Accumulation of prion triggers the enhanced glycolysis via activation of AMKP pathway in prion infected rodent and cell models

doi:10.21203/rs.3.rs-2595947/v1

Download PDF

Research Article

Accumulation of prion triggers the enhanced glycolysis via activation of AMKP pathway in prion infected rodent and cell models

https://doi.org/10.21203/rs.3.rs-2595947/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 20 Sep, 2023

Read the published version in Molecular Neurobiology →

You are reading this latest preprint version

Mitochondrial dysfunction is one of hallmarks in pathophysiology of prion disease and other neurodegenerative diseases. Various metabolic dysfunctions are identified and considered to contribute to the progression of some types of neurodegenerative diseases. In this study, we evaluated the status of glycolysis pathway in prion infected rodent and cell models. The levels of the key enzymes, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were significantly increased, accompanying with markedly downregulated mitochondrial complexes. Double stained IFAs revealed that the increased HK2 and PFK distributed widely in GFAP-, Iba1- and NeuN-positive cells. We also identified increased levels of AMP-activated protein kinase (AMPK) and the downstream signaling. Changes of AMPK activity in prion infected cells by the AMPK-specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Transient removal or complete clearance of prion propagation in the prion infected cells partially but significantly reversed the increases of the key enzymes in glycolysis, the upregulation of AMPK signaling pathway, and the decreases of the mitochondrial complexes. Measurements of the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) showed lower OCR and higher ECAR in prion infected cell line, which were sufficiently reversed by clearance of prion propagation. Those data indicate a metabolic reprogramming from oxidative phosphorylation to glycolysis in the brains during the progression of prion disease. Accumulation of PrP^Sc is critical for the switch to glycolysis, largely via activating AMPK pathway.

Prion

glycolysis

mitochondrial complex

AMPK signaling

Prion diseases, also termed as transmissible spongiform encephalopathy (TSE), are a group of incurable fatal neurodegenerative diseases affecting human and various species animals [1]. Besides of large amounts of deposits of the misfolded protein PrP^Sc in central nerve system (CNS), extensive aberrances and dysfunctions in the brains of prion diseases continually progress and deteriorate along with the long incubation time [2]. One of the leading disturbances in pathophysiology of prion disease, as well as other neurodegenerative diseases, is mitochondrial dysfunction that may reflect in many aspects, e.g., functional molecules, signaling pathways, oxidative phosphorylation and energy metabolism, structural and morphological abnormality [2–4]. Those mitochondrial impairments in neurons may induce competition for a limited energetic resource, and further, lead to deleterious consequences on the neighbor healthy neurons, which is believed to be responsible for spreading neurodegeneration from normal aging to neurodegeneration [5, 6].

It is well known that the glycolysis pathway metabolizes glucose into pyruvate or lactate and generates 4 molecules of ATP. Three kinases, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), are the main rate-limiting enzymes in glycolysis [7], amongst PK has two isoenzymes, PKM1 and PKM2. Glycolysis is common in almost all types of cells, allowing for rapid generation of ATP than oxidative phosphorylation. Physiologically, glycolysis is the only metabolic pathway for ATP in red blood cells. Brain cells, white blood cells and bone marrow cells also obtain energy via glycolysis in aerobatic. During strenuous exercise or prolonged exercise, glycolysis becomes the main pathway to provide much needed energy for living body. Pathologically, such as respiratory or circulatory dysfunction, severe anemia, and massive blood loss, glycolysis is accelerated or even excessive. Most malignant tumor cells undergo high rate of glycolysis in the cytosol even in the situation of sufficient oxygen, represented by overexpressions of three key kinases in glycolysis [7].

Aberrant alteration of metabolic function is one of the common pathophysiological changes in neurodegenerative diseases, i.e., Alzheimer disease, (AD) Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) [8]. Although there are controversial results, the increased glycolysis rate and increased PFK are frequently described in the brains from the patients and transgenic mouse models of AD, PD and ALS, particularly in fibroblasts [8, 9]. The decreases of the capacity of mitochondrial oxidative phosphorylation due to the impairment of mitochondria is proposed to result in increasing glycolysis rate [8, 10, 11]. However, the status and role of glycolysis in CNS tissues of prion disease remained much less addressed.

In this study, we found that the levels of HK2, PFKFB, and PKM2 were significantly increased in the brains of several rodent prion models at clinically terminal stage and in a prion infected cell model. The increased PFKFB and HK2 were detectable in GFAP-, Iba1- and NeuN-positive cells. Markedly downregulated mitochondrial complexes were also noticed in prion hamster and cell models. We also identified increased levels of AMP-activated protein kinase (AMPK) and its phosphorylated form in prion infected animals and cell line. Changes of AMPK activity in prion infected cells by specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Clearance of prion propagation in the prion infected cells reversed the increases of the key enzymes in glycolysis and the decreases of the mitochondrial complexes, largely via downregulating AMPK signaling pathway.

Ethics Statement

Usage of animal specimens in this study was approved by the Ethical Committee of the National Institute for Viral Disease Prevention and Control (IVDC), China CDC. Animal housing and experimental protocols were in accordance with the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals.

