology ,Cell Biology ,Leptomycin ,Immunohistochemistry ,Fusion protein ,Choline acetyltransferase ,Protein Transport ,chemistry ,Cytoplasm ,Mutagenesis, Site-Directed ,Nuclear localization sequence - Abstract
We demonstrated previously that 69- and 82-kDa human choline acetyltransferase are localized predominantly to the cytoplasm and the nucleus, respectively. We have now identified a nuclear localization signal common to both forms of enzyme using confocal microscopy to study the subcellular compartmentalization of choline acetyltransferase tagged with green fluorescent protein in living HEK 293 cells. To identify functional nuclear localization and export signals, portions of full-length 69-kDa choline acetyltransferase were cloned into the vector peGFP-N1 and the cellular distribution patterns of the fusion proteins observed. Of the nine constructs studied, one yielded a protein with nuclear localization and another produced a protein with cytoplasmic localization. Mutation of the critical amino acids in this novel putative nuclear localization signal in the 69- and 82-kDa enzymes demonstrated that it is functional in both proteins. Moreover, 69-kDa choline acetyltransferase but not the 82-kDa enzyme is transported out of the nucleus by the leptomycin B-sensitive Crm-1 export pathway. By using bikaryon cells expressing both 82-kDa choline acetyltransferase and the nuclear protein heterogeneous nuclear ribonucleoprotein with green and red fluorescent tags, respectively, we found that the 82-kDa enzyme does not shuttle out of the nucleus in measurable amounts. These data suggest that 69-kDa choline acetyltransferase is a nucleocytoplasmic shuttling protein with a predominantly cytoplasmic localization determined by a functional nuclear localization signal and unidentified putative nuclear export signal. For 82-kDa choline acetyltransferase, the presence of the unique amino-terminal nuclear localization signal plus the newly identified nuclear localization signal may be involved in a process leading to predominantly nuclear accumulation of this enzyme, or alternatively, the two nuclear localization signals may be sufficient to overcome the force(s) driving nuclear export.
- Published
- 2003
26. Phosphorylation of 69-kDa Choline Acetyltransferase at Threonine 456 in Response to Amyloid-β Peptide 1–42
- Author
-
R. Jane Rylett, Tomas Dobransky, Dyanne Brewer, and Gilles A. Lajoie
- Subjects
Phosphopeptides ,Threonine ,Spectrometry, Mass, Electrospray Ionization ,Biochemistry ,Mass Spectrometry ,Choline O-Acetyltransferase ,MAP2K7 ,Neuroblastoma ,Ca2+/calmodulin-dependent protein kinase ,Tumor Cells, Cultured ,medicine ,Humans ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Protein Kinase C ,Protein kinase C ,Amyloid beta-Peptides ,Chemistry ,Kinase ,Circular Dichroism ,Cell Biology ,Molecular biology ,Choline acetyltransferase ,Peptide Fragments ,Recombinant Proteins ,Kinetics ,Microscopy, Electron ,Phosphothreonine ,Mutagenesis, Site-Directed ,Acetylcholine ,medicine.drug - Abstract
Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.
- Published
- 2003
27. [Untitled]
- Author
-
Tomas Dobransky and R. Jane Rylett
- Subjects
Kinase ,General Medicine ,Biology ,Biochemistry ,Choline acetyltransferase ,Cell biology ,Cellular and Molecular Neuroscience ,nervous system ,Ca2+/calmodulin-dependent protein kinase ,mental disorders ,medicine ,Cholinergic ,Phosphorylation ,Cholinergic neuron ,health care economics and organizations ,Protein kinase C ,Acetylcholine ,medicine.drug - Abstract
Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.
- Published
- 2003
28. Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells
- Author
-
R. Jane Rylett, Wanda L. Davis, Nicholas A. Bock, and Bettina E. Kalisch
- Subjects
medicine.medical_specialty ,biology ,Neurite ,Biochemistry ,Choline acetyltransferase ,Nitric oxide ,Cell biology ,Nitric oxide synthase ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Nerve growth factor ,Endocrinology ,nervous system ,chemistry ,Cell culture ,Internal medicine ,medicine ,biology.protein ,Acetylcholine ,medicine.drug ,Neurotrophin - Abstract
NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor l-NAME (N ω-nitro-l-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, l-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both l-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.
