SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins.

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.

Infiltrating Hematogenous Macrophages Aggregate Around ß-Amyloid Plaques in an Age- and Sex-Dependent Manner in a Mouse Model of Alzheimer Disease.

ß-Amyloid (Aß) 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 Aß1-42, area of Aß plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aß accumulation, paralleled by the accumulation of hMϕ around Aß plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aß plaques was age- and sex-dependent and preceded cognitive impairment.

La COVID-19 et les priorités de recherche sur le vieillissement.

Cet 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.

The Comprehensive Assessment of Neurodegeneration and Dementia: Canadian Cohort Study.

BACKGROUND: The Comprehensive Assessment of Neurodegeneration and Dementia (COMPASS-ND) cohort study of the Canadian Consortium on Neurodegeneration in Aging (CCNA) is a national initiative to catalyze research on dementia, set up to support the research agendas of CCNA teams. This cross-country longitudinal cohort of 2310 deeply phenotyped subjects with various forms of dementia and mild memory loss or concerns, along with cognitively intact elderly subjects, will test hypotheses generated by these teams. METHODS: The COMPASS-ND protocol, initial grant proposal for funding, fifth semi-annual CCNA Progress Report submitted to the Canadian Institutes of Health Research December 2017, and other documents supplemented by modifications made and lessons learned after implementation were used by the authors to create the description of the study provided here. RESULTS: The CCNA COMPASS-ND cohort includes participants from across Canada with various cognitive conditions associated with or at risk of neurodegenerative diseases. They will undergo a wide range of experimental, clinical, imaging, and genetic investigation to specifically address the causes, diagnosis, treatment, and prevention of these conditions in the aging population. Data derived from clinical and cognitive assessments, biospecimens, brain imaging, genetics, and brain donations will be used to test hypotheses generated by CCNA research teams and other Canadian researchers. The study is the most comprehensive and ambitious Canadian study of dementia. Initial data posting occurred in 2018, with the full cohort to be accrued by 2020. CONCLUSION: Availability of data from the COMPASS-ND study will provide a major stimulus for dementia research in Canada in the coming years.

Évaluation complète d'une étude de cohorte canadienne portant sur la démence et la neuro-dégénérescence. Contexte : L'évaluation globale de la neuro-dégénérescence et de la démence (COMPASS-ND), étude de cohorte du Consortium canadien en neuro-dégénérescence associée au vieillissement (CCNV), représente une initiative nationale visant à promouvoir la recherche portant sur la démence et à soutenir les programmes de recherche des équipes du CCNV. Totalisant 2310 sujets recrutés partout au pays, cette cohorte longitudinale regroupe des individus fortement « phénotypés ¼ qui présentent diverses formes de démence et de pertes de mémoire légères. En plus de sujets âgés dont les fonctions cognitives sont intactes, ces 2310 sujets ont permis de valider les hypothèses formulées par les équipes du CCNV. Méthodes : Nous avons utilisé de nombreux documents pour décrire cette étude : le protocole de la COMPASS-ND ; la demande initiale de subvention ; le cinquième rapport d'étape semi-annuel du CCNV soumis aux Instituts de recherche en santé du Canada (IRSC) en décembre 2017 ; ainsi que d'autres documents produits à la suite de modifications consécutives à la mise en œuvre de ce projet. Résultats: L'étude de cohorte COMPASS-ND du CCNV inclut des participants de partout au Canada dont les divers états cognitifs sont associés à des maladies neurodégénératives ou au risque d'en souffrir. Ils feront l'objet d'un large éventail d'examens expérimentaux, cliniques, génétiques et d'imagerie afin d'aborder de manière spécifique les causes, le diagnostic, le traitement et la prévention de ces états cognitifs chez les personnes âgées. Les données obtenues à la suite d'évaluations cliniques et cognitives, ainsi que celles issues d'échantillons biologiques, d'imagerie cérébrale, de tests génétiques et de dons de cerveaux, seront utilisées pour tester les hypothèses générées par les équipes de recherche du CCNV et d'autres chercheurs canadiens. Cette étude constitue donc à ce jour l'étude canadienne la plus complète et la plus ambitieuse au sujet de la démence. La présentation des données initiales ayant eu lieu en 2018, la cohorte devrait atteindre sa taille maximale d'ici à 2020.Conclusion : La disponibilité des données de l'étude COMPASS-ND stimulera considérablement la recherche sur la démence au Canada au cours des prochaines années.