Antibodies and reagents

Commercially supplied the antibodies in this study include mAbs for PrP (Santa Cruz, sc-58581, USA), NeuN (Merck Millipore, MAB377, USA), GFAP (GeneTex, GTX34759, USA), Iba1 (Sigma, SAB2702364, USA), HK2 (Abcam, ab209847, UK; Cell Signaling Technology, 2867, USA), PFKFB3 (Abcam, ab181861, UK), PKM1 (Cell Signaling Technology, 7067, USA), PKM2 (Cell Signaling Technology, 4053, USA), AMPKa (Cell Signaling Technology, 2532, USA), pAMPKa (Cell Signaling Technology, 2535, USA), YAP (Cell Signaling Technology, 14074, USA), pYAP (Cell Signaling Technology, 4911, USA), OXPHOS (Abcam, ab110413, UK), PI3 (Abcam, 191606, UK), AKT (Santa cruz, sc-5298, USA), pAKT (Cell Signaling Technology, 4060, USA), mTOR (Cell Signaling Technology, 2983, USA), pmTOR (Cell Signaling Technology, ab2974, USA).

The reagents used in this study include PBS (Sinodetech scientific, CBS004-BR500, China), DMEM (GIBCO, 25200056, USA), penicillin streptomycin (Thermo Fisher Scientific, 15140122, USA), trypsin (GIBCO, 25200056, USA), AICAR (Selleck, S1802, USA), Dorsomorphin (Selleck, S7306, USA), resveratrol (Sigma, PHL89539, USA), RIPA (Beyotime, P0013B, China), Protease Inhibitor Cocktail (Merck Millipore, 535140-1SET, Germany), XFe96 FluxPaks (Agilent, 102416-100, USA), 96-well cell culture plate (Nunc, 165306, USA), Seahorse XF Calibrant Solution (Agilent, 100840-000, USA), Seahorse XF Base Medium (Agilent, 102353-100, USA), Glucose (Sigma, G7528, USA), L-glutamine (Sigma, G8540, USA), Sodium pyruvate (Sigma, S8636, USA), Oligomycin (Abcam, ab141829, UK), 2-DG (Sigma, D8375, USA), FCCP (Sigma, C2920, USA), Antimycin (Abcam, ab141904, UK), Rotenone (Sigma, R8875, USA).

Scrapie infected rodent models

The brain samples of Syrian golden hamsters inoculated intracerebrally with hamster-adapted scrapie agent 263K, those of C57BL/6 (C57) mice inoculated intracerebrally with two types of mice-adapted scrapie strains 139A and ME7, were enrolled in this study [12]. The bioassay procedures, the clinical, neuropathological, and pathogenic features of these infected animals were described previously [ref]. The average incubation periods of 263K-infected hamsters, 139A- and ME7-infected mice were 80.1 ± 5.7, 183.9 ± 23.1 and 184.2 ± 11.8 days, respectively. Age-matched healthy hamsters and mice were used as control.

Preparation of brain homogenates

Brain homogenates were prepared according to the procedure described previously [ref]. Brain tissues from the scrapie-infected and healthy rodents were washed in PBS for three times, and 10% (w/v) brain homogenates were prepared in cold lysis buffer (100 mM NaCl, 10mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.5) containing a mixture of protease inhibitors (Merck, 539134, US). The tissue debris were removed with low-speed centrifugation at 2000 g for 10 min and the supernatants were collected for further study.

Cell culture and cell lysates

Cell line SMB-S15 and its normal partner cell line SMB-PS were provided by Roslin institute, U.K. The cell line SMB-S15 was established originally by culture from brains taken from mice clinically affected by Chandler scrapie strain, in which PrP^Sc replicates consistently itself along with cell passages [ref]. Cell line SMB-PS was established from the SMB-S15 cells treated with pentosane sulfate (PS) that completely removed PrP^Sc propagation and its infectivity in bioassays [ref]. SMB cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 33°C humidified atmosphere with 5% CO₂.

The preparation of cellular lysates of SMB cells were described elsewhere [ref]. Briefly, cells were washed with PBS, digested by pancreatic enzyme, and harvested by centrifugation at 5000 rpm for 3 min. The cell pellets were lysed for 2 h in RIPA buffer with protease inhibitor cocktail set III, followed by centrifugation at 5000 rpm for 3 min and the supernatants were collected.

Drug treatment upon cultured cells

SMB-S15 cells were passaged on a 6-well cell plates. 24 h after passage, cells were exposed to 1000 µM AICAR (AMPK activator) or 10 µM Dorsomorphin (AMPK inhibitor) for 6 h and 12 h. The drug treated SMB-S15 cells, we well as untreated SMB-S15 and SMB-PS cells, were harvested for further study.

Mitochondrial isolation

Isolations of the mitochondrial fractions were performed on ice or at 4°C. Approximately 8 × 10⁶ cells were scraped, washed once with mitochondrial isolation buffer (MIB, 10 mM Tris, 200 mM sucrose, 1 mM EGTA, pH 7.5) containing freshly prepared 1 × protease inhibitor cocktail. Cells were resuspended in 1 ml MIB and broken about 35 times by 2 ml needles. The lysates were centrifuged at 500 × g for 10 min. The supernatants were collected and centrifuged at 7000 × g for 10 min. The mitochondria in the spherules were washed once with MIB and centrifuged at 10000 × g for 10 min. The pellets were suspended in 300 µl MIB and the protein concentrations were determined by BCA method.

Western blot

Protein samples were separated by 12% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked in TBS containing 0.1% Tween 20 (TBST) with 5% skimmed milk at room temperature (RT) for 2 h and then incubated with primary antibodies at 4 ℃ overnight. After washing with TBST three times, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at RT for 2 h. The blots were developed using enhanced chemiluminescence (ECL) system. Images were captured by ChemiScope 6000 (CLiNX) and quantified by Image J software.