- Published
- 2002
29. PC12nnr5 cells expressing TrkA receptors undergo morphological but not cholinergic phenotypic differentiation in response to nerve growth factor
- Author
-
Wanda L. Davis, R. Jane Rylett, Bettina E. Kalisch, Jacqueline C. Baskey, and Susan O. Meakin
- Subjects
Proto-Oncogene Proteins c-jun ,JUNB ,Electrophoretic Mobility Shift Assay ,Tropomyosin receptor kinase A ,Biology ,Nitric Oxide ,PC12 Cells ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Choline O-Acetyltransferase ,Cellular and Molecular Neuroscience ,Genes, Reporter ,Nerve Growth Factor ,Animals ,Low-affinity nerve growth factor receptor ,Receptor, trkA ,Transcription factor ,Neurons ,Cell Differentiation ,Transfection ,Choline acetyltransferase ,Acetylcholine ,Rats ,Cell biology ,Transcription Factor AP-1 ,Phenotype ,Nerve growth factor ,Cancer research ,Cholinergic ,Proto-Oncogene Proteins c-fos - Abstract
We investigated mechanisms underlying nerve growth factor-mediated morphological differentiation and expression of cholinergic neuronal phenotype. In PC12, but not PC12nnr5 cells, nerve growth factor induces neurite-like outgrowths and enhances cholinergic phenotype; stable expression of TrkA receptors in nnr5 cells (called B5P cells) restores morphological differentiation but not expression of choline acetyltransferase. Transfection with an AP-1 luciferase reporter gene revealed that PC12 but not B5P cells expressed nerve growth factor-induced functional AP-1 activity. RT-PCR analysis of nerve growth factor-mediated changes in AP-1 transcription factors showed rapid increases in c-fos and junB mRNA in PC12 and B5P cells, while increases in c-jun were small. Using DNA-protein gel shift assays we determined that nerve growth factor stimulates AP-1 binding in both PC12 and B5P cells, and identified c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD in AP-1 complexes. In Fos/Jun functional luciferase reporter assays, nerve growth factor stimulated phosphorylation of c-Fos in both PC12 and B5P cells, but phosphorylation of c-Jun only in PC12, and not in B5P cells. These data indicate that mechanisms relating to AP-1 transcription factor complexes underlying nerve growth factor-mediated enhancement of cholinergic gene expression may differ from those required for morphological differentiation.
- Published
- 2002
30. Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C
- Author
-
Wanda L. Davis, Tomas Dobransky, and R. Jane Rylett
- Subjects
PRKCQ ,Molecular Sequence Data ,Mitogen-activated protein kinase kinase ,Biochemistry ,Catalysis ,Cell Line ,Choline O-Acetyltransferase ,MAP2K7 ,Serine ,Humans ,Amino Acid Sequence ,c-Raf ,Phosphorylation ,Molecular Biology ,Protein Kinase C ,Protein kinase C ,DNA Primers ,MAPK14 ,Base Sequence ,biology ,Cyclin-dependent kinase 2 ,Cell Biology ,Molecular biology ,Choline acetyltransferase ,Acetylcholine ,Enzyme Activation ,biology.protein ,Tetradecanoylphorbol Acetate ,Subcellular Fractions - Abstract
Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.
- Published
- 2001
31. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity
- Author
-
Tomas DOBRANSKY, Wanda L. DAVIS, Gong-Hua XIAO, and R. Jane RYLETT
- Subjects
Cell Biology ,Molecular Biology ,Biochemistry - Abstract
Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and α-calcium/calmodulin-dependent protein kinase II (α-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or α-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.
- Published
- 2000
32. Absence of p75NTR expression reduces nerve growth factor immunolocalization in cholinergic septal neurons
- Author
-
Karmen M. Krol, Michael D. Kawaja, Bettina E. Kalisch, Keith A. Crutcher, and R. Jane Rylett
- Subjects
musculoskeletal diseases ,Genetically modified mouse ,medicine.medical_specialty ,Basal forebrain ,General Neuroscience ,Hippocampus ,Biology ,Hippocampal formation ,Tropomyosin receptor kinase A ,Choline acetyltransferase ,biological factors ,Endocrinology ,Nerve growth factor ,nervous system ,Internal medicine ,medicine ,Cholinergic ,sense organs ,skin and connective tissue diseases - Abstract
Septal axons provide a cholinergic innervation to the nerve growth factor (NGF)-producing neurons of the mammalian hippocampus. These cholinergic septal afferents are capable of responding to target-derived NGF because they possess trkA and p75(NTR), the two transmembrane receptors that bind NGF and activate ligand-mediated intracellular signaling. To assess the relative importance of p75(NTR) expression for the responsiveness of cholinergic septal neurons to hippocampally derived NGF, we used three lines of mutant and/or transgenic mice: p75(-/-) mice (having two mutated alleles of the p75(NTR) gene), NGF/p75(+/+) mice (transgenic animals overexpressing NGF within central glial cells and having two normal alleles of the p75(NTR) gene), and NGF/p75(-/-) mice (NGF transgenic animals having two mutated alleles of the p75(NTR) gene). BALB/c and C57B1/6 mice (background strains for the mutant and transgenic lines of mice) were used as controls. Both lines of NGF transgenic mice possess elevated levels of NGF protein in the hippocampus and septal region, irrespective of p75(NTR) expression. BALB/c and C57Bl/6 mice display comparably lower levels of NGF protein in both tissues. Despite differing levels of NGF protein, the ratios of hippocampal to septal NGF levels are similar among BALB/c, C57B1/6, and NGF/p75(+/+) mice. Both p75(-/-) and NGF/p75(-/-) mice, on the other hand, have markedly elevated ratios of NGF protein between these two tissues. The lack of p75(NTR) expression also results in a pronounced absence of NGF immunoreactivity in cholinergic septal neurons of p75(-/-) and NGF/p75(-/-) mice. BALB/c, C57B1/6, and NGF/p75(+/+) mice, on the other hand, display NGF immunoreactivity that appears as discrete granules scattered through the cytoplasm of cholinergic septal neurons. Elevated levels of NGF in the hippocampus and septal region coincide with hypertrophy of cholinergic septal neurons of NGF/p75(+/+) mice but not of NGF/p75(-/-) mice. Levels of choline acetyltransferase (ChAT) enzyme activity are, however, elevated in the septal region and hippocampus of both NGF/p75(+/+) and NGF/p75(-/-) mice, compared with control mice. These data indicate that an absence of functional p75(NTR) expression disrupts the normal cellular immunolocalization of NGF by cholinergic septal neurons but does not affect the ability of these neurons to respond to elevated levels of NGF, as determined by ChAT activity.
