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Transmissible spongiform encephalopathies (TSE) are characterized by the misfolding of the host encoded prion protein (PrPC) into a pathogenic isoform (PrPSc) which leads to the accumulation of β-sheet-rich fibrils and subsequent loss of neurons and synaptic functions. Although many compounds have been identified which inhibit accumulation or dissolve fibrils and aggregates in vitro there is no therapeutic treatment to stop these progressive neurodegenerative diseases. Here we describe the effects of the traditional medicinal herb Scutellaria lateriflora (S. lateriflora) and its natural compounds, the flavonoids baicalein and baicalin, on the development of prion disease using in vitro and in vivo models. S. lateriflora extract as well as both constituents reduced the PrPres accumulation in scrapie-infected cell cultures and cell-free conversion assays and lead to the destabilization of pre-existing PrPSc fibrils. Moreover, tea prepared from S. lateriflora, prolonged significantly the incubation time of scrapie-infected mice upon oral treatment. Therefore S. lateriflora extracts as well as the individual compounds can be considered as promising candidates for the development of new therapeutic drugs against TSEs and other neurodegenerative diseases like Alzheimer’s and Parkinson’s disease.
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases which are characterized by the accumulation and deposition of a pathogenic isoform (PrPSc) of the host encoded cellular prion protein (PrPC) designated PrPSc. Both isoforms share the same amino acid sequence but differ in conformation, resistance to proteinase K (PK), and pathogenicity. PrPSc tends to oligomerize by a seeded polymerization mechanism followed by the formation of multimers and eventually of fibril structures. This aggregation can be reproduced in an analogous manner in vitro, using cell-based as well as cell-free assays. The evolved PrPSc like isoforms – termed PrPres – harbor similar biochemical characteristics like resistance to Proteinase K and detection by same antibodies.
Accumulation of PrPSc in the central nervous system (CNS) is accompanied by neurological dysfunctions, neuronal vacuolation, and astrocytic gliosis. Although the exact disease causing mechanism is unknown to date, there is evidence for a general neurotoxicity of these aggregates, which deteriorate synaptic function and induce oxidative stress and membrane disruption (Soto and Estrada, 2008). Prion diseases belong to the group of protein misfolding diseases like Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s disease (HD), which are generally characterized by an incorrect folding process of a host encoded protein with a conformation different from its native structure. The misfolding is followed by a self-aggregation and polymerization of the protein according to a “seeding-nucleation” process (Jarrett and Lansbury, 1993).
As PrPSc formation and aggregation is the central event in prion diseases, the inhibition of oligomer formation and fibril extension as well as the enhancement of fibril degradation are major targets for the development of therapeutic strategies against TSEs. Several substances have been identified which inhibit PrPres formation and accumulation in vitro and prolonged survival in scrapie-infected animals: Congo red (Caughey and Race, 1992), branched polyamines (Supattapone et al., 2001), porphyrins and phthalocyanines (Priola et al., 2000; Caughey et al., 2007), heparan sulfate mimetics (Adjou et al., 2003), amphotericin (Mange et al., 2000), curcumine (Caughey et al., 2003; Yang et al., 2009), and tetracyclines (De Luigi et al., 2008). However, for various reasons none of these compounds has been included in prevention and treatment regimes for humans yet. Most recently two new substance classes, benzothiazoles (Geissen et al., 2011) and diphenyl-pyrazoles (Leidel et al., 2011), were identified by high-throughput screening approaches that inhibit PrPres accumulation in cell culture models and prolong incubation times in scrapie-infected mice.
Other therapeutic strategies rely on passive immunization (White et al., 2003), RNA interference (Pfeifer et al., 2006), RNA aptamers (Proske et al., 2002), copper chelating antibiotics (Murakami-Kubo et al., 2004), or on the induction of autophagy by Lithium (Heiseke et al., 2009).
Recent studies on AD suggest that phenolic compounds like green tea epigallocatechin gallate (Rezai-Zadeh et al., 2005), herb extracts like grape seed polyphenolic extract (Wang et al., 2009; Liu et al., 2011), or medicinal herbs like Paeonia suffruticosa (Fujiwara et al., 2009) can be used for the treatment of neurodegenerative diseases.
