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Alzheimer's disease (AD) is characterized by amyloidosis, neuroinflammation, cholinergic dysfunction and cognitive impairment. In AD, the cholinergic neuronal marker choline acetyltransferase (ChAT) is reduced and the primate-specific nuclear isoform, 82-kDa ChAT, is mislocalized to cytoplasm. Cell-based studies suggest a role for 82-kDa ChAT in regulating expression of AD-related genes with potential reductions in β-amyloid (Aβ) levels. To study this further, we crossed transgenic mice expressing human 82-kDa ChAT with the AD mouse model APP NL-G-F and used molecular techniques and neurobehavioral tests to study the impact of 82-kDa ChAT on AD pathology. These mice had altered expression of genes linked to Aβ clearance and inflammation, and reduced cognitive decline, amyloidosis and gliosis. These effects were inversely related to age and Aβ plaque load and correlated best with 82-kDa ChAT localized to nuclei of neurons. The study suggests a role for 82-kDa ChAT in decreasing AD-like pathology., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The inaugural Canadian Conferences on Translational Geroscience were held as 2 complementary sessions in October and November 2023. The conferences explored the profound interplay between the biology of aging, social determinants of health, the potential societal impact of geroscience, and the maintenance of health in aging individuals. Although topics such as cellular senescence, molecular and genetic determinants of aging, and prevention of chronic disease were addressed, the conferences went on to emphasize practical applications for enhancing older people's quality of life. This article summarizes the proceeding and underscores the synergy between clinical and fundamental studies. Future directions highlight national and global collaborations and the crucial integration of early-career investigators. This work charts a course for a national framework for continued innovation and advancement in translational geroscience in Canada., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The E4 allele of apolipoprotein E (ApoE4) is a key genetic risk factor for late-onset Alzheimer's disease (AD), increasing the risk of developing the disease by up to three-fold. However, the mechanisms by which ApoE4 contributes to AD pathogenesis are poorly understood. Here, we utilize a mouse model expressing either human ApoE3 or human ApoE4 to examine the effects of the E4 allele on a wide range of genetic and molecular pathways that are altered in early AD pathology. We demonstrate that ApoE4-expressing mice begin to show early differential expression of multiple genes, leading to alterations in downstream pathways related to neural cell maintenance, insulin signaling, amyloid processing and clearance, and synaptic plasticity. These alterations may result in the earlier accumulation of pathological proteins such as β-amyloid that may build up within cells, leading to the accelerated degeneration of neurons and astrocytes as observed in ApoE4-positive individuals. We also examine the metabolic effects associated with a high-fat diet (HFD) in male ApoE4-expressing mice compared with regular chow diet (RD) fed mice at different ages. We found that young ApoE4-expressing mice fed HFD developed metabolic disturbances, such as elevated weight gain, blood glucose, and plasma insulin levels that cumulatively have been observed to increase the risk of AD in humans. Taken together, our results reveal early pathways that could mediate ApoE4-related AD risk and may help identify more tractable therapeutic targets for treating ApoE4-associated AD., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) is an important cholinergic neuronal marker whose levels and/or activity are reduced in physiological and pathological aging. One isoform of ChAT, 82-kDa ChAT, is expressed only in primates and found primarily in nuclei of cholinergic neurons in younger individuals, but this protein becomes mostly cytoplasmic with increasing age and in Alzheimer's disease (AD). Previous studies suggest that 82-kDa ChAT may be involved in regulating gene expression during cellular stress. Since it is not expressed in rodents, we developed a transgenic mouse model that expresses human 82-kDa ChAT under the control of an Nkx2.1 driver. Behavioral and biochemical assays were used to phenotype this novel transgenic model and elucidate the impact of 82-kDa ChAT expression. The 82-kDa ChAT transcript and protein were expressed predominantly in basal forebrain neurons and subcellular distribution of the protein recapitulated the age-related pattern found previously in human necropsy brains. Older 82-kDa ChAT-expressing mice presented with better age-related memory and inflammatory profiles. In summary, we established a novel transgenic mouse expressing 82-kDa ChAT that is valuable for studying the role of this primate-specific cholinergic enzyme in pathologies associated with cholinergic neuron vulnerability and dysfunction., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morey, Esmaeili, Duennwald and Rylett.)

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.

This article describes priority areas for research on the impact of the Covid-19 pandemic on older adults that have been identified by the CIHR Institute of Aging (CIHR-IA). The process used by CIHR-IA consists of several iterative phases and thus far has resulted in identification of three key areas for Covid-19 research needs and four cross-cutting thematic areas. The key research priority areas are as follows: response of older adults to disease, vaccination, and therapeutics; mental health and isolation; and supportive care environments. The four cross-cutting themes are equity, diversity, and inclusion (EDI); ethical/moral considerations; evidence-informed practices; and digital health technologies. The priorities outlined in this article will inform CIHR-IA's responses to Covid-19 research needs.

