Calcium-dependent serine-threonine phosphatase, calcineurin inactivation mediated by baicalein attenuates prion protein-mediated neuronal cell damage

Background: Prion diseases are a group of fatal neurodegenerative disorders characterized by neuronal cell death. Calcineurin and autophagy mediate prion-induced neurodegeneration, suggesting that inhibition of calcineurin and autophagy could be a target for therapy. Baicalein has been reported to exert neuroprotective effects against calcium-dependent neuronal cell death. Results: In this study, we investigated the effects of baicalein on the development of prion diseases. We found that baicalein treatment inhibits prion protein-induced apoptosis. Baicalein inhibited calcium up-regulation and protected the cells against prion peptide ‐ induced neuron cell death by calcineurin inactivation. Furthermore, baicalein increased p62 protein levels and decrease LC3-II protein levels indicating autophagic ux inhibition and baicalein inhibited prion protein-induced neurotoxicity through autophagy ux inhibition. Conclusions: Taken together, this study demonstrated that baicalein attenuated prion peptide-induced neurotoxicity via calcineurin inactivation and autophagic ux reduction, and also suggest that baicalein may be an effective therapeutic drug against neurodegenerative diseases, including prion diseases.


Background
Prion diseases, known as transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders. They include kuru and sporadic Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathies in cattle, and scrapie in sheep and goats [1,2]. Prion diseases are characterized by misfolding of normal cellular prion protein (PrP C ) into scrapie prion protein (PrP Sc ), which is an abnormal protease-resistant misfolded isoform [3,4]. PrP C is soluble, with a predominant alpha-helical conformation, but a disease-causing, infectious form (PrP Sc ) is insoluble, β-sheet-rich, and protease-resistant [5]. Because of β-sheet-rich conformation, PrP Sc is highly pathogenic and neurotoxic when compared with the largely α-sheet-rich structure of PrP C [6,7]. PrP Sc is a misfolded protein aggregate accumulating within endosomes or on the neuronal cell surface. It leads to cytotoxicity and cell death [8][9][10].
Calcineurin or protein phosphatase 2B (PP2B) is highly abundant in the brain and regulates synaptic plasticity and neuronal death [20]. Calcineurin immunoreactivity occurs exclusively in the neurons throughout brain [21,22]. Overactivation of calcineurin by amyloid-beta and prion proteins induces neurotoxicity [3,23]. Tacrolimus or FK506 is a well-known immunosuppressive drug, which binds to the FK506-binding protein (FKBP). The FK506-FKBP12 complex binds to calcineurin and inhibits its activity [24].
Autophagy is a highly conservative cellular process in which cells degrade and recycle bulk cytosolic proteins and damaged organelles via lysosomal degradation [25,26]. Importantly, autophagy plays an important role in the survival of neural cells [27,28]. Autophagy plays a key role in in ammatory diseases, and protein misfolding diseases [29]. However, the role of autophagy in baicalein-mediated neuroprotective effects is not fully understood.
Our previous study showed that baicalein inhibits prion peptide-induced neuronal apoptosis by inhibiting JNK activation. However, the effect of baicalein on prion protein-induced neuronal cell damage via calcineurin and autophagy has yet to be reported. In this study, we investigated the protective effects of baicalein against PrP (106-126)-mediated apoptosis, calcineurin and autophagy activation.
We investigated whether baicalein affected prion peptide-induced neurotoxicity. The results showed that prion protein induced neuronal cell death, and baicalein reduced prion protein-mediated neurotoxicity ( Figures 1A and B). Using TUNEL assay, we demonstrated that the apoptosis in the PrP (106-126)-treated cells led to the emission of green uorescence, indicating DNA strand breakage ( Figure 1C). These results indicate that baicalein is effective in preventing prion peptide-induced neuronal apoptosis.

Baicalein inhibits PrP (106-126)-induced apoptosis by calcineurin inactivation
To determine the effect of baicalein on prion peptide expression in neuronal cells, we investigated the role of prion peptide in altering Ca 2+ levels in neuronal cells. PrP (106-126) treatment resulted in a rapid increase in Ca 2+ , and baicalein reduced PrP-induced Ca 2+ (Figures 2A and B). Fluorescence imaging also indicated that baicalein inhibited calcium increase ( Figure 2C). The activity of calcineurin was evaluated by measuring the formation of free phosphate ( ), using a speci c kit assay. The results showed that prion protein elevated calcineurin activity and baicalein reduced prion peptide-induced calcineurin activity ( Figure 2D). Next, to investigate whether the inhibition of calcineurin exerts neuroprotective effects against PrP (106-126)-induced neurotoxicity, we analyzed the levels of calcineurin and cell viability using a calcineurin inhibitor (FK506). We also found that baicalein and calcineurin inhibitor blocked the PrP (106-126)-induced neuronal apoptosis ( Figures 3A and B). TUNEL assay demonstrated that baicalein and calcineurin inhibitor reduced apoptosis in PrP (106-126)-treated cells resulting in the emission of green uorescence, indicating DNA strand breakage ( Figure 3C). The results showed that baicalein and calcineurin inhibitor reduced prion protein-induced calcineurin activity ( Figure 3D).