The here presented study shows that the American skullcap Scutellaria lateriflora, a traditional medical herb in North America, exhibits strong anti-prion activity. Extracts of this plant have been traditionally used as sedative and for the treatment of insomnia, anxiety, or neuralgia (Foster, 1996). Moreover, we can show here that the crude aqueous extract inhibits prion propagation and dissolves prion aggregates in vitro. Continuous oral administration of S. lateriflora tea (water extract) significantly prolonged incubation times in scrapie-infected mice. Notably, the natural constituents, the flavonoids baicalein and baicalin-hydrate, exhibit the same inhibitory effects on prion propagation and dissolve existing fibrils.
Baicalein and baicalein-hydrate were purchased from Sigma-Aldrich and solubilized in DMSO. The dried herb S. lateriflora was purchased from Goldener Zweig (Buchholz, Germany). Water extracts (tea) were prepared by mixing 30g of sliced dry herbs in 1.0l boiling water for 30min. After cooling the tea was administered directly to mice. For in vitro experiments tea was lyophilized to yield dried extracts. In cell-based assays the dried material was resuspended in H2O, concentrated by a factor of 10 (termed tea concentrate) and added to the cell culture medium. In cell-free conversion assay the dried extract was directly resuspended in the conversion buffer in a final volume of 20μl.
C57Bl/6 mice were inoculated with cloned mouse scrapie strains RML intracerebrally (i.c.; 30μl of 1% mouse brain homogenate) or intraperitoneally (i.p.; 50μl of 1% brain homogenate). The health status of the mice was inspected daily, and their body weights were recorded weekly. After the onset of TSE-associated clinical symptoms (and weight loss, abnormal tail tonus, hind limb paralysis), the animals were euthanized. The incubation times were calculated as the time between inoculation and death. The brains were removed and kept as following: one half of each brain was stored at −20°C, and the other half was fixed in 4% neutral buffered formalin.
Pathogenic isoform for PrP conversion reactions was purified from brains of C57/Bl6 mice i.c. inoculated with mouse scrapie strain Me7. Brains were taken at the beginning of clinical symptoms in the animals and stored at −20°C. PrPSc was purified according to Eiden et al. (2006).
Before embedding in paraffin the formalin fixed brain was cut at five different levels to reveal caudal medulla, rostral medulla, midbrain, thalamic, and frontal slices. Lesion profile scoring was carried out on Hematoxylin and Eosin (H&E) stained sections according to standard methodology (Fraser and Dickinson, 1968). For immunohistochemistry samples were processed (with minor modifications) as described previously (Hardt et al., 2000). Three micrometers section were de-paraffinized and rehydrated. Pre-treatment included an incubation step with 98% formic acid (15min) and blocking of the endogenous peroxidase activity with 3% H2O2/methanol (30min) followed by autoclaving in citrate buffer (pH 6.0, 20min). The monoclonal antibody SAF 84 was applied at a dilution of 1:2000 for 2h at room temperature and detected by avidin–biotin–horseradish peroxidase (Vector Elite). Color reactions were finally developed using diaminobenzidine (DAB) substrate.
Mouse brain samples 10% (w/v) were prepared in lysis buffer (0.42mM sucrose solution containing 0.5% deoxycholic acid sodium salt (DOC) and 0.5% Non-idet P40) and incubated with PK (50μg/ml, final concentration) for 60min at 55°C. Reactions were stopped with pefabloc (Roche) followed by incubation with 0.3% (w/v) phosphotungstic acid precipitation (PTA) and selective precipitation of PrPSc according to Gretzschel et al. (2005).