- Published
- 2000
33. Inhibitors of serine/threonine phosphatases increase membrane-bound choline acetyltransferase activity and enhance acetylcholine synthesis
- Author
-
Lara Cooke and R. Jane Rylett
- Subjects
Phosphatase ,Choline ,Choline O-Acetyltransferase ,Rats, Sprague-Dawley ,Serine ,chemistry.chemical_compound ,Phosphoprotein Phosphatases ,medicine ,Animals ,Enzyme Inhibitors ,Molecular Biology ,Cholinesterase ,biology ,General Neuroscience ,Cell Membrane ,Biological Transport ,Okadaic acid ,Choline acetyltransferase ,Acetylcholine ,Rats ,Biochemistry ,chemistry ,biology.protein ,Cholinergic ,Female ,Neurology (clinical) ,Choline transport ,Subcellular Fractions ,Developmental Biology ,medicine.drug - Abstract
The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129 +/- 11% and 137 +/- 10% of control, respectively. High-affinity choline transport was reduced to 77 +/- 6% and 76 +/- 7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73 +/- 6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.
- Published
- 1997
34. An In Vivo Proton Magnetic Resonance Spectroscopy Study of Schizophrenia Patients
- Author
-
Ashok Malla, R. Jane Rylett, Peter C. Williamson, Jeffrey A. Stanley, Dick J. Drost, Thomas J. Carr, and R. Terry Thompson
- Subjects
Adult ,Male ,Psychosis ,medicine.medical_specialty ,Magnetic Resonance Spectroscopy ,Adolescent ,Glutamine ,Central nervous system ,Glutamic Acid ,Prefrontal Cortex ,Glutamatergic ,In vivo ,Internal medicine ,medicine ,Humans ,Prefrontal cortex ,Psychiatric Status Rating Scales ,Aspartic Acid ,Glutamate receptor ,Middle Aged ,medicine.disease ,Magnetic Resonance Imaging ,Psychiatry and Mental health ,Endocrinology ,medicine.anatomical_structure ,Schizophrenia ,Chronic Disease ,Female ,Schizophrenic Psychology ,Psychology ,Neuroscience ,Antipsychotic Agents - Abstract
The level of the ]H metabolites in the left dorsolateral prefrontal region of schizophrenia patients at different stages of illness were measured in vivo using a short echo time spectroscopy technique. During both the early onset and chronic stages, normal A/-acetylaspartate levels were observed, which suggests that these patients had no significant neuronal cell damage and/or loss. The in vivo measurements of glutamate in the first-episode, drugnaive patients failed to provide convincing evidence for the involvement of the glutamatergic system in the dorsolateral prefrontal region. Significant differences in the glutamine levels were observed in the acutely medicated and chronic patients; however, the interpretation of these differences requires further study. Schizophrenia Bulletin, 22(4): 597-609,1996.
- Published
- 1996
35. Identification and partial characterization of the high-affinity choline carrier from rat brain striatum
- Author
-
R. Jane Rylett, Wanda L. Davis, and Sandra A. Walters
- Subjects
Central nervous system ,Striatum ,Biology ,Choline ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Basal ganglia ,medicine ,Animals ,Molecular Biology ,Hemicholinium 3 ,Ligand (biochemistry) ,Corpus Striatum ,Rats ,Molecular Weight ,Kinetics ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Cholinergic ,Neuromuscular Blocking Agents ,Choline transport ,Carrier Proteins ,Acetylcholine ,Synaptosomes ,medicine.drug - Abstract
[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.