Baicalein treatment inhibits PrP (106-126)-induced apoptosis via autophagy
We investigated whether baicalein affects prion peptide-induced autophagy. We found that baicalein increased SQSTM1/p62 and decreased LC3-II ( Figure 4A). Orange uorescence was recovered by treatment with calcineurin inhibitor ( Figure 4B). Prion peptide-induced autophagic ux was abolished by baicalein and con rmed by the up-regulation of SQSTM1/p62 protein and the down-regulation of LC3-II ( Figure 4C). We also conducted TEM to establish the effect of lysosomal inhibition of autophagy by baicalein. As shown in Figure 4D, multiple vesicles including double-membraned autophagosomes (arrowheads) were induced by treatment of cells with baicalein, which indicated inhibition of autophagic ux and lysosomal degradation ( Figure 4D). Next, to investigate whether the inhibition of autophagy exerted neuroprotective effects against PrP (106-126)-induced neurotoxicity, we analyzed the levels of autophagy and cell viability using an autophagy inhibitor (chloroquine). We also found that baicalein and autophagy inhibitor blocked PrP (106-126)-induced neuronal apoptosis ( Figures 5A and B). TUNEL assay demonstrated that exposure to baicalein and autophagy inhibitor reduced apoptotic process in PrP (106-126)-treated cells that led to the emission of green uorescence, indicating DNA strand breakage ( Figure 5C). Prion peptide-induced autophagic ux was abolished by baicalein and was con rmed by the upregulation of SQSTM1/p62 protein and the downregulation of LC3-II, and inhibition of prion peptideinduced autophagy was con rmed by the upregulation of SQSTM1/p62 protein and LC3-II ( Figure 5D).

Discussion
The study was to investigate the role of baicalein in calcineurin inactivation and autophagy, and the regulation of PrP (106-126)-induced apoptosis by baicalein in neuronal cells. The results suggest that the decrease in calcineurin and autophagy by baicalein and the consequent reduction in prion proteininduced neurotoxicity may be the key mechanisms underlying the neuroprotective effects of baicalein.
It has been reported that baicalein inhibits intracellular Ca 2+ concentrations by reducing phospholipase C activity in C6 rat glioma cells [30]. Wang et al suggested that the protective effect of baicalein is mediated via calcineurin pathway in angiotensin II-treated mice. Baicalein inhibits cardiac remodeling by attenuating calcineurin signaling pathways in mice [19]. Consistent with these ndings, our results also demonstrated that baicalein attenuates calcineurin activity. Together, these ndings suggest that baicalein effectively inhibits prion protein-induced calcium and calcineurin activity.
Because baicalein has neuroprotective and antioxidant properties, It has been reported to exert a neuroprotective effect against ß-amyloid peptide [31,32], 6-OHDA-induced PD [15,18] and human prion protein fragment 106-126 (PrP) [33]. A medicinal herb Scutellaria lateri ora is known to inhibit PrP replication in vitro and delay the onset of prion disease in Mice [34]. The neuroprotection of baicalein has been linked to its anti-apoptotic, anti-in ammatory and pro-differentiation mechanisms. This study explored the neuroprotective role of baicalein against prion protein-induced neurotoxicity.
Although prion peptide 106-126 was used more than 20 years as a model of prion disorders, the relevance of pathogenic mechanism is still controversial. The prion peptide PrP106-126 has been widely used as a suitable model peptide because this fragment is soluble in water and exhibits several physicochemical and biological properties similar to those of PrP Sc , such as aggregation in solution, antiproteinase K digestion, and neurotoxicity [42,43]. However, an important argument about PrP peptide 106-126 is the facts that it is not actually a fragment found in the brains of humans or animals with prion diseases [44]. Even we investigated baicalein effect in this study, we will further study the issue using longer fragments or full-length PrP mutant models to examine therapeutic potential of baicalein in prion disease.

Conclusions
In the future, we will further study the neuroprotective effects of baicalein, autophagy and the calcineurin pathway in mouse models to examine baicalein's potential therapeutic role in prion disease  Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magni cation, x10,000) installed at the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University.

Statistical analysis
All data were expressed as mean ± standard error, and compared using the one-way ANOVA followed by the Tukey test. All statistical analysis was performed using GraphPad Prism software. Results were considered signi cant at * p < 0.05, ** p < 0.01 or *** p < 0.001, as appropriate.