Cell-free conversion studies were performed as described before (Eiden et al., 2006; Kupfer et al., 2007). For the conversion reaction 400ng of recombinant ovine PrPC, expressed in E. coli, was incubated with 200–400ng of purified PrPSc in a conversion buffer [50mM citrate buffer (pH 6.0), 200mM KCl, 5mM MgCl2, and 1.25% sarkosyl; Horiuchi et al., 2000]. Standard conversion reactions were carried out for 3days at 37°C. Afterward, samples were incubated with PK (final concentration of 30μg/ml) for 1h at 37°C. PK was diluted in TN-buffer (0.15M NaCl, 0.05M Tris/HCl pH 7.4). The reaction was stopped with PMSF (phenyl methansulfonyl fluoride; 10mM). Twenty micrograms of a carrier protein (thyroglobulin) was added and the samples were incubated with a fourfold volume of methanol at –20°C to precipitate the proteins. After centrifugation at 12000g for 15min, the proteins were pelleted and resolubilized by heating to 95°C for 5min in SDS-PAGE loading buffer [1% (w/v) SDS, 25mM Tris/HCl pH 7.4, 0.5% mercaptoethanol, and 0.001% bromophenol blue].
Samples were separated on SDS-polyacrylamide gels containing 16% (w/v) acrylamide/bisacrylamide, 0.375M Tris/HCl (pH 8.8), 0.5% (w/v) SDS (sodium dodecyl sulfate), 0.06% (w/v) APS (ammonium peroxydisulfate), and 0.06% (v/v) TEMED (N,N,N′,N′-tetramethylethylenediamine) with a 4% stacking gel [4% (w/v) acrylamide/bisacrylamide, 0.12M Tris/HCl (pH 6.8), 0.5% (w/v) SDS, 0.2% APS, 0.2% (v/v) TEMED].
For immunoblotting proteins were transferred in a semi-dry chamber to a polyvinylidene difluoride (PVDF) membrane. Membranes were then incubated in blocking buffer (PBS-0.1% Tween 20, 5% non-fat dry milk powder) followed by incubation for 60min with the monoclonal antibody (mab) P4 (R-Biopharm), mab SAF-70 (SPIbio), or polyclonal antibody (pab) Ra 10 (Groschup et al., 1994, 1997). In contrast to mab P4, which detects ovine PrPC/res, pab Ra10 detects only murine PrPC/Sc. Membranes were washed three times for 10min with PBS containing 0.1% Tween 20 and then incubated with a secondary antibody bound to alkaline phosphatase (Goat-anti-mouse-alkaline phosphatase or goat anti-rabbit-peroxidase). After washing, the chemiluminescence substrate CDP-Star (Tropix) was applied and membranes were incubated for 5min before the light signals were recorded on a Versadoc Imaging System (Biorad). Visualization was carried out with the Biorad VersaDoc™ quantification software Quantity One. The percentage of converted substrate was calculated as: signal volume of PrPres digested with PK/signal volume of PrPC without PK*10. Conversion rates were calculated for each time point as a mean value from four independent reactions. For stripping, membranes were incubated twice for 15min with a buffer containing 0.2M glycine (pH 2.0) and 1% SDS.
Two scrapie-infected mouse cell lines were used: ScN2A-cells [RML infected neuroblastoma-(N2A)-cells] and SMB-cells (“scrapie-mouse-brain,” infected with strain 22F, a gift of TSE Resource Centre at the Institute for Animal Health, UK). ScN2A-cells were cultured in Opti-MEM, 10% fetal calf serum (FCS), and 5% CO2 at 37°C. SMB-cells were cultured in MEM+ [MEM199, 10% newborn calf serum (NCS), 5% FCS] and 5% CO2 at 35°C.
The cell-based assay was carried out according to Leidel et al. (2011) with two scrapie-infected mouse cell lines: ScN2A-cells and SMB-cells. These cells were adapted for dot blot procedure in 96-well plate format to identify potential inhibitors of PrPSc formation. Compounds were resolved in DMSO and added to cells in eight replicates in concentrations of 10, 100, and 1000μM (final concentration). After 3days of incubation in a CO2-incubator, the supernatants were removed and cells lysed in 100μl lysis buffer (50mM Tris/HCl, 150mM NaCl, 0.5% Na-deoxycholate, 0.5% TX-100, pH 8.0). Lysates were or electrophoresed and immunoblotted as described in the previous section or dot blotted on PVDF membranes using a 96-well dot blot apparatus (Transblotter, Roth). Dot blotting membranes were dried for 1h at 37°C and fixed sample incubated with DNAse (100μg/ml) for 1h at 37°C. After rinsing the membranes twice with distilled water, the membrane was incubated with PK (25μg/ml) for 90min at 37°C. The digestion was terminated using PMSF (2mM) and membranes were rinsed in 3M guanidium thiocyanate for 10min followed by washing them five times in distilled water.
