Efficacy and Mechanism of a Glycoside Compound Inhibiting Abnormal Prion Protein Formation in Prion-Infected Cells: Implications of Interferon and Phosphodiesterase 4D-Interacting Protein

Abstract

A new type of antiprion compound, Gly-9, was found to inhibit abnormal prion protein formation in prion-infected neuroblastoma cells, in a prion strain-independent manner, when the cells were treated for more than 1 day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were downregulated after more than 2 days of treatment, and the phosphodiesterase 4D-interacting protein gene, a gene involved in microtubule growth, was upregulated after more than 1 day of treatment. A supplement of interferon given to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of the Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D-interacting protein restored or increased both the abnormal prion protein level and the normal prion protein level, without transcriptional alteration of the prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D-interacting protein might affect prion protein levels by altering the trafficking of prion protein-containing structures. Interferon and phosphodiesterase 4D-interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein levels independently. Although the in vivo efficacy of Gly-9 was limited, the findings for Gly-9 provide insights into the regulation of abnormal prion protein in cells and suggest new targets for antiprion compounds. IMPORTANCEThis report describes our study of the efficacy and potential mechanism underlying the antiprion action of a new antiprion compound with a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the compound's mechanism of action: interferon and a microtubule nucleation activator, phosphodiesterase 4D-interacting protein. In particular, phosphodiesterase 4D-interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein but also for the implication of new targets for therapeutic development.

A Single Subcutaneous Injection of Cellulose Ethers Administered Long before Infection Confers Sustained Protection against Prion Diseases in Rodents

“…Intracerebral or intraperitoneal infection was performed by inoculation with 20 or 100 μL, respectively, of a 1% (w/v) brain homogenate obtained from a terminally ill 263K prion-infected hamster. In case of continuous intracerebroventricular infusion, continuous infusion into the cerebral third ventricle was performed using an osmotic minipump and brain infusion kit, as previously described [49]. Mice were monitored every day until the time of terminal disease, at which time the mice were akinetic (with a lack of grooming behavior, coordination, and parachute reaction) or exhibited a rigid tail, an arched back, and weight loss of approximately 10% within one week.…”

“…Mice were monitored every day until the time of terminal disease, at which time the mice were akinetic (with a lack of grooming behavior, coordination, and parachute reaction) or exhibited a rigid tail, an arched back, and weight loss of approximately 10% within one week. The mice were killed at this stage and disease was confirmed by immunoblotting and immunohistochemical analyses of abnormal PrP deposition in the brain, as previously described [12,49]. Because it was difficult to determine the time of symptom onset in the animal model used in this study, survival time was defined as the time from prion infection to terminal disease.…”

“…Total RNA was extracted and cDNA was synthesized using a first-strand cDNA synthesis kit (Takara Bio, Inc., Kyoto, Japan). The PrP mRNA level was measured by real-time polymerase chain reaction, as previously described [49]. …”

“…To detect PrP C and β-actin, cell lysate was used without further treatments and mixed with a concentrated loading buffer. Immunoblotting analysis was performed using the anti-PrP monoclonal antibody SAF83 (SPI-Bio, Massy, France) and anti-β-actin monoclonal antibody, as described previously [49]…”

Insights from Therapeutic Studies for PrP Prion Disease

“…Among these models, prion-infected cell models have been the most frequently used since Congo red and polyanionic glycans were found to possess antiprion activity (Caughey and Race 1992;Caughey and Raymond 1993). In fact, our research group has identified dozens of antiprion compounds, including quinacrine and compB, using prion-infected cell models (Doh-ura et al 2000(Doh-ura et al , 2007bIshikawa et al 2004Ishikawa et al , 2006Murakami-Kubo et al 2004;Kawatake et al 2006;Kawasaki et al 2007;Nguyen et al 2008Nguyen et al , 2011Hamanaka et al 2011Hamanaka et al , 2015Teruya and Doh-ura 2013;Nishizawa et al 2014). However, these cell models are not fully compatible with in vivo prion-infected neuronal cells.…”