Immunohistochemical assay (IHC)

Brain tissues were fixed in 10% buffered formalin solution and paraffin sections (5 µm in thickness) were prepared routinely. Sections were repaired under high temperature with 1% sodium citrate solution in microwave for 30 min and quenched for endogenous peroxidases with methanol containing 3% H₂O₂ for another 10 min. After blocking in 5% bovine serum albumin (BSA) at RT for 15 min, the sections were incubated with various primary antibodies, including 1:300-diluted mAb for PFKFB3 and HK2 at 4°C overnight. Subsequently, the sections were incubated with 1:200-diluted HRP-conjugated goat anti-mice or HRP-conjugated goat anti-rabbit secondary antibody at 37 ℃ for 1 h and visualized by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB). The sections were counterstained with hematoxylin, dehydrated, and mounted in permount. For detection of PrP^Sc, the brain sections were exposed to the buffer containing 4 M guanidinium isothiocyanate (GdnSCN) at 4 ℃ for 1 h prior to the routine IHC process.

Immunofluorescent assay (IFA)

To label cell membrane structure, cell lines SMB-PS and -S15 were incubated with 5.0 µg/ml Texas Red®-X-conjugated wheat germ agglutinin (WGA) at 33°C for 1 h. After washing with PBS, SMB cells were fixed with 4% paraformaldehyde at RT for 20 min, followed by treatment with 0.4% Triton X-100 for 5 min and blocked with PBS containing 5% BSA for 2 h. Subsequently, fixed cells were incubated with primary antibodies, including 1:200-diluted mAb for PFKFB3, 1:200-diluted pAb for HK2, 1:200-diluted mAb for pAMPKa at 4 ℃ overnight. After washing, fixed cells were incubated with 1:200-diluted Alexa Fluor 488-labeled goat-derived anti-rabbit or Alexa Fluor 568-labeled goat-derived anti-mice antibodies at RT for 1 h. Further, sections were incubated with 1 µg/ml DAPI at RT for 5 min. The sections were sealed, and the images of the targeting proteins were analyzed by high-solution confocal microscope (Leica, Germany).

Brain tissue slices were permeabilized with 0.4% Triton X-100 for 30 min and blocked with BSA at RT for 1 h. After blocked, sections were incubated with 1:200-diluted mAb for PFKFB3, 1:200-diluted pAb for HK2, 1:200-diluted mAb for pAMPKa, 1:200 diluted mAb for NeuN, 1:200 diluted pAb for Iba1, 1:200 diluted mAb for GFAP at 4°C overnight. Slices were incubated with corresponding secondary antibodies and DAPI according to the above procedures. The images were analyzed by high-solution confocal microscope (Leica, Germany).

Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

Cellular OCR and ECAR were determined by the Seahorse XF96 Extracellular Flux Analyzer (Agilent Seahorse Bioscience) in accordance with manufacturer’s protocol. Cell lines SMB-S15, -PS and -RES were separately inoculated on XF96 microplates (10000 cells/well) till approximately 80% abundance. The culture medium was changed and washed with the test medium (Seahorse XF Base Medium with 25 mM glucose, 4 mM L-glutamine and 1 mM sodium pyruvate) for 3 times. Cells were stored in a non-CO₂ incubator in an unbuffered assay medium (Antitron) for 60 min before measurement. Cellular OCR was detected with XF Cell Mito Stress Test Kit (Agilent). After baseline recording, cells were received with 2 µM oligomycin, 0.5 µM FCCP and 1 µM rotenone/1 µM antimycin A successively. Cellular ECAR was evaluated with XF Cell Glycolysis Test Kit (Agilent, 103020). The culture medium was changed and washed with the test medium (Seahorse XF Base Medium, 4 mM L-glutamine) for 3 times. After baseline recording, cells were received with 25 mM glucose, 2 µM oligomycin and 50 mM 2-DG successively. The data each preparation was automatically counted by the Analyzer and the values of the cellular OCR and ECAR were normalized with the cell numbers.

Statistical analysis

Statistical analysis was conducted using the SPSS 22.0 statistical package. Quantitative analysis of immunoblot images, the positive staining in IHC and fluorescent intensity in IF assays were carried out using software Image J. Quantification of IFA was represented as integral optical density (IOD) that is the sum of the reaction intensities of all selected objects in the field of view. All experiments in the present study were conducted at least three times with consistent results. All data were presented as the mean + SEM. The p values for differences between two groups were determined by two-tailed Student’s t test. The p values were described as *** (p < 0.001), ** (p < 0.01), * (p < 0.05) and ns (not significant).

Increases of HK2, PFKFB3, PKM2 in the brains of scrapie infected rodents at terminal stage

To evaluate the potential alterations of the three key kinases for glycolysis (HK, PFK ad PK) in the brain tissues during prion infection, the stored brain samples of several scrapie infected experimental rodents were enrolled into this study. Western blot of the brain homogenates from three scrapie agent 263K infected hamsters at terminal stage revealed stronger bands of HK isoenzyme 2 (HK2) and PFK isoenzyme FB3 (PFKFB3) in 263K infected hamsters compared to the age matched normal ones, with statistical difference in the digital analysis of the gray values of blotted signals (Fig. 1A). PK isoenzyme M1 and M2 (PKM1 and PKM2) specific Western blots identified an increased PKM2 but insignificantly changed PKM1 in the brains of 263K infected hamsters (Fig. 1A). Similar changing trends of those three kinases were also observed in the brain tissues of other two scrapie infected mice models, 139A and ME7, which revealed significantly higher levels of HK2, PFKFB3 and PKM2, whilst similar level of PKM1 compared to the normal controls (Fig. 1B).