The incubation of the membrane with blocking buffer (PBS-0.1% Tween 20, 5% non-fat dry milk powder) was followed by an incubation for 60min with polyclonal antibody (pab) Ra 10. Membranes were washed three times for 10min with PBS containing 0.1% Tween 20 and then incubated with a secondary antibody bound to peroxidase (Goat anti-rabbit-PO). Afterward, the chemiluminescence substrate ECL (Roche) was applied and incubated on the membrane for 1min before the light signals on the membrane were detected directly in a camera. The intensity of the PrPSc signal of each well was quantified by using the Versadoc imaging system. The half maximal (50%) inhibitory concentration (IC50) of each compound was calculated from eight independent reactions.
Survival times were analyzed by Kaplan–Meier Survival analysis using the log-rank test to compare the curves. The Statistical analysis was done using SigmaBlot statistical software (San Jose, CA, USA). Survival times are expressed as mean±SD.
The effect of S. lateriflora tea on the PrPres formation was first analyzed in vitro using two different scrapie-infected cell lines (SMB and ScN2A-cells), which permanently accumulate PrPres. When tea concentrate was added to the culture medium, a dose dependent reduction of PrPres accumulation was observed in SMB as well as ScN2A-cells (Figure (Figure1).1). Cells were cultivated in T25 flasks and incubated with increasing amounts of S. lateriflora tea for 3days. After this time, cells were harvested and the level of PK-resistant PrPres was assessed by Western blotting. A dilution of 1:500 induced a reduction of PrPres to 58% for ScN2A (Figure (Figure1A,1A, lane 2) and to 64% for SMB (Figure (Figure1B,1B, lane 2) cells compared to the control (Figures (Figures1A,B,1A,B, lane 1). Dilutions of 1:50 resulted in a reduction to 33% for ScN2A-cells (Figure (Figure1A,1A, lane 3) and 57% for SMB-cells (Figure (Figure1B,1B, lane 3). About 1:20 dilutions displayed reduction to 28% for ScN2A (Figure (Figure1A,1A, lane 4) and 47% for SMB-cells (Figure (Figure1B,1B, lane 4) and reached values of 28.7% for ScN2A-cells (Figure (Figure1A,1A, lane 5) and 42.3% for SMB-cells (Figure (Figure1B,1B, lane 5). Relative inhibition by tea is depicted in Figure Figure11C.
To test the direct effects of tea on the conversion of PrPC to PrPres, a cell-free conversion assay was used in which a purified murine PrPSc template induced the conversion of recombinant cellular PrPC into a PK-resistant PrPres fragment (Eiden et al., 2006; Kupfer et al., 2007). Newly converted PrPres-fragments were detected by mab P4 (Figure (Figure2A,2A, lanes 1–2), while PrPSc aggregates were visualized by the polyclonal antibody Ra10 (Figure (Figure2B,2B, lanes 1–2). Dried tea extract (3, 0.3, and 0.03mg respectively) was added to the conversion assay, inhibited PrPres formation and led to clearance of PrPres-fragments (Figure (Figure2A,2A, lanes 3–6). Even 0.03mg caused a reduced PrPres formation compared to the control (Figure (Figure2A,2A, lanes 7–8) by 23%.
Similar results were seen in the case of dissolution of PrPSc aggregates after PK digestion (Figure (Figure2B).2B). In contrast to the control (Figure (Figure2B,2B, lanes 1–2), PrPSc aggregates were completely digested if co-incubated with 3.0mg dried tea extract (Figure (Figure2B,2B, lanes 3–4), as well as with 0.3mg (Figure (Figure2B,2B, lanes 5–6). About 0.03mg dried tea extract induced a partial disaggregation of PrPSc aggregates (Figure (Figure2B,2B, lanes 7–8) by 40% compared to the control.
