Dataset Information

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

ABSTRACT: The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for brain CHT capacity, we isolated T cells from the spleen, and synaptosomes from cortex and striatum, of wild type and CHT-overexpressing mice (CHT-OXP). Choline uptake capacity in T cells from CHT-OXP mice was two-fold higher than in wild type mice, mirroring the impact of CHT over-expression on synaptosomal CHT-mediated choline uptake. Monitoring T lymphocyte CHT protein and activity may be useful for estimating human CNS cholinergic capacity and for testing hypotheses concerning the contribution of CHT and, more generally, ACh signaling in cognition, neuroinflammation and disease.

SUBMITTER: Koshy Cherian A

PROVIDER: S-EPMC5524217 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

Koshy Cherian Ajeesh A Parikh Vinay V Wu Qi Q Mao-Draayer Yang Y Wang Qin Q Blakely Randy D RD Sarter Martin M

Neurochemistry international 20170531

The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for br ...[more]

PMID: 28577989

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Project description:ObjectiveThe elevated choline transporters (ChT), choline kinase (ChK), choline uptake, and phosphorylation in certain tumor cells have influenced the development of radiolabeled choline derivatives as diagnostic probes for imaging cell membrane proliferation. We, therefore, aimed to develop a choline-based moiety for imaging choline kinase-overexpressed tumors by single-photon emission tomography (SPECT). A novel choline-based diagnostic probe was synthesized and evaluated preclinically in various ChT- and ChK-overexpressed tumor models for SPECT imaging applications.MethodsThe synthesis of diethylenetriaminepentaacetic acid-bis-choline ethylamine [DTPA-bis(ChoEA)] featured the conjugation of dimethylaminoethanol to a bifunctional chelator DTPA anhydride. [99mTc]Tc-DTPA-bis(ChoEA) was prepared, and its in vivo characteristics were evaluated in BALB/c mice and tumor-xenografted PC3, A549, and HCT116 athymic mouse models. The in vitro parameters, including cell binding and cytotoxicity, were assessed in PC3, A549, and HCT116 cell lines. To evaluate the specificity of the radioprobe, competitive binding studies were performed. Small-animal SPECT/CT diagnostic imaging was performed for in vivo evaluation. The mouse biodistribution data was further investigated to estimate the radiation dose in humans.ResultsIn silico studies suggested high binding with enhanced specificity. A standard radiolabeling procedure using stannous chloride as a reducing agent showed a labeling yield of 99.5 ± 0.5%. The in silico studies suggested high binding with enhanced specificity. [99mTc]Tc-DTPA-bis(ChoEA) showed high in vitro stability and specificity. The pharmacokinetic studies of [99mTc]Tc-DTPA-bis(ChoEA) in mice showed an increased tumor-to-background ratio after few minutes of intravenous administration. The first-in-human trial was also conducted. The effective dose was estimated to be 0.00467 mSv/MBq (4.67 mSv/GBq), resulting in a radiation dose of up to 1.73 mSv for the 370 MBq injection of [99mTc]Tc-DTPA-bis(ChoEA).ConclusionsThe synthesized radioprobe [99mTc]Tc-DTPA-bis(ChoEA) accumulates specifically in choline kinase-overexpressed tumors with a high signal-to-noise ratio. The preclinical and first-in-man data suggested that [99mTc]Tc-DTPA-bis(ChoEA) could potentially be used as a diagnostic SPECT tracer in the monitoring and staging of cancer.

Project description:Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

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Dataset Information

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

Publications

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

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