To obtain more evidence of abnormal changes of those kinases, the brain sections of 263K-infected hamsters at final stage were subjected to HK2 and PFKFB3 specific IHC assays separately, controlled with normal ones. Stronger brown signals of HK2 (Fig. 1C) and PFKFB3 (Fig. 1D) were detected in the brain regions of 263K infected mice, including cortex, cerebellum, hippocampus and thalamus, particularly in the latter two regions, which deposited in large round and polygon morphisms. The brain slices of 263K-infected mice and normal mice were also employed to the HK2 and PFKFB3 specific IFAs. As expected, stronger green signals of HK2 (Fig. 1E) and PFKFB3 (Fig. 1F) were observed in the brains of 263K-infected mice compared to the normal control, which displayed round and polygon morphisms. Those data highlight the increases of HK2, PFKFB3 and PKM2 in the brains of prion infection at terminal stage.

Colocalization of the increased PFKFB3 and HK2 with different types of cells in brains

To explore the cell types with increased PFKEB3 in the brain tissues, the brain sections of normal and 263K-infected hamsters were subjected into double-stained IFAs of anti-PFKFB3 together with anti-Iba1, anti-GFAP or anti-NeuN separately. More PFKFB3 signals (green) were observed in various brain regions of 263K-infected hamsters, accompanying with more Iba1 (red) and GFAP (red) signals. Amounts of yellow signals were identified in the Iba1-stained cells (Fig. 2A) and GAFP stained cells (Fig. 2B) in the merged images. Obvious yellow signals were also observed in the NeuN-stained cells in the merged images (Fig. 2C). Further double stained IFAs of anti-PFKFB3 together with anti-Iba1, anti-GFAP or anti-NeuN also identified more HK2 (red) signals in the regions of cortex, cerebellum and hippocampus of 263K infected animals, which colocalized with the signals (green) of NeuN, GFAP and Iba1 in the enlarged merged images (yellow, Fig. 2D-F). It highlights that the increased PFKFB3 and HK2 signals distribute widely in the proliferative microglia and astrocytes and the damage neurons in the brains of scrapie infected hamsters at terminal stage.

Increases of HK2, PFKFB3, PKM2 in the prion infected cell line SMB-S15

The possible changes of those three kinases in a prion infected cell line SMB-S15 were measured by individual Western blots. Compared with the data of its normal partner cell line SMB-PS, stronger HK2 and PFKFB3 specific signals were detected in the lysates of SMB-S15 cells, showing significantly higher levels of relatively gray values of blots after normalized with that of the individual actin, whereas the levels of PKM1 and PKM2 looked comparable between those cell lines (Fig. 3A).

Downregulation of mitochondrial complexes in prion infected cells and experimental hamsters

To evaluate the possible changes of mitochondrial complexes in prion infected cells, the lysates of the whole cells and the fraction of mitochondria of SMB-S15 and -PS cells were prepared separately. Western blots showed that in the preparations of the whole cellular lysates, the complexes I, III, IV and V in SMB-S15 cells were weaker than those in SMB-PS cells, with statistical differences after quantitative calculation (Fig. 4A). Similar profile was also noticed in the test of the mitochondrial fractions of those two cells, showing significantly decreases in complexes I, III, IV and V in SMB-S15 cells (Fig. 4B). The levels of those mitochondrial complexes in the brain tissues of scrapie 263K infected hamsters at terminal stage were also analyzed together with that of the age-matched normal control. The signals of the complexes I, II, IV, V in the brain homogenates of 263K infected animals were weaker than that of normal ones (Fig. 4C). Those data highlight a downregulation of the mitochondrial complexes of the tissues and cells of prion infection.

Increase of p-AMPKa in the scrapie infected rodents and cultured cells

The glycolysis process is regulated by various biological pathways, in which the AMPK pathway plays important role via phosphorylation of AMPK. Compared with that of normal hamsters, Western blots of 10% brain homogenates of 263K infected hamsters showed almost unchanged level of AMPKa, but significantly higher level of phosphorylated form of AMPKa (pAMPKa) (Fig. 5A). pAMPKa specific IFAs of brain sections from normal and infected hamsters revealed more brilliant and stronger green signals in the regions of cortex, cerebellum, hippocampus and thalamus of 263K infected animals (Fig. 5B). Furthermore, the brain samples of 263K infected hamsters collected at various time-points post-infection were enrolled. Western blots showed that the levels of brain pAMPKa gradually increased in the period of early and middle stages of 263K infection (from 20 to 60 days post infection, dpi), and markedly increased in the late stage (from 60 to 80 dpi) (Fig. 5C). In line with the increase of brain pAMPKa, the levels of PFKFB3 in the brains of 263K infected animals also displayed significantly increased in the late stage of infection.

To test the status of AMPK in prion infected cells, the levels of AMPKa and pAMPKa in two SMB cell lines were evaluated by the individual Western blots. Similar as the observations in scrapie infected animals, the level of AMPKa were comparable between those two cell lines, whereas the level of pAMPKa in SMB-S15 cells was significantly higher than that of SMB-PS cells (Fig. 5D). Protein YAP is one of the down-stream targets of AMPK pathway, whose phosphorylation is positively mediated by activation of AMPK pathways. Western blots of the lysates of two SMB cell lines showed that both levels of YAP and phosphor-YAP (pYAP) in SMB-S15 were higher than that of SMB-PS cells (Fig. 5E). Further, the levels of phosphorylated AKT (pAKT) positively regulated by AMPK and phosphorylated mTOR at position of aa. 2481 (pmTOR2481) negatively regulated by AMPK in SMB cells were measured by Western blots. Markedly higher levels of AKT and pAKT and lower levels of mTOR and pmTOR2481 were identified in SMB-S15 cells (Fig. 5F). Compared with the change of AKT and mTOR, the increase of pAKT and decrease of pmTOR2481 in SMB-S15 cells were much more significant. Above data reflect a higher activity of AMPK during prion infection.

