Effect of combination drug treatment on PrPSc in cell culture mode

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are always fatal infectious neurodegenerative disorders that affect animals and humans. The main feature of these diseases is the conversion of the normal prion protein (PrPc) found in the host into the pathological isoform PrPSc through profound conformational changes. The infectious prion protein is aggregate-prone, very stable and cannot be fully digested by proteases. As in other neurodegenerative diseases, the main hallmark of prion diseases is loss of neurons. It has been very difficult to find a potent treatment for the various types of prion diseases. TSEs are characterized by long incubation times and a short clinical phase that adds to the complication of not only finding a treatment but also for detecting the disease before symptoms start. Autophagy is a basic cellular program that involves degradation of intracellular organelles or cytoplasmic proteins when a cell is e.g. undergoing starvation. Imbalances of the level of autophagy have been shown to be associated with diseases like cancer, myopathies and neurodegenerative diseases like Huntington's, Parkinson's and prion diseases. Hence, it is important to exploit autophagy and its relationship with prion diseases to possibly find a way to treat them. This will not only prove beneficial to prion research but also to other neurodegenerative diseases characterized with abnormal protein aggregation. Research in our and others laboratories has shown that treating prion-infected cells and animals with autophagy-inducing drugs like Glivec, Tamoxifen, Lithium, and Rapamycin helps in clearing PrPSc and decreasing prion infection. However, these drugs have problems in vivo like effectively crossing the blood-brain barrier and side effects. This study was focused on using combinations of autophagy-inducing compounds to achieve additive effects in clearing PrPSc and hence having therapeutic potential without side-effects. The in-vitro model used in these studies were N2a22L cells, a persistently prion infected neuroblastoma cell line. Various autophagy-inducing drugs in different combinations were tested on N2a22L cells for 3, 6 and 10 days at sub-effective concentrations for individual drugs. The best combination consisted of all four drugs- Glivec, Rapamycin, Tamoxifen and Lithium together. This combination was not toxic to the cells and decreased PrPSc levels substantially after a 6 day treatment. However, this additive effect was transient and PrPSc levels started to increase again when cells were treated for 10 days. A possible explanation for this transient anti-prion effect was that cells developed a form of resistance against drug treatment. To explore further if cells treated with all four drugs start becoming resistant to the same drugs, experiments were performed with the cells that were previously exposed to these drugs. In a parallel study, where the drug combinations were given to the pre-exposed cells and the naive N2a22L cells, it was observed that naive cells displayed elevated levels of autophagy upon treatment; hence more clearance of PrPSc compared to pre-exposed cells was found. These results indicate that autophagy is a main mechanism through which these drugs are acting to decrease the prion infection. However, since the N2a22L cells have the potential to develop resistance against autophagy-inducing drugs, it was important to explore other drugs that might have different mode of actions to help reduce the prion infection. Thus, Suramin, a compound which targets PrPSc biogenesis, was added as one of the drugs combined with autophagy inducing drugs. From all tested combinations, Suramin with Glivec worked the best at 3, 6 and 10 days and there was no indication of reappearance of infection in 10 days treatment as observed in previous experiments. This combination was not toxic to N2a22L cells and might work to potentially decrease the prion infection. Hence, drugs that work through two different pathways- Glivec through induction of autophagy and Suramin through re-routing the aggregated abnormal prion proteins to acidic compartments for digestion, completely bypassing the plasma membrane, have the most potential anti-prion effect. If this combination has similar effects in other prion infected cell culture and animal models, it could be used as a possible treatment for peripheral prion infections. For future experiments, it would be important to test these combinations on different prion infected cell lines to confirm their effects. The duration of drug treatment can also be increased to observe if prion infection disappears, reappears or fluctuates. If successful, they should then be tried in prion infected animal models to observe if there is any improvement in crossing the blood-brain barrier in therapeutic models of prion infection and a prolongation of incubation time in prophylactic and post-exposure models of peripheral prion infection.