- Published
- 1996
36. Overexpression of pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A in nerve cells confers resistance to amyloid β and other toxins by decreasing mitochondrial respiration and reactive oxygen species production
- Author
-
Jordan T. Newington, Daisy Y. Wong, Robert C. Cumming, Tim Rappon, R. Jane Rylett, and Shawn Albers
- Subjects
Male ,Gene Expression ,Mitochondrion ,medicine.disease_cause ,Biochemistry ,Pediatrics ,Mice ,Adenosine Triphosphate ,chemistry.chemical_classification ,Aged, 80 and over ,Cerebral Cortex ,Membrane Potential, Mitochondrial ,Neurons ,education.field_of_study ,Molecular Bases of Disease ,Warburg effect ,Mitochondria ,Isoenzymes ,Female ,Pyruvate dehydrogenase kinase ,Cellular respiration ,Lactate dehydrogenase A ,Cell Respiration ,Mice, Transgenic ,Biology ,Protein Serine-Threonine Kinases ,Cell Line ,Oxygen Consumption ,Alzheimer Disease ,medicine ,Animals ,Humans ,education ,Molecular Biology ,Aged ,Reactive oxygen species ,Amyloid beta-Peptides ,L-Lactate Dehydrogenase ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,Cell Biology ,Hydrogen Peroxide ,Staurosporine ,Molecular biology ,Rats ,chemistry ,Anaerobic glycolysis ,Case-Control Studies ,Lactate Dehydrogenase 5 ,Reactive Oxygen Species ,Oxidative stress - Abstract
We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease.
- Published
- 2012
37. Peroxynitrite donor SIN-1 alters high-affinity choline transporter activity by modifying its intracellular trafficking
- Author
-
Alexis C. Gordon, Leah K. Cuddy, Stephen S. G. Ferguson, Ewa Jaworski, Stefanie A. G. Black, and R. Jane Rylett
- Subjects
Neurotransmitter transporter ,Time Factors ,genetic structures ,Endosome ,Leupeptins ,Endosomes ,Biology ,Cysteine Proteinase Inhibitors ,Endocytosis ,Nitric Oxide ,Transfection ,Tritium ,Choline ,chemistry.chemical_compound ,Neuroblastoma ,Lysosome ,Cell Line, Tumor ,Peroxynitrous Acid ,medicine ,Humans ,Nitric Oxide Donors ,Cell Line, Transformed ,rab5 GTP-Binding Proteins ,Dose-Response Relationship, Drug ,General Neuroscience ,Ubiquitination ,Membrane Transport Proteins ,Hemicholinium 3 ,Articles ,Clathrin ,Cell biology ,Choline transporter ,Luminescent Proteins ,Protein Transport ,medicine.anatomical_structure ,chemistry ,Molsidomine ,Mutation ,Cholinergic ,Lysosomes ,Peroxynitrite - Abstract
Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO−) donor]; ONOO−can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative–nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamin-dependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle- and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative–nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.
- Published
- 2012
38. Effect of cellular differentiation on nucleoside transport in human neuroblastoma cells
- Author
-
R. Jane Rylett, Kenneth W. Jones, and James R. Hammond
- Subjects
Cellular differentiation ,Biology ,Nucleoside transporter ,Neuroblastoma ,Thioinosine ,Tumor Cells, Cultured ,medicine ,Humans ,Cytotoxic T cell ,Molecular Biology ,General Neuroscience ,Biological Transport ,Cell Differentiation ,Nucleosides ,medicine.disease ,Molecular biology ,In vitro ,Chemically defined medium ,Biochemistry ,biology.protein ,Neurology (clinical) ,Formycins ,Nucleoside ,Intracellular ,Developmental Biology - Abstract
The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroblastoma cells were compared through measurement of the cellular accumulation of [3H]formycin B in the absence and presence of specific nucleoside transport blockers such as dipyridamole and nitrobenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity probe to obtain an estimate of the number of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]formycin B (25 microM) via a NBMPR/dipyridamole sensitive, Na(+)-independent, nucleoside transport system (Vi = 1.52 pmol/microliters/s; maximum intracellular concentration = 45 pmol/microliters cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR binding sites (135,000 sites/cell; KD = 0.4 nM). When cell differentiation was induced by exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]formycin B uptake increased to 1.92 pmol/microliters/s, and the steady-state intracellular concentration of [3H]formycin B also increased significantly to 73 pmol/microliters. However, there was no concomitant change in the number of [3H]NBMPR binding sites, and the additional uptake was not Na(+)-dependent. This enhanced uptake in the differentiated cells appeared to be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhibition by NBMPR at concentrations that blocked transport completely in the undifferentiated cells. This cell model may prove useful for basic studies on regulation of nucleoside transporter subtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (+/- specific transport blockers) in the treatment of neuroblastoma.