To further characterize the underlying inhibitory mechanisms, two main polyphenolic compounds of this herbal extract, the flavonoids Baicalin (baicalein 7-O-glucuronide) and its aglycone baicalein (5,6,7-trihydroxyflavone), were studied with regard to their inhibitory properties. Both components have been isolated from this herb previously (Awad et al., 2003) und were also identified in this study as ingredients in the tea by LCMS–IT–TOF hybrid mass spectrometry (data not shown). In addition, a structural analog to baicalein, the flavonoid epicatechin and quercetin were analyzed.
The potentially inhibitory effects on the PrPres accumulation and conversion were assayed by a cell-based dot blot assay system (Geissen et al., 2011) and the cell-free conversion assay. Structures, assay results, and half maximal (50%) inhibitory concentrations (IC50) of both compounds are summarized in Table Table1.1. The cell-based dot blot assay was carried out with two different scrapie-infected SMB and ScN2A-cells again. Cells were seeded in 96 wells and incubated with decreasing concentrations of the corresponding flavonoids (1mM, 100 and 10μM). IC50 values of Baicalein ranged from 138μM (SMB-cells) to 63.7μM (ScN2A-cells). Baicalin showed significantly lower inhibitory effects compared to baicalein in SMB-cells (IC50: >1000μM) as well as in ScN2A-cells (IC50: 239.8μM). Two additional flavonoids with similar structure, epicatechin, and quercetin, showed also strong inhibitory effects in both cell-types: epicatechin IC50 values were 96.6μM (ScN2A-cells) and 106.8μM (SMB-cells) and quercetin IC50 values were 36.8μM (ScN2A-cells) and 274.3μM (SMB-cells; Table Table1).1). Baicalein displayed a strong dose dependent inhibitory effects to PrPres formation (IC50: 18.48μM) and dissolution of PrPSc aggregates (IC50: 41.56μM) in the cell-free conversion assay, in contrast to baicalin which yielded IC50 values of >1000μM for PrPres inhibition and 440μM for PrPSc dissolution. Epicatechin and quercetin, although highly similar in structural to baicalein, showed no inhibitory effects in cell-free conversion with regards to PrPres formation and PrPSc disintegration.
In summary, these data demonstrate that tea of S. lateriflora contains two active compounds, baicalein and baicalin, which are specific inhibitors of PrPC conversion and PrPres amplification in vitro as well as enhance the degradation of PrPSc fibrils.
In another set of experiments in vivo effects of S. lateriflora tea were assessed. Wild-type mice were infected either by intracerebrally (i.c.) or intraperitoneally (i.p.) inoculation with mouse scrapie strain RML and the drinking water was replaced by freshly brewed tea starting 2weeks prior to their inoculation and for the whole lifetime of the mice. Survival times are depicted in Figure Figure3.3. Six Bl6/C57 mice were used for the treatment and compared to six intracerebrally inoculated mice which received normal drinking water. The mean life spans of the control group (Figure (Figure5A,5A, bold line) was 147±3.1dpi (Figure (Figure3B)3B) whereas the tea treated mice (Figure (Figure5A5A dashed line) survived significantly longer (184±26.7dpi; Figure Figure3B).3B). Three of the treated mice survived up to 60days compared to the control mice. In the case of i.p. inoculation of RML strain, a mean prolongation of 15days was achieved (Figures (Figures3C,D).3C,D). The mean incubation time of the control group (Figure (Figure3C,3C, bold line) was 191±8.6dpi (Figure (Figure3D)3D) whereas the tea treated mice (Figure (Figure3C,3C, dashed line) survived significantly longer (206±8.6dpi; Figure Figure33D).
Brains of the i.c. infected mice were subjected to comparative biochemical and immunohistochemical investigations. PrP immunoblot analysis revealed no differences of PrPSc banding patterns and total amounts of PrPSc in both groups. The PrPSc content in all brains was analyzed after PK digestion and selective precipitation of PrPSc from corresponding mouse brain homogenates with sodium phosphotungstic acid (Figure (Figure4).4). No differences in PrPSc banding patterns or PK resistances was detected in mice which eventually developed scrapie in the tea treated and control group (Figure (Figure4,4, lanes 2–13). The brain lesion profiles and PrPSc deposition patterns in the different mouse brain regions were similar for the treated and the untreated mice (Figures (Figures55A–D).