Changes of AMPK activity in SMB-S15 cells notably affect the cellular levels of HK2 and PFKFB3

To see the influence of changing AMPK activity on the levels of the kinases in glycolysis, SMB-S15 cells were exposed to 10 µM of dorsomorphin which is AMPK inhibitor. Cells were collected pre-exposure and 6, 12 h post-exposure and cellular lysates were prepared, respectively. Western blots illustrated that compared to the data of pre-exposure (indicated as 0 h), the levels of AMPKa in the dorsomorphin treated SMB-S15 cells did not change, whereas that of pAMPKa dropped down remarkably (Fig. 6A). Accompanying with the decrease of pAMPKa, the levels of YAP and pYAP in SMB-S15 cells treated with dorsomorphin were also reduced (Fig. 6A). Subsequently, the levels of three enzymes for glycolysis in dorsomorphin treated SMB-S15 cells were evaluated. As expected, the cellular levels of HK2 and PFKFB3 were down-regulated, particularly in the reactions of 12 h post-exposure with statistical difference, meanwhile, that of PKM1 and PKM2 seemed not to be notably changed (Fig. 6B). Subsequently, the SMB cells were exposed to 1000 µM of AICAR that is an AMPK agonist. Western blots of the cellular lysates revealed that the despite of unchanged AMPKa, levels of pAMPKa, as well as that of YAP and pYAP were increased 6 and 12 h post-exposure (Fig. 6C). The levels of HK2, PFKFB3 and PKM2 in the AICAR treated SMB-15 cells were also upregulated in different degree, showing statistically increases after quantitative assays (Fig. 6D). It suggests that the changes of the cellular AMPK activity affect the levels of the key enzymes of glycolysis in the cells with prion propagation.

Removal of prion replication in SMB-S15 cells decreases the levels of glycolysis

The assess the possible influence of removal of prions on cellular AMPK pathway and glycolysis, SMB-S15 cells were exposed to 10 mM resveratrol (RES). PrP^Sc was remarkably reduced after exposing to RES for 3 days (R-3d) and almost undetectable in that of 7 days (R-7d), whereas remained unchanged in the cells exposing to DMSO for 3 and 7 days (D-3d and D-7d) (Fig. 7A). The viability of the SMB-S15 cells treated with RES or DMSO were same as the mock (data not shown). Compared to the data of DMSO-treated cells, the levels of AMPK and pAMPKa in RES-treated cells were lower, particularly in the preparation of R-7d, meanwhile, the levels of YAP and pYAP in RES-treated cells were also reduced (Fig. 7B). Subsequent Western blots for the kinases in glycolysis revealed reduced HK2 and PFKFB3 levels and almost unchanged of PKM1 and PKM2 in RES-treated cells (Fig. 7C).

To assess the possible influence of withdraw of resveratrol on the cellular glycolysis, cell line SMB-RES that is the continually passaged R-7d cells (SMB-S15 cells after exposed to resveratrol for 7 days) was employed into study. PK-treated Western blot verified that SMB-RES cells was free of detectable PrP^Sc (Fig. 8A). Compared to that of SMB-S15 cells, the levels of AMPK, pAMPK, YAP, pYAP and pAKT of SMB-RES cells were significantly decreased, while that of pmTOR2481 did not change markedly (Fig. 8B and C). The levels of HK2 and PFKFB3 in SMB-RES cells were also lower than SMB-S15 cells (Fig. 8D). Further, the levels of mitochondrial complexes in SMB-RES cells were tested together with SMB-S15 and SMB-PS cells. In the preparation of whole cellular lysates, the decreased complexes I, III, IV and V in SMB-S15 cells were recovered in SMB-RES cells showing the comparable levels as SMB-PS cells (Fig. 8E). Similar reactive patterns were also identified in the reactions of mitochondrial fractions of those SMB cells, that SMB-RES cells had the similar levels of mitochondrial complexes as SMB-PS cells (Fig. 8F). Those results indicate that removal of prion replication in SMB-S15 cells can reverse the aberrantly increased AMPK signaling activity, the increased levels of HK2 and PFKFB3, and the decreased levels of mitochondrial complexes.

Removal of prion reversed the aberrant cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

The cellular OCRs and ECARs of SMB-S15, -PS and -RES cells were determined with Seahorse XF96 Extracellular Flux Analyzer. OCR represents mitochondrial oxidative phosphorylation (Fig. 9A). The average basal oxygen consumption rate (before administration of oligomycin), oxygen consumption rate of oxidative phosphorylation (before FCCP) and maximum oxygen consumption rate (before rotenone/antimycin A) of SMB-S15 cells (red line) were remarkably lower than that of SMB-PS cells (green line). Removal of prions by resveratrol (SMB-RES, blue line) partially but significantly increased the cellular basal oxygen consumption rate and maximum oxygen consumption rate. ECAR reflects glycolysis metabolic condition (Fig. 9B). SMB-S15 cells (red line) showed higher average values of glycolysis (after administration of glucose) and glycolytic capacity (after oligo) than SMB-PS cells (green line), while SMB-RES cells showed decreased average values of glycolysis and glycolytic capacity. It supplies the direct data of low mitochondrial oxidative phosphorylation and high glycolysis metabolic condition in the cells with prion propagations, whilst clearance of prions sufficiently reverses those abnormalities.