- Published
- 1994
39. Impact of Oxidative - Nitrosative Stress on Cholinergic Presynaptic Function
- Author
-
R. Jane Rylett and Stefanie A. G. Black
- Subjects
0303 health sciences ,Basal forebrain ,Substantia innominata ,Hippocampus ,Biology ,Nucleus basalis ,03 medical and health sciences ,0302 clinical medicine ,nervous system ,medicine ,Cholinergic ,Cholinergic neuron ,Neuroscience ,030217 neurology & neurosurgery ,Acetylcholine ,030304 developmental biology ,medicine.drug ,Mesopontine - Abstract
Cholinergic neurotransmission plays an essential role in a variety of physiological processes in both the central and peripheral nervous systems. Cholinergic neurons use the classical neurotransmitter acetylcholine (ACh) to communicate with their target cells. In the periphery, ACh is the neurotransmitter used at the skeletal neuromuscular junction, at all preand postganglionic parasympathetic synapses and at preganglionic sympathetic synapses. In the central nervous system, the actions of ACh are widespread with cholinergic neurotransmission involved in attention, learning and memory, cognition, sleep, wakefulness, and modulation of sensory information (Hasselmo, 2006; Sarter & Parihk, 2005; Woolf & Butcher, 2010). Dysfunction of cholinergic neurotransmission in the central nervous system is apparent in a number of neurological disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, schizophrenia and amyotrophic lateral sclerosis (Bohnen & Albin, 2010; Mesulam, 2004; Oda, 1999). Cholinergic neurons innervate almost all areas of the brain, where this can be mediated by either intrinsic interneurons or by extrinsic projection neurons. Cholinergic interneurons localized in the striatum are involved in motor function, cognition, and behavior (Woolf & Butcher, 2010). The basal forebrain, which is comprised of the nucleus basalis of Meynert, medial septum, diagonal band of Broca, the magnocellular preoptic nucleus, and substantia innominata, contains the cell bodies of cholinergic neurons that project to the hippocampus, amygdala, olfactory bulb, and all areas of the cerebral cortex (Woolf & Butcher, 2010). Collectively, basal forebrain cholinergic neuron activity plays a role in attention, learning, memory, perception, and consciousness (Sarter et al., 2003; Woolf, 1998; Woolf & Butcher, 2010). Cholinergic neurons in the mesopontine region (the pedunculopontine and laterodorsal nuclei) project to the thalamus, hypothalamus, basal forebrain, medial frontal cortex, brainstem and spinal cord (Woolf & Butcher, 2010). Descending cholinergic projections from the mesopontine area decrease muscle tone during rapid eye movement sleep while ascending cholinergic projections are involved in cognitive functions and consciousness (Woolf & Butcher, 2010). Cholinergic projections to the interpeduncular nucleus originate from neurons with cell bodies in the medial habenula; these neurons regulate electroencephalogram patterns and rapid eye movement sleep (Woolf & Butcher, 2010). The cycle of ACh synthesis, storage, release and degradation has been well-characterized at the cellular and molecular levels and is depicted in Figure 1. ACh is synthesized in the
- Published
- 2011
40. Amyloid-beta oligomers increase the localization of prion protein at the cell surface
- Author
-
Fabiana A, Caetano, Flavio H, Beraldo, Glaucia N M, Hajj, Andre L, Guimaraes, Sofia, Jürgensen, Ana Paula, Wasilewska-Sampaio, Pedro H F, Hirata, Ivana, Souza, Cleiton F, Machado, Daisy Y-L, Wong, Fernanda G, De Felice, Sergio T, Ferreira, Vania F, Prado, R Jane, Rylett, Vilma R, Martins, and Marco A M, Prado
- Subjects
Neurons ,Analysis of Variance ,Amyloid beta-Peptides ,Microscopy, Confocal ,Mitogen-Activated Protein Kinase 3 ,Time Factors ,Cell Membrane ,Green Fluorescent Proteins ,Embryo, Mammalian ,Flow Cytometry ,Transfection ,Hippocampus ,Peptide Fragments ,Mice ,Protein Transport ,Animals ,Humans ,Biotinylation ,PrPC Proteins ,Cells, Cultured ,rab5 GTP-Binding Proteins - Abstract
In Alzheimer's disease, the amyloid-β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aβ oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aβ oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.
- Published
- 2011
41. Modulation of high-affinity choline carrier activity following incubation of rat hippocampal synaptosomes with hemicholinium-3
- Author
-
Sandra A. Walters, R. Jane Rylett, and Wanda L. Davis
- Subjects
Biology ,Hippocampus ,Choline ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Hemicholinium-3 ,medicine ,Extracellular ,Animals ,Molecular Biology ,Incubation ,Synaptosome ,General Neuroscience ,Membrane Transport Proteins ,Hemicholinium 3 ,Acetylcholine ,Rats ,chemistry ,Biochemistry ,Biophysics ,Cholinergic ,Female ,Neurology (clinical) ,Choline transport ,Carrier Proteins ,Synaptosomes ,Developmental Biology ,medicine.drug - Abstract
Membrane carriers display structural and functional asymmetry with a substrate binding site which can be oriented alternately, but not simultaneously, to the extracellular and intracellular environment. Hemicholinium-3 is an inhibitor of the high-affinity choline carrier in cholinergic nerve terminals which binds to the transporter at the outer membrane surface but is not taken up into the cell. In the present study, we investigated the decline in choline transport which occurs during the first few minutes cholinergic nerve terminals are incubated in physiological salt solutions. Following incubation of rat hippocampal synaptosomes with hemicholinium-3, samples were washed free of the inhibitor and high-affinity choline uptake was measured. Choline uptake into hemicholinium-treated nerve terminals was significantly greater than control (132 +/- 4%). This effect appeared not to be due to an increase in uptake of choline above initial values in the hemicholinium-treated synaptosomes, but to a decrease in choline carrier activity in control samples by more than 25% during the first few minutes of incubation. Addition of hemicholinium-3 to samples after the preincubation induced decrease in choline uptake, followed by a wash period to remove the inhibitor resulted in elevation of choline uptake levels to initial levels. The effect of hemicholinium-3 was concentration-dependent, requiring near saturating concentrations of the inhibitor to elicit the effect. Measurement of acetylcholine content of synaptosomes at different points during the incubation procedure revealed that there was a trend for transmitter levels to vary inversely compared to choline uptake activity, but the differences were not statistically significant during treatments when significant changes in transport activity were measured.
