Therapeutic Efficacy Of FK506 And Minocycline Alone And In Combination Against Prion Infection In Hamsters

BACKGROUND:Prion diseases,also known as transmissible spongiform encephalopathies(TSEs),are a group of fatal neurodegenerative diseases affecting both humans and animals worldwide.Recent advanced research has shown that accumulation of misfolded prion proteins result in endoplasmic reticulum stress and ultimately a crosstalk between endoplasmic reticulum and mitochondria initiates to rescue cells from damage.The main event behind endoplasmic reticulum stress is thought to be dysregulated calcium and its downstream effects on mitochondrial dynamics.Prion infections of the central nervous system(CNS)are characterized by initial reactive gliosis followed by overt neuronal death.Gliosis is likely to be initially caused by the deposition of misfolded,proteinase K-resistant,isoforms(termed PrPSc)of the normal cellular prion protein(PrPc)in the brain.Proinflammatory cytokines and chemokines released by PrPSc-activated glia and stressed neurons as well may contribute directly or indirectly to the disease development by enhancing gliosis and inducing neurotoxicity.Recent studies have illustrated that early neuroinflammation activate the calcineurin nuclear factor of activated T-cells(NFAT)signaling cascade,resulting in nuclear translocation of nuclear factor kappa-b(NF-kB)to promote apoptosis.Hence,useful therapeutic approaches to slow down the course of prion disease development should control early neuroinflammation to suppress NFAT signaling.Here we used a hamster model of prion diseases to test,for the first time,the neuroprotective and NFAT-suppressive effect of a second-generation semisynthetic tetracycline derivative,minocycline in comparison to a calcineurin inhibitor FK506,with known NFAT suppressive activity.RESULTS PART-1:We used FK506 and minocycline alone in different stages of prion disease in hamsters in first part of our project.The basic aim was to see the effect of two mentioned drugs on early prion disease anomalies such as neuroinflammation and synaptic dysfunction and later stage changes leading to neuronal degeneration.Our results indicate that prolonged treatment with minocycline,starting from the pre-symptomatic stage of prion disease was more effective than FK506 given either during pre-symptomatic or symptomatic stage of prion disease.We showed that FK506 earlier stage therapy had some side effects as seen by reducing the weight of hamsters and cell cycle arrest.These adverse effects were not visible after late symptomatic stage treatment with FK506.Specifically,minocycline treatment reduced the expression of the astrocytes activation marker GFAP and of the microglial activation marker IBA-1,subsequently reducing the level of pro-inflammatory cytokines interleukin 1 beta(IL-1?)and tumor necrosis factor alpha(TNF-a).We further found that minocycline and FK506 treatment inhibited mitogen-activated protein kinase(MAPK)p38 phosphorylation and NF-kB nuclear translocation in a caspase dependent manner,and enhanced phosphorylated cAMP response element-binding protein(pCREB)and phosphorylated Bcl2-associated death promoter(pBAD)levels to reduce cognitive impairment and apoptosis.RESULTS PART-2:In our second project,based on our initial results of treatment with FK506 and minocycline alone in different stages of prion infection,we used a cocktail of FK506 and minocycline for the treatment of prion disease in hamsters in clinical stage.Here we shown that combinatory therapy with FK506+minocycline successfully alleviated prion induced astrogliosis and cytotoxicity in hamsters.Combinatory therapy with FK506+minocycline did not show any effect on the vacuolation profile as we saw slightly significant change in the vacuolation profile but we found intact dentate gyrus neurons in FK506+minocycline treated animals as compared to prion only group hamsters.We further showed that FK506+minocycline reduced the levels of proinflammatory cytokines IL-? and TNF-?,and increased the levels of anti-inflammatory cytokines IL-10 and IL-27.FK506+minocycline modulated MAPK-p38 pathway in caspase dependent manner.The cocktail treatment alleviated prion induced mitochondrial fragmentation in hamsters via maintaining mitochondrial membrane integrity.FK506+minocycline therapy significantly rescued prion infected hamsaters from mitochondrial damage by reducing cyctoplasmic translocation of cytochrome C and inhibiting mithochondrial translocation of apoptosis regulator bcl-2-like protein 4(BAX)from cytoplasm.Survival and cognition was increased through maintaining the levels of pBAD and pCREB via MAPK-NRF2-HO-1 pathway.Taken together our results indicate that,therapeutic remedy with FK506 and minocycline is a better choice for prolonged use in prion diseases and encourage its further clinical development as a possible treatment for this disease.CONCLUSIONS:The findings from our first project indicate that FK506 was effective in clinical phase of prion infection while minocycline was effective in early presymptomatic phase of prion infection.Futhermore,FK506 is necessary for cell survival and growth and its inhibition in early presymptomatic phase of prion infection leads to cell cycle arrest and stunted growth.These intriguing results lead us to the second part of our project where we used a cocktail of FK506+minocycline in clinical phase of prion infection,and we found very beneficial effects of cocktail treatment in hamsters infected with prions.We demonstrated that FK506+minocycline rescued prion induced neuronal and synaptic dysfunction via MAPK-NF-kB-NRF2-HO-1 pathway and enhanced memory and survival via pBAD and pCREB pathway.