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Development of fluorescence imaging probes for nicotinic acetylcholine ?4?2? receptors.

ABSTRACT: Nicotinic acetylcholine ?4?2? receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity ?4?2? nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging ?4?2? nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the ?4?2? nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in ?4?2? nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface ?4?2? nAChRs, labeled with antibodies specific for a ?2 subunit extracellular epitope indicating that nifrofam labels ?4?2? nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of ?4?2? nAChRs in brain regions.

SUBMITTER: Samra GK

PROVIDER: S-EPMC6004331 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Development of fluorescence imaging probes for nicotinic acetylcholine α4β2∗ receptors.

Samra Gurleen K GK Intskirveli Irakli I Govind Anitha P AP Liang Christopher C Lazar Ronit R Green William N WN Metherate Raju R Mukherjee Jogeshwar J

Bioorganic & medicinal chemistry letters 20171217 3

Nicotinic acetylcholine α4β2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synth ...[more]

PMID: 29277457

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Project description:The positron emission tomography (PET) radioligand (-)-[(18)F]flubatine is specific to α4β2(⁎) nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4β2(⁎) nAChR quantification with bolus plus constant infusion (B/I) (-)-[(18)F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n=8) or B/I (n=4) administration of (-)-[(18)F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n=6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5mg over 60min beginning 5min prior to radiotracer injection); 2. A single scan B/I paradigm (n=7) lasting up to 240min with 1.5mg physostigmine administered over 60min beginning at 125min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8±3.3mL/cm(3) in thalamus, 12.9±1.6mL/cm(3) in cerebellum, and ranged from 9.8 to 12.5mL/cm(3) in other gray matter regions. The B/I paradigm with equilibrium analysis at 120min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (-)-[(18)F]flubatine VT/fP in 120min, and suggest possible sensitivity of (-)-[(18)F]flubatine binding to physostigmine-induced changes in acetylcholine levels.

Project description:Nicotine, the principal reinforcing compound in tobacco, acts in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). This review summarizes our current knowledge regarding how the α5 accessory nAChR subunit, encoded by the CHRNA5 gene, differentially modulates α4β2* and α3β4* receptors at the cellular level. Genome-wide association studies have linked a gene cluster in chromosomal region 15q25 to increased susceptibility to nicotine addiction, lung cancer, chronic obstructive pulmonary disease, and peripheral arterial disease. Interestingly, this gene cluster contains a non-synonymous single-nucleotide polymorphism (SNP) in the human CHRNA5 gene, causing an aspartic acid (D) to asparagine (N) substitution at amino acid position 398 in the α5 nAChR subunit. Although other SNPs have been associated with tobacco smoking behavior, efforts have focused predominantly on the D398 and N398 variants in the α5 subunit. In recent years, significant progress has been made toward understanding the role that the α5 nAChR subunit-and the role of the D398 and N398 variants-plays on nAChR function at the cellular level. These insights stem primarily from a wide range of experimental models, including receptors expressed heterologously in Xenopus oocytes, various cell lines, and neurons derived from human induced pluripotent stem cells (iPSCs), as well as endogenous receptors in genetically engineered mice and-more recently-rats. Despite providing a wealth of available data, however, these studies have yielded conflicting results, and our understanding of the modulatory role that the α5 subunit plays remains incomplete. Here, we review these reports and the various techniques used for expression and analysis in order to examine how the α5 subunit modulates key functions in α4β2* and α3β4* receptors, including receptor trafficking, sensitivity, efficacy, and desensitization. In addition, we highlight the strikingly different role that the α5 subunit plays in Ca2+ signaling between α4β2* and α3β4* receptors, and we discuss whether the N398 α5 subunit variant can partially replace the D398 variant.

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Dataset Information

Development of fluorescence imaging probes for nicotinic acetylcholine ?4?2? receptors.

Publications

Development of fluorescence imaging probes for nicotinic acetylcholine α4β2<sup>∗</sup> receptors.

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