RESUMO
Lynx1 is a GPI-tethered protein colocalized with nicotinic acetylcholine receptors (nAChRs) in the brain areas important for learning and memory. Previously, we demonstrated that at low micromolar concentrations the water-soluble Lynx1 variant lacking GPI-anchor (ws-Lynx1) acts on α7-nAChRs as a positive allosteric modulator. We hypothesized that ws-Lynx1 could be used for improvement of cognitive processes dependent on nAChRs. Here we showed that 2 µM ws-Lynx1 increased the acetylcholine-evoked current at α7-nAChRs in the rat primary visual cortex L1 interneurons. At higher concentrations ws-Lynx1 inhibits α7-nAChRs expressed in Xenopus laevis oocytes with IC50 ~ 50 µM. In mice, ws-Lynx1 penetrated the blood-brain barrier upon intranasal administration and accumulated in the cortex, hippocampus, and cerebellum. Chronic ws-Lynx1 treatment prevented the olfactory memory and motor learning impairment induced by the α7-nAChRs inhibitor methyllycaconitine (MLA). Enhanced long-term potentiation and increased paired-pulse facilitation ratio were observed in the hippocampal slices incubated with ws-Lynx1 and in the slices from ws-Lynx1-treated mice. Long-term potentiation blockade observed in MLA-treated mice was abolished by ws-Lynx1 co-administration. To understand the mechanism of ws-Lynx1 action, we studied the interaction of ws-Lynx1 and MLA at α7-nAChRs, measured the basal concentrations of endogenous Lynx1 and the α7 nAChR subunit and their association in the mouse brain. Our findings suggest that endogenous Lynx1 limits α7-nAChRs activation in the adult brain. Ws-Lynx1 partially displaces Lynx1 causing positive modulation of α7-nAChRs and enhancement of synaptic plasticity. Ws-Lynx1 and similar compounds may constitute useful hits for treatment of cognitive deficits associated with the cholinergic system dysfunction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Inibidores da Colinesterase/toxicidade , Disfunção Cognitiva/prevenção & controle , Disfunção Cognitiva/psicologia , Plasticidade Neuronal/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Acetilcolina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/farmacocinética , Alcaloides/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , Disfunção Cognitiva/induzido quimicamente , Interneurônios/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Olfato/efeitos dos fármacos , Córtex Visual/efeitos dos fármacos , Xenopus laevisRESUMO
Differentially expressed in ovarian cancer-2/disabled 2 (DOC-2/DAB2) protein, often lost in prostate cancer and other cancer types, is a part of homeostatic machinery in normal prostate epithelium. DOC-2/DAB2 modulates mitogen-elicited mitogen-activated protein kinase (MAPK) signal transduction by sequestering several adaptor or effector molecules, such as growth factor receptor bound protein 2 and c-Src. We have shown that the proline-rich sequence in DOC-2/DAB2 is the key functional domain for this action. In this study, we further synthesized peptide based on the functional proline-rich domain and examined its biological function in prostate cancer using cell-permeable peptide (CPP) as a delivery system. From screening of several CPPs in prostate cancer cell lines, a polyarginine peptide (R11) seemed to be the best delivery vehicle because of its highly efficient uptake. In addition, we also observed a similar in vitro half-life and cellular location of R11 in four different prostate cancer cell lines. By conjugating a proline-rich sequence (PPL) or control sequence (AAL) derived from DOC-2/DAB2 to the COOH terminus of R11, we showed that R11PPL but not R11 or R11AAL was able to suppress either serum- or androgen-induced cell proliferation in prostate cancer cells without endogenous DOC-2/DAB2 expression. Consistently, the activation status of MAPK elicited by these mitogens was significantly inhibited by R11PPL but not by R11AAL or R11. Taken together, we conclude that a functional peptide derived from proline-rich domain in DOC-2/DAB2 has growth-inhibitory activity as its native protein, and CPP seems to be an efficient delivery system in prostate cancer cells.