Both glycolysis and oxidative phosphorylation are the major metabolic pathways for ATP production in living cell and living body. In this study, we have found significantly upregulated glycolysis processes both in the brains of prion rodent models and in the prion infected cell line, accompanying with decreased mitochondrial complexes and downregulation of oxidative phosphorylation. Those aberrances of the energy metabolism seems to be closely associated with the prion infection, as removal of prion in the prion infected cells in vitro partially but significantly restores the abnormally increased glycolysis and the decreased mitochondrial complexes. It highlights that under the prion pathology, brain tissues and cells may expend more of the glycolysis pathway for energy supply through the way of metabolic reprogramming, to overcome the prion induced mitochondrial dysfunctions.

Impairment of mitochondrial function and disturbance of glucose metabolism have been described in many neurodegenerative diseases, such as AD, PD, ALS, Huntington disease (HD), frontotemporal dementia (FTD), reflecting a common pathophysiological phenomenon [13, 14] [15–18]. Extensive mitochondrial damage and dysfunction closely link with the oxidative stress and Aβ toxicity in AD [19–21]. Reduced mitochondrial complex II, III, and IV activities in the regions of striatum of HD cases strongly indicate a reduced oxidative phosphorylation [22]. The impairment of mitochondrial function is also repeatedly detected in neuronal cells after prion infection [3, 23, 24]. Although some studies have identified reductions of both glycolysis and oxidative phosphorylation resulting in synaptic dysfunction and neuronal death [25, 26], overwhelming data indicates that metabolic remodeling from oxidative phosphorylation to glycolysis occurs in various neurodegenerative diseases. For example, exposure to amyloid beta (Aβ) elicits acute microglial inflammation accompanied by metabolic reprogramming from oxidative phosphorylation to glycolysis [27]. The microglia cells in 5 ⋅ FAD transgenic mouse reveal higher glucose transporter and glycolysis activity but remarkable defeat in oxidative phosphorylation, which worsens along with disease progression [28]. A shift from oxidative phosphorylation to glycolysis HD cases is related to the oxidative stress and cell death [29]. Obviously, the mitochondrial dysfunction and damage during the pathological processes of neurodegenerative diseases are very likely to trigger such metabolic reprogramming.

AMPK is an essential molecule in the regulation of biological energy metabolism and glucose catabolism [30, 31]. AMPK is also a key protein involving in a variety of signaling pathways. Through its different functional pathways, AMPK plays the key role in maintaining energy homeostasis in normal cells and in various abnormal situations, i.e., inflammation, malignant carcinoma, metabolic disorders, neurodegenerative diseases [32–35]. In prion disease, upregulated AMPK signaling has been also detected, which is responsible for activation of autophagic flux [36, 37]. Our data here propose more direct linkage between the activated AMPK and increased glycolysis in prion pathology. Clearance of prion propagation in prion infected cell line in cultured cells downregulates the activated AMPK pathway and restores the cellular mitochondrial function and glycolysis level imply that the cells with prion aggregation can, at least partially, maintain the energy homeostasis via metabolic reprogramming from oxidative phosphorylation to glycolysis.

Our IFA results illustrate that the increased key enzymes of glycolysis (PFKFB and HK2) distribute widely in the neurons, astrocytes, and microglia in the prion infected brain tissues at clinical final stage. One may raise a question whether the damaged neuronal cells and proliferative and activated glial cells in the prion infected brains undergo the same metabolic reprogramming. Aggregations of pathological misfolded proteins in degenerative diseases, e.g., Aβ, PrP^Sc, may locate intra- and intercellularly in brains. Gene co-expression modules in the directly converted induced neurons (iNs) from AD patient derived fibroblasts and the postmortem brains of AD cases highlight aberrant metabolic regulation, while the Exon10/Exon9 ratios of PKM2/PKM1 in iNs and in AD brains are increased, strongly indicating a metabolic switch to aerobic glycolysis [38]. Close association between the accumulations of PrP^Sc and the increase of glycolysis in cultured cells in this study verifies occurrence of metabolic reprogramming in the cells with misfolded PrP proteins. One may assume that the neurons and other types of cells having aggregated prions insides are more likely to take advantage of glycolysis for their energy demands. On the other hand, enhance glycolysis is often observed in inflammatory cells and carcinoma cells. Activation of microglia is usually accompanied by a metabolic switch from oxidative phosphorylation to aerobic glycolysis, whilst inhibition of glycolysis may ameliorate neuroinflammatory diseases associated with microglia activation [39]. Increased aerobic glycolysis in carcinoma cells, known as Warburg effect, is common abnormal metabolic mechanism to evade normal cell apoptosis process and enhance the proliferation and migration [40, 41]. Usually, majority cancer cells have intact mitochondrial functions, i.e., mitochondrial respiration [42][43][44]. It is reasonable to speculate that the increased glycolysis program in microglia, and possibly part of astrocytes, in prion brains is a pathophysiological flection of fast proliferative inflammatory cells.