Into the Fourth Dimension: Dysregulation of Genome Architecture in Aging and Alzheimer's Disease.

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.

Integrating sex and gender into neurodegeneration research: A six-component strategy.

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.

Amino-Terminal ß-Amyloid Antibody Blocks ß-Amyloid-Mediated Inhibition of the High-Affinity Choline Transporter CHT.

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 ß-amyloid peptides (Aß) 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 Aß peptides released from neural cells. The Aß-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 Aß-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aß on CHT activity. Importantly, neutralizing Aß using an anti-Aß antibody directed at the N-terminal amino acids 1-16 of Aß, but not by an antibody directed at the mid-region amino acids 22-35 of Aß, attenuates the effect of Aß on CHT activity and trafficking. This indicates that a specific N-terminal Aß epitope, or specific conformation of soluble Aß, may impair CHT activity. Therefore, Aß 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.

Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP.

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.

82-kDa choline acetyltransferase and SATB1 localize to ß-amyloid induced matrix attachment regions.

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-ß1-42 (Aß1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aß1-42 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 Aß1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aß1-42-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 Aß1-42 -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 Aß-exposure.

Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

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.

Mobility and Cognition in Seniors. Report from the 2008 Institute of Aging (CIHR) Mobility and Cognition Workshop.

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.

Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

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.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to ß-amyloid peptides (Aß), 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 Aß 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 Aß1-42 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 Aß1-42 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 Aß production. This decreased formation of Aß could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aß. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aß production, with this potentiating the decline in function of cholinergic neurons.

Overexpression of pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A in nerve cells confers resistance to amyloid ß and other toxins by decreasing mitochondrial respiration and reactive oxygen species production.

We previously demonstrated that nerve cell lines selected for resistance to amyloid ß (Aß) 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 Aß and other neurotoxins. Treatment of Aß-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 Aß treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aß 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 Αß 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 Aß deposition without developing Alzheimer disease.

Choline transporter CHT regulation and function in cholinergic neurons.

Choline uptake into cholinergic nerve terminals by the sodium-dependent high-affinity choline transporter CHT is essential for providing choline as substrate for synthesis of acetylcholine (ACh); ACh is used by cholinergic neurons to communicate information to a wide range of tissues in central and peripheral nervous systems. CHT is expressed almost exclusively in cholinergic neurons, and is subject to transcriptional and post-translational control by factors that promote or diminish cholinergic neurotransmission. The distribution of CHT proteins within cholinergic presynaptic terminals is dynamically regulated. Thus, choline uptake activity is determined largely by the plasma membrane CHT level, and this is finely controlled by a balance between internalization and recycling of CHT proteins in endosomal compartments. CHT proteins are also in synaptic vesicle membranes, thereby allowing cell surface CHT levels to increase rapidly in conjunction with exocytotic transmitter release to provide enhanced choline for ACh re-synthesis. Little is known about post-translational modification of CHT, although data is emerging that CHT activity and subcellular trafficking is modulated by kinase-mediated phosphorylation. Recent studies have also identified proteins with which CHT interacts, but this requires further investigation to reveal the role of other proteins in regulating CHT function and activity. Polymorphisms in CHT protein and modifications in its expression are linked to neurological and psychiatric disorders, and can alter function of peripheral systems that are regulated by cholinergic innervation, such as the cardiovascular system. The critical role of CHT in maintaining cholinergic transmission indicates that it could be a target for therapeutic intervention to promote ACh synthesis, but mechanisms by which this can be accomplished have not been adequately addressed.

Peroxynitrite donor SIN-1 alters high-affinity choline transporter activity by modifying its intracellular trafficking.

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.

Amyloid-beta oligomers increase the localization of prion protein at the cell surface.

In Alzheimer's disease, the amyloid-ß peptide (Aß) 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 Aß. We show here that Aß oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aß oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aß 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 Aß oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aß oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aß oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aß oligomers. Our experiments show for the first time that Aß oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.

Role of alpha7 nicotinic acetylcholine receptor in calcium signaling induced by prion protein interaction with stress-inducible protein 1.

The prion protein (PrP(C)) is a conserved glycosylphosphatidylinositol-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 PrP(C)-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 α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR 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)·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Oligomeric aggregates of amyloid beta peptide 1-42 activate ERK/MAPK in SH-SY5Y cells via the alpha7 nicotinic receptor.

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.