RESUMO
Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: ⢠Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. ⢠New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. ⢠Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Priônicas/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Pirazinas/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eletrorretinografia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Jejum , Feminino , Glucose/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Priônicas/química , Agregação Patológica de Proteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/fisiopatologia , Método Simples-Cego , Solubilidade , Organismos Livres de Patógenos Específicos , Transcrição Gênica/efeitos dos fármacosRESUMO
The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.