Warburg or Warburg-like effect has been already described in AD studies, in which neuron cells appear to prefer the low-energy glycolysis, similar as the malignant cells in cancer, to resist to Aβ-dependent apoptosis [6, 38]. Mitochondrial damage and dysfunction may force the neuronal cells to reprogram the energy metabolism [26, 45]. Our data here highlight a similar metabolic reprogramming process in prion brains as AD brains. Although the metabolic reprogramming from oxidative phosphorylation to glycolysis and mitochondrial dysfunction can be reversed by clearance of prion in the cell model, obviously, such reversions are impossible in the prion infected brain since complete removal of prions in living body is still not realizable yet. Besides of glycolysis dysfunction, other metabolic deficits also contribute to the pathology of neurodegenerative diseases, e.g., impairment in glucose metabolism, tricarboxylic acid (TCA) cycle and oxidative phosphorylation, pentose phosphate pathway [11]. Long lasing of those metabolic dysfunctions will result in the reduction of ATP generation, which causes the disturbance in energy supply subsequently and induces neuron damage and death ultimately. Efforts on the metabolic changes, especially in the early stage of prion disease and other neurodegenerative diseases are deserved.

Acknowledgement

We gratefully acknowledge the support from Chinese National Natural Science Foundation Grants (81630062), and Grant (2019SKLID501, 2019SKLID603, 2019SKLID307) from the State Key Laboratory for Infectious Disease Prevention and Control, China CDC.

Statements & Declarations

Funding

This work was supported by Chinese National Natural Science Foundation Grants (81630062), and Grant (2019SKLID501, 2019SKLID603, 2019SKLID307) from the State Key Laboratory for Infectious Disease Prevention and Control, China CDC.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation by Qin Fan, Kang Xiao, Ruhan A, Li-Ping Gao, data collection by Qin Fan, Yue-Zhang Wu, Dong-Dong Chen, Chao Hu, Xiao-Xi Jia, and analysis were performed by Qin Fan, Chu-Mou Liu, Xin Liu, Chen Cao, Qi Shi. The first draft of the manuscript was written by Xiao-Ping Dong and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and analysed during the current study are not publicly available due to data security but are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Chinese Laboratory Animal Management. Approval was granted by the Research Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that human research participants provided informed consent for publication of the images in Figures.