- Published
- 1993
42. Role of alpha7 nicotinic acetylcholine receptor in calcium signaling induced by prion protein interaction with stress-inducible protein 1
- Author
-
Camila P. Arantes, Regina P. Markus, Nicolle G.T. Queiroz, Marco A. M. Prado, Vilma R. Martins, R. Jane Rylett, Flavio H. Beraldo, Kirk Young, and Tiago G. Santos
- Subjects
Male ,alpha7 Nicotinic Acetylcholine Receptor ,animal diseases ,Apoptosis ,Receptors, Nicotinic ,Biochemistry ,Hippocampus ,Mice ,Cells, Cultured ,Heat-Shock Proteins ,Calcium signaling ,TRANSGENIC MICE ,Neurons ,CELLULAR PRION ,NEUROPROTECTION ,Mitogen-Activated Protein Kinase 3 ,Reverse Transcriptase Polymerase Chain Reaction ,Molecular Bases of Disease ,MOUSE MODEL ,AMYLOID-BETA ,Recombinant Proteins ,Cell biology ,ALZHEIMERS-DISEASE ,medicine.anatomical_structure ,Female ,Signal transduction ,Anatomy ,Intracellular ,Protein Binding ,EXPRESSION ,Immunoprecipitation ,MAP Kinase Signaling System ,Blotting, Western ,CROSS-TALK ,Biology ,Neuroprotection ,Cell and Developmental Biology ,mental disorders ,medicine ,Animals ,Humans ,PrPC Proteins ,Calcium Signaling ,RNA, Messenger ,Protein kinase A ,Molecular Biology ,Cell Proliferation ,HEK 293 cells ,Cell Biology ,Embryo, Mammalian ,LAMININ ,nervous system diseases ,Mice, Inbred C57BL ,BETA OLIGOMERS ,Neuron - Abstract
The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.
- Published
- 2010
43. The vesicular acetylcholine transporter is required for neuromuscular development and function
- Author
-
Marco A. M. Prado, Braulio M. de Castro, Ricardo F. Lima, Martín Cammarota, Patricia Lima, Marcus Vinicius Gomez, Marc G. Caron, Ernani Amaral, Christopher Kushmerick, Cintia M. L. Neves, Cristina Guatimosim, Thomas W. Gould, Ronald W. Oppenheim, Ian Welch, Ivan Izquierdo, R. Jane Rylett, Rita Gomes Wanderley Pires, Cristina Martins-Silva, Cristiane Alves da Silva Menezes, Vania F. Prado, and Xavier De Jaeger
- Subjects
Vesicular Acetylcholine Transport Proteins ,QUANTAL TRANSMITTER SECRETION ,Molecular Sequence Data ,CRE TRANSGENE EXPRESSION ,Neuromuscular Junction ,Biology ,Muscle Development ,Synaptic vesicle ,Neuromuscular junction ,PROGRAMMED CELL-DEATH ,Cell Line ,Cell and Developmental Biology ,Mice ,Vesicular acetylcholine transporter ,medicine ,Animals ,Humans ,AFFINITY CHOLINE TRANSPORTER ,PROTEIN-KINASE-C ,Muscle, Skeletal ,Molecular Biology ,Gene knockout ,MOTOR-NERVE TERMINALS ,Mice, Knockout ,Motor Neurons ,2-(4-PHENYLPIPERIDINO) CYCLOHEXANOL AH5183 ,TORPEDO ELECTRIC ORGAN ,Base Sequence ,Cell Biology ,SYNAPTIC VESICLES ,Articles ,Embryo, Mammalian ,Choline acetyltransferase ,Acetylcholine ,Cell biology ,Choline transporter ,medicine.anatomical_structure ,BRAIN CORTICAL SLICES ,Biochemistry ,Knockout mouse ,Synaptic Vesicles ,Anatomy ,medicine.drug - Abstract
The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.