The here presented study demonstrates that solubilized ingredients of S. lateriflora not only inhibit the scrapie fibril formation but also destabilize pre-formed PrPSc fibrils in a concentration dependent manner. Main constituents of the herb were the polyphenolic flavonoids baicalin and baicalein, which were found at high concentrations in corresponding extracts. Both components in purified form showed dose dependent inhibitory properties and thus were the active constituents responsible for the herbs effects on scrapie fibril regulation.
The strongest effects displayed baicalein, the main metabolite of baicalin, which inhibited PrPres accumulation in both cell-based and cell-free assays and promoted the destabilization of PrPSc fibrils at low concentrations. A possible molecular mechanism is based on direct binding to the PrPC and the formation of a stable PrP–baicalein/baicalin complex, which blocks PrP conversion and/or accumulation. In the same way the destabilization of pre-existing fibrils would be caused by a specific intercalation of baicalein/baicalin into β-sheet structures of the PrPSc aggregates which may lead to a forced PrPSc degradation by lysosomal proteases. Similar effects by Baicalein have been described for the inhibition of α-synuclein (Zhu et al., 2004) and amyloid-β peptide (Aβ) fibrillization (Lu et al., 2011).
In our present study oral administration of S. lateriflora tea significantly prolonged incubation of mice either infected intracerebral or intraperitoneal. These effects can be explained by direct interference of baicalein to evolving PrP aggregates in the CNS. Baicalein is able to cross the blood brain barrier and ultimately reach the CNS (Tsai et al., 2002). Baicalin, on the other hand, may act as pro-drug: After oral uptake, the sugar residue is cleaved by enzymes in the intestinal tract to generate baicalein. The more lipophilic baicalein is better absorbed in the gastrointestinal tract and can then enter the brain via the blood system (Tarragó et al., 2008).
Within the CNS, the inhibition and dissolution of prion aggregates by Baicalein could protect neuronal cells from membrane disruption and subsequent neuronal cell death. Additional anti-oxidant properties of baicalein on reactive oxygen species (ROS), which evolve during protein aggregation, may reinforce this neuroprotective effect (Li et al., 2010). Baicalein is able to minimize ROS by directly quenching free radicals or indirectly inducing anti-oxidant enzymes like superoxide dismutase and catalase (Shieh et al., 2000; Kang et al., 2011).
Direct effects on the prion conversion were also seen in other plant derived polyphenols like curcumin which is a strong inhibitor of prion conversion replication in vitro (Caughey et al., 2003). It is in vivo efficacy, however, remains controversial: the oral administration of curcumin had no significant effect on the onset of scrapie in hamsters (Caughey et al., 2003), whereas scrapie-infected mice significantly survived longer after oral treatment (Riemer et al., 2008). Effects of other polyphenols (e.g., tannic acid, katacine, and 2′-2′′-bis epigallocatechin gallate) that interfere with PrP conversion in vitro were also studied in vivo (Kocisko et al., 2003). However, neither these substances nor mixtures of natural compounds including polyphenolic tea extract and pine bark extracts displayed any effects on incubation periods in scrapie-infected animals (Kocisko et al., 2004).
Up to now only few compounds have been reported that delay onset on prion diseases after oral treatment. This includes pravastatin (Vetrugno et al., 2009), which is involved in cholesterol biosynthesis and influences membrane structure and function, fucoidan (Doh-Ura et al., 2007), as well as amyloid dye derivatives (Kawasaki et al., 2007). However, possible drug associated side-effects have to be further evaluated. Recently, diphenyl-pyrazoles (Leidel et al., 2011) were identified as a new class of anti-prion compounds, which prolong incubation time of scrapie-infected mice even after oral application.
In conclusion, beneficial effects of S. lateriflora tea can be explained by the anti-aggregatory and potential anti-oxidative effects of its natural constituents, baicalein and baicalin. Only few studies were conducted with herbal extracts of S. lateriflora for prevention and therapy of protein misfolding diseases to date. Results shown here for prion diseases are promising also in the context of other diseases in this group.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The LCMS–IT–TOF analysis was carried out by Andreas Link and Andrea Bodtke, Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.