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., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

The sodium-coupled high-affinity choline transporter CHT plays a critical role in acetylcholine (ACh) synthesis by taking up the substrate choline from the synaptic cleft after neurotransmitter release; this conservation mechanism is the rate-limiting step for production of ACh, thereby facilitating communication by subsequent action potentials. Mice carrying a null mutation for CHT die within an hour of birth due to respiratory failure, indicating the essential role of CHT proteins for sustaining cholinergic transmission. Choline uptake activity is regulated dynamically by CHT proteins undergoing rapid trafficking between subcellular compartments and the plasma membrane where they are functionally active. CHT proteins internalize from the cell surface into the endolysosomal pathway by a clathrin-mediated mechanism, but can undergo ubiquitination and proteosomal degradation under conditions such as cellular oxidative stress. Over the years, functionally-relevant CHT polymorphisms have been linked to a range of neurological and psychiatric disorders, including ADHD and depression; the impact of these mutations and the extent to which they alter cholinergic signaling have not been addressed fully. Recent studies have identified compounds that can either promote or diminish cholinergic neurotransmission by modulating CHT function, thus having the potential to serve as pharmacological tools or therapeutic prototypes. Here, we review regulation of CHT activity, trafficking and subcellular disposition of CHT proteins, alteration of transporter function in genetic, neurological and psychiatric diseases, and investigations of compounds that modulate activity of the transporter., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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.

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 14 PKLP V PP 20 . We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val 18 (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.

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.

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 (BafA 1 ). BafA 1 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.

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., (© 2016 International Society for Neurochemistry.)

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.

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.

The high-affinity choline transporter (CHT) is responsible for choline uptake into cholinergic neurons, with this being the rate-limiting step for acetylcholine production. Altering CHT protein disposition directly impacts choline uptake activity and cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild-type APP (APPwt) and determine if this differs with Swedish mutant APP (APPSwe) in SH-SY5Y human neuroblastoma cells. APPSwe differs from APPwt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APPSwe than with APPwt. Surprisingly, however, CHT cell surface levels and choline uptake activity are decreased to the same extent and CHT co-localization to early endosomes increased similarly in cells expressing either APPwt or APPSwe. A critical observation is that CHT co-immunoprecipitates with βCTF from APPSwe-expressing cells. We propose that decreased CHT function is mediated differently by APPwt and APPSwe; APPwt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APPSwe on CHT is mediated indirectly potentially by binding to the βCTF fragment or by Aβ released from cells. High-affinity choline transporter (CHT) takes choline into cholinergic neurons for acetylcholine synthesis. Amyloid precursor protein (APP) interacts with CHT proteins, but this is decreased for Swedish mutant APP (APPSwe). CHT cell surface levels and localization to early endosomes, and choline uptake activity are changed similarly by APPwt or APPSwe. APPSwe mediates effects indirectly potentially by βCTF or Aβ., (© 2015 International Society for Neurochemistry.)

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.

Purpose: Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer's. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells., Methods: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status., Results: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry., Conclusions: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis., (© 2013 International Society for Neurochemistry.)

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.

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.

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.

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., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

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.

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) and is a phenotypic marker for cholinergic neurons. Cholinergic neurons in brain are involved in cognitive function, attentional processing and motor control, and decreased ChAT activity is found in several neurological disorders including Alzheimer's disease. Dysregulation of ChAT and cholinergic communication is also associated with some spontaneous point-mutations in ChAT that alter its substrate binding kinetics, or by disruption of signaling pathways that could regulate protein kinases for which ChAT is a substrate. It has been identified recently that the catalytic activity and subcellular distribution of ChAT, and its interaction with other cellular proteins, can be modified by phosphorylation of the enzyme by protein kinase-C and Ca2+/calmodulin-dependent protein kinase II; these kinases appear also to mediate some of the effects of beta-amyloid peptides on cholinergic neuron functions, including the effects on ChAT. This review outlines a new model for the regulation of cholinergic transmission at the level of the presynaptic terminal that is mediated by hierarchically-regulated, multi-site phosphorylation of ChAT.

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

Maintenance of acetylcholine synthesis depends on the effective functioning of a high-affinity sodium-dependent choline transporter (CHT1). Recent studies have shown that this transporter is predominantly localized inside the cell, unlike other neurotransmitter transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylcholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis.

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.

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.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

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.