- Published
- 2009
44. Oligomeric aggregates of amyloid beta peptide 1-42 activate ERK/MAPK in SH-SY5Y cells via the alpha7 nicotinic receptor
- Author
-
Kirk F. Young, R. Jane Rylett, and Stephen H. Pasternak
- Subjects
MAPK/ERK pathway ,Cell signaling ,Time Factors ,alpha7 Nicotinic Acetylcholine Receptor ,Amyloid beta ,MAP Kinase Signaling System ,Polymers ,MAP Kinase Kinase 1 ,Peptide ,Nicotinic Antagonists ,Receptors, Nicotinic ,Cellular and Molecular Neuroscience ,Alzheimer Disease ,Cell Line, Tumor ,mental disorders ,Humans ,Enzyme Inhibitors ,Phosphorylation ,chemistry.chemical_classification ,Neurons ,Amyloid beta-Peptides ,Mitogen-Activated Protein Kinase 3 ,biology ,Dose-Response Relationship, Drug ,Activator (genetics) ,Chemistry ,P3 peptide ,Neurofibrillary Tangles ,Cell Biology ,Molecular biology ,Peptide Fragments ,nervous system diseases ,Cell biology ,Enzyme Activation ,nervous system ,Mitogen-activated protein kinase ,biology.protein - Abstract
The production and aggregation of amyloid beta peptides (Abeta) has been linked to the development and progression of Alzheimer's disease. It is apparent that the various structural forms of Abeta can affect cell signalling pathways and the activity of neurons differently. In this study, we investigated the effects of oligomeric and fibrillar aggregates of Abeta 1-42 (Abeta42) and non-aggregated peptide upon activation of the ERK/MAPK signalling pathway. In SH-SY5Y cells, acute exposure to oligomeric Abeta42 led to phosphorylation of ERK1/2 at concentrations as low as 1 nM and up to 100 nM. These changes were detected as early as 5 min following exposure to 100 nM oligomeric Abeta42, reaching a maximum level after 10 min. Phosphorylation of ERK1/2 subsequently declined to and remained at basal levels after 30 min to 2h of exposure. Fibrillar aggregates of Abeta42 did not significantly induce phosphorylation of ERK1/2 and non-aggregated Abeta42 did not activate the pathway. The effects of oligomeric Abeta42 to increase ERK phosphorylation above basal levels were inhibited by MLA, a specific antagonist of the alpha7 nAChR. U0126, an inhibitor of MEK, the upstream activator of ERK1/2, completely blocked induction of ERK1/2 phosphorylation. Oligomeric aggregates of Abeta42 are the principal structural form of the peptide that activates ERK/MAPK in SH-SY5Y cells and these effects are mediated by the alpha7 nAChR.
- Published
- 2009
45. Identification of a novel Zn2+-binding domain in the autosomal recessive juvenile Parkinson-related E3 ligase parkin
- Author
-
R. Jane Rylett, Ventzislava A. Hristova, S. Beasley, and Gary S. Shaw
- Subjects
Protein Structure ,Protein Folding ,Recombinant Fusion Proteins ,Ubiquitin-Protein Ligases ,Molecular Sequence Data ,Sequence alignment ,medicine.disease_cause ,Biochemistry ,Models, Biological ,Parkin ,Conserved sequence ,Protein structure ,Parkinsonian Disorders ,Models ,Structural Biology ,medicine ,Animals ,Humans ,Trypsin ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Protein Processing ,Conserved Sequence ,Genetics ,Mutation ,biology ,Protein Stability ,Serine Endopeptidases ,Post-Translational ,Neurosciences ,Ubiquitination ,Cell Biology ,Biological ,Ubiquitin ligase ,nervous system diseases ,Protein Structure, Tertiary ,Rats ,Zinc ,Protein Structure and Folding ,biology.protein ,Protein Processing, Post-Translational ,Sequence Alignment ,Tertiary ,Binding domain - Abstract
Missense mutations in park2, encoding the parkin protein, account for approximately 50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a approximately 160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys(150)-Cys(169) and Cys(196)-His(215) consisting of CX(2)-(3)CX(11)CX(2)C and CX(4-6)CX(10-16)-CX(2)(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn(2+) ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.
