Fluorescence-labeled receptor ligands have emerged as valuable molecular tools, being indispensable for studying receptor−ligand interactions by fluorescence-based techniques such as high-content imaging, fluorescence microscopy, and fluorescence polarization. Through application of a new labeling strategy for peptides, a series of fluorescent neurotensin(8−13) derivatives was synthesized by attaching red-emitting fluorophores (indolinium-and pyridinium-type cyanine dyes) to carbamoylated arginine residues in neurotensin(8−13) analogues, yielding fluorescent probes with high NTS 1 R affinity (pK i values: 8.15−9.12) and potency (pEC 50 values (Ca 2+ mobilization): 8.23−9.43). Selected fluorescent ligands were investigated by flow cytometry and high-content imaging (saturation binding, kinetic studies, and competition binding) as well as by confocal microscopy using intact CHO-hNTS 1 R cells. The study demonstrates the applicability of the fluorescent probes as molecular tools to obtain, for example, information about the localization of receptors in cells and to determine binding affinities of nonlabeled ligands.
Background
Physiological homeostasis decline, immunosenescence, and increased risk for multiple diseases, including neurodegeneration, are all hallmarks of ageing. Importantly, it is known that the ageing process is sex-biased. For example, there are sex differences in predisposition for multiple age-related diseases, including neurodegenerative and autoimmune diseases. However, sex differences in age-associated immune phenotypes are not clearly understood.
Results
Here, we examined the effects of age on immune cell phenotypes in both sexes of C57BL/6J mice with a particular focus on NK cells. We found female-specific spleen weight increases with age and concordant reduction in the number of splenocytes per gram of spleen weight compared to young females. To evaluate sex- and age-associated changes in splenic immune cell composition, we performed flow cytometry analysis. In male mice, we observed an age-associated reduction in the frequencies of monocytes and NK cells; female mice displayed a reduction in B cells, NK cells, and CD8 + T cells and increased frequency of monocytes and neutrophils with age. We then performed a whole blood stimulation assay and multiplex analyses of plasma cytokines and observed age- and sex-specific differences in immune cell reactivity and basal circulating cytokine concentrations. As we have previously illustrated a potential role of NK cells in Parkinson’s disease, an age-related neurodegenerative disease, we further analyzed age-associated changes in NK cell phenotypes and function. There were distinct differences between the sexes in age-associated changes in the expression of NK cell receptors, IFN-γ production, and impairment of α-synuclein endocytosis.
Conclusions
This study demonstrates sex- and age-specific alterations in splenic lymphocyte composition, circulating cytokine/chemokine profiles, and NK cell phenotype and effector functions. Our data provide evidence that age-related physiological perturbations differ between the sexes which may help elucidate sex differences in age-related diseases, including neurodegenerative diseases, particularly Parkinson’s disease, where immune dysfunction is implicated in their etiology.
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