- Published
- 2009
46. Assessing the severity of perinatal hypoxia-ischemia in piglets using near-infrared spectroscopy to measure the cerebral metabolic rate of oxygen
- Author
-
Daisy Y.L. Wong, R. Jane Rylett, Keith St. Lawrence, Ting-Yim Lee, Kenneth M. Tichauer, and Jennifer Hadway
- Subjects
Pathology ,medicine.medical_specialty ,Time Factors ,animal diseases ,Sus scrofa ,Ischemia ,Cerebral metabolic rate ,chemistry.chemical_element ,Near-Infrared Spectrometry ,macromolecular substances ,Biology ,Oxygen ,fluids and secretions ,Internal medicine ,medicine ,Animals ,Cerebrum ,Spectroscopy, Near-Infrared ,integumentary system ,Perinatal hypoxia ,Hypoxia (medical) ,medicine.disease ,chemistry ,Recien nacido ,Pediatrics, Perinatology and Child Health ,Hypoxia-Ischemia, Brain ,Cardiology ,medicine.symptom - Abstract
Reduced cerebral function after neonatal hypoxia-ischemia is an early indicator of hypoxic-ischemic encephalopathy. Near-infrared spectroscopy offers a clinically relevant means of detecting impaired cerebral metabolism from the measurement of the cerebral metabolic rate of oxygen (CMRO2). The purpose of this study was to determine the relationship between postinsult CMRO2 and duration of hypoxia-ischemia in piglets. Twelve piglets were subjected to randomly selected durations of hypoxia-ischemia (5-28 min) and five animals served as controls. Measurements of CMRO2 were taken before and for 24 h after hypoxia-ischemia. Histology was carried out in nine piglets (six insults, three controls) to estimate brain injury. In the first 4 h after the insult, average CMRO2 of the insult group was significantly depressed (33 +/- 3% reduction compared with controls) and by 8 h, a significant correlation developed, which persisted for the remainder of the study, between CMRO2 and the duration of ischemia. Histologic staining suggested little brain damage resulted from shorter insult durations and considerable damage from more prolonged insults. This study demonstrated that near-infrared spectroscopy could detect early changes in CMRO2 after hypoxia-ischemia for a range of insult severities and CMRO2 could be used to distinguish insult severity by 8 h after the insult.
- Published
- 2008
47. Quantitative Cerebral Metabolism Measurements with NIRS: Application to Neonatal Brain Injury
- Author
-
Jennifer Hadway, Kenneth M. Tichauer, Ting-Yim Lee, Keith St. Lawrence, Daisy Y. Wong, and R. Jane Rylett
- Subjects
Pathology ,medicine.medical_specialty ,business.industry ,Neonatal brain ,medicine ,Cerebral metabolic rate ,Cerebral metabolism ,business ,Near infrared radiation - Abstract
A near-infrared technique has been developed to measure cerebral metabolic rate of oxygen quantitatively and non-invasively at the bedside. The ability of these measurements to diagnose hypoxic-ischemic insult severity was assessed in the newborn piglet.
- Published
- 2008
48. Regulated recycling and plasma membrane recruitment of the high-affinity choline transporter
- Author
-
Fabiola M, Ribeiro, Metta, Pinthong, Stefanie A G, Black, Alexis C, Gordon, Vania F, Prado, Marco A M, Prado, R Jane, Rylett, and Stephen S G, Ferguson
- Subjects
Time Factors ,Staining and Labeling ,Symporters ,Cell Membrane ,Immunoblotting ,Fluorescent Antibody Technique ,Humans ,Biological Transport ,Endosomes ,Transfection ,Binding, Competitive ,Cell Line ,Potassium Chloride - Abstract
The high-affinity choline transporter (CHT1) is responsible for uptake of choline from the synaptic cleft and supplying choline for acetylcholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K(+)-depolarization promotes Ca(2+)-dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K(+)-depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular transporters to the recycling pool.
- Published
- 2007
49. Activity and subcellular trafficking of the sodium-coupled choline transporter CHT is regulated acutely by peroxynitrite
- Author
-
Stefanie A. G. Black, R. Jane Rylett, Stephen S. G. Ferguson, Fabiola M. Ribeiro, Metta Pinthong, and Chumpol Pholpramool
- Subjects
Luminescence ,Time Factors ,Cell ,Cell Culture Techniques ,Cholinergic Agents ,Kidney ,Choline ,Membrane Potentials ,chemistry.chemical_compound ,Neuroblastoma ,Enzyme Inhibitors ,Internalization ,media_common ,Superoxide ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Biochemistry ,Data Interpretation, Statistical ,Molecular Medicine ,Peroxynitrite ,Subcellular Fractions ,Nitrogen ,media_common.quotation_subject ,Transfection ,Nitric oxide ,Cell Line ,Inhibitory Concentration 50 ,Cell Line, Tumor ,Peroxynitrous Acid ,medicine ,Animals ,Humans ,Biotinylation ,Reactive nitrogen species ,Pharmacology ,Dose-Response Relationship, Drug ,L-Lactate Dehydrogenase ,Cell Membrane ,Sodium ,Membrane Transport Proteins ,Hemicholinium 3 ,Culture Media ,Rats ,Choline transporter ,Kinetics ,Oxidative Stress ,chemistry ,Gene Expression Regulation ,Molsidomine ,Cholinergic ,Tyrosine - Abstract
Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.
- Published
- 2007
50. Substrate binding and catalytic mechanism of human choline acetyltransferase
- Author
-
Brian H. Shilton, R. Jane Rylett, and Ae-Ri Kim
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Entropy ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Choline ,Choline O-Acetyltransferase ,Serine ,chemistry.chemical_compound ,medicine ,Transferase ,Humans ,Coenzyme A ,Amino Acid Sequence ,Cholinergic neuron ,chemistry.chemical_classification ,Binding Sites ,Substrate (chemistry) ,Choline acetyltransferase ,Recombinant Proteins ,Enzyme ,chemistry ,Acetylcholine ,medicine.drug - Abstract
Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.
- Published
- 2006
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.