Chen C, Dong XP (2016) Epidemiological characteristics of human prion diseases. Infect Dis poverty 5(1):47. 10.1186/s40249-016-0143-8PubMed PMID: 27251305; PubMed Central PMCID: PMC4890484
Shi Q, Chen C, Xiao K, Zhou W, Gao C, Gao L et al (2022) Extensive Disturbances of Intracellular Components and Dysfunctions of Biological Pathways in the Brain Tissues During Prion Infection— China’s Studies. China CDC Weekly 4(10):741–747. 10.46234/ccdcw2022.154
Zhu T, Chen JL, Wang Q, Shao W, Qi B (2018) Modulation of Mitochondrial Dynamics in Neurodegenerative Diseases: An Insight Into Prion Diseases. Front Aging Neurosci 10:336 Epub 2018/11/21. 10.3389/fnagi.2018.00336
Beard E, Lengacher S, Dias S, Magistretti PJ, Finsterwald C (2021) Astrocytes as Key Regulators of Brain Energy Metabolism: New Therapeutic Perspectives. Front Physiol 12:825816 Epub 2022/01/29. 10.3389/fphys.2021.825816
Demetrius LA, Driver JA (2015) Preventing Alzheimer's disease by means of natural selection. J R Soc Interface 12(102):20140919 Epub 2015/01/01. 10.1098/rsif.2014.0919
Demetrius LA, Magistretti PJ, Pellerin L (2014) Alzheimer's disease: the amyloid hypothesis and the Inverse Warburg effect. Front Physiol 5:522 Epub 2015/02/03. 10.3389/fphys.2014.00522
Li XB, Gu JD, Zhou QH (2015) Review of aerobic glycolysis and its key enzymes - new targets for lung cancer therapy. Thorac Cancer 6(1):17–24 Epub 2015/08/15. 10.1111/1759-7714.12148
Bell SM, Burgess T, Lee J, Blackburn DJ, Allen SP, Mortiboys H (2020) Peripheral Glycolysis in Neurodegenerative Diseases. International journal of molecular sciences. ;21(23). Epub 2020/12/02. doi: 10.3390/ijms21238924. PubMed PMID: 33255513; PubMed Central PMCID: PMCPMC7727792
Bigl M, Bleyl AD, Zedlick D, Arendt T, Bigl V, Eschrich K (1996) Changes of activity and isozyme pattern of phosphofructokinase in the brains of patients with Alzheimer's disease. Journal of neurochemistry. ;67(3):1164-71. Epub 1996/09/01. doi: 10.1046/j.1471-4159.1996.67031164.x. PubMed PMID: 8752123
Sonntag KC, Ryu WI, Amirault KM, Healy RA, Siegel AJ, McPhie DL et al (2017) Late-onset Alzheimer's disease is associated with inherent changes in bioenergetics profiles. Sci Rep 7(1):14038 Epub 2017/10/27. 10.1038/s41598-017-14420-x
Yan X, Hu Y, Wang B, Wang S, Zhang X (2020) Metabolic Dysregulation Contributes to the Progression of Alzheimer's Disease. Front Neurosci 14:530219 Epub 2020/12/01. 10.3389/fnins.2020.530219
Xiao K, Zhang BY, Zhang XM, Wang J, Chen C, Chen LN et al (2016) Re-infection of the prion from the scrapieinfected cell line SMB-S15 in three strains of mice, CD1, C57BL/6 and Balb/c. Int J Mol Med 37(3):716–726 PubMed PMID: 26820255; PubMed Central PMCID: PMC4771105
Illarioshkin SN, Klyushnikov SA, Vigont VA, Seliverstov YA, Kaznacheyeva EV Molecular Pathogenesis in Huntington's Disease. (1608–3040 (Electronic))
Singh AA-O, Kukreti R, Saso LA-O, Kukreti S Oxidative Stress: A Key Modulator in Neurodegenerative Diseases. LID – 10.3390/molecules24081583 [doi] LID – 1583. (1420–3049 (Electronic)).
Cai QA-O, Jeong YY Mitophagy in Alzheimer's Disease and Other Age-Related Neurodegenerative Diseases. LID – 10.3390/cells9010150 [doi] LID – 150. (2073–4409 (Electronic))
Monzio Compagnoni GA-O, Di Fonzo A, Corti S, Comi GP, Bresolin N, Masliah E The Role of Mitochondria in Neurodegenerative Diseases: the Lesson from Alzheimer's Disease and Parkinson's Disease. (1559 – 1182 (Electronic))
Butterfield DA, Boyd-Kimball D, Oxidative, Stress Amyloid-β Peptide, and Altered Key Molecular Pathways in the Pathogenesis and Progression of Alzheimer's Disease. (1875–8908(Electronic)).
Harley J, Clarke BE, Patani RA-O The Interplay of RNA Binding Proteins, Oxidative Stress and Mitochondrial Dysfunction in ALS. LID – 10.3390/antiox10040552 [doi] LID – 552. (2076–3921 (Print)).
Wang X, Su B, Zheng L, Perry G, Smith MA, Zhu X (2009) The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer's disease. J Neurochem 109(1):153–159 Epub 2009/05/07. 10.1111/j.1471-4159.2009.05867.x
Wang X, Wang W, Li L, Perry G, Lee HG, Zhu X (2014) Oxidative stress and mitochondrial dysfunction in Alzheimer's disease. Biochim Biophys Acta 1842(8):1240–1247 Epub 2013/11/06. 10.1016/j.bbadis.2013.10.015
Wang W, Zhao F, Ma X, Perry G, Zhu X (2020) Mitochondria dysfunction in the pathogenesis of Alzheimer's disease: recent advances. Mol Neurodegener 15(1):30 Epub 2020/05/31. 10.1186/s13024-020-00376-6
Powers WJ, Videen TO, Markham J, McGee-Minnich L, Antenor-Dorsey JV, Hershey T et al (2007) Selective defect of in vivo glycolysis in early Huntington's disease striatum. Proc Natl Acad Sci U S A 104(8):2945–2949 Epub 2007/02/15. 10.1073/pnas.0609833104
Shah SZA, Zhao D, Hussain T, Sabir N, Mangi MH, Yang L (2018) p62-Keap1-NRF2-ARE Pathway: A Contentious Player for Selective Targeting of Autophagy, Oxidative Stress and Mitochondrial Dysfunction in Prion Diseases. Front Mol Neurosci 11:310. 10.3389/fnmol.2018.00310PubMed PMID: 30337853; PubMed Central PMCID: PMCPMC6180192 Epub 2018/10/20
Li C, Wang D, Wu W, Yang W, Ali Shah SZ, Zhao Y et al (2018) DLP1-dependent mitochondrial fragmentation and redistribution mediate prion-associated mitochondrial dysfunction and neuronal death. Aging Cell 17(1). 10.1111/acel.12693PubMed PMID: 29166700; PubMed Central PMCID: PMCPMC5771399 Epub 2017/11/23
Butterfield DA, Halliwell B (2019) Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease. Nat Rev Neurosci 20(3):148–160. 10.1038/s41583-019-0132-6PubMed PMID: 30737462; PubMed Central PMCID: PMCPMC9382875 Epub 2019/02/10
Cunnane SC, Trushina E, Morland C, Prigione A, Casadesus G, Andrews ZB et al (2020) Brain energy rescue: an emerging therapeutic concept for neurodegenerative disorders of ageing. Nat Rev Drug Discov 19(9):609–633 Epub 2020/07/28. 10.1038/s41573-020-0072-x
Baik SH, Kang S, Lee W, Choi H, Chung S, Kim JI et al (2019) A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease. Cell Metab 30(3):493–507 e6. Epub 2019/07/02. 10.1016/j.cmet.2019.06.005
Choi H, Choi Y, Lee EJ, Kim H, Lee Y, Kwon S et al (2021) Hippocampal glucose uptake as a surrogate of metabolic change of microglia in Alzheimer's disease. J Neuroinflammation 18(1):190 Epub 2021/09/02. 10.1186/s12974-021-02244-6
Sameni S, Syed A, Marsh JL, Digman MA (2016) The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease. Sci Rep 6:34755 Epub 2016/10/08. 10.1038/srep34755
Herzig S, Shaw RJ (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol 19(2):121–135 Epub 2017/10/05. 10.1038/nrm.2017.95
Cantó C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC et al (2009) AMPK regulates energy expenditure by modulating NAD + metabolism and SIRT1 activity. Nature 458(7241):1056–1060 Epub 2009/03/06. 10.1038/nature07813
Ulland TK, Song WM, Huang SC, Ulrich JD, Sergushichev A, Beatty WL et al (2017) TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease. Cell 170(4):649– 63.e13. Epub 2017/08/13. 10.1016/j.cell.2017.07.023
Ciccarese F, Zulato E, Indraccolo S LKB1/AMPK Pathway and Drug Response in Cancer: A Therapeutic Perspective. Oxid Med Cell Longev. 2019;2019:8730816. Epub 2019/11/30. doi: 10.1155/2019/8730816. PubMed PMID: 31781355; PubMed Central PMCID: PMCPMC6874879
Garcia D, Shaw RJ (2017) AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance. Mol Cell 66(6):789–800 Epub 2017/06/18. 10.1016/j.molcel.2017.05.032
Zhang S, Lachance BB, Mattson MP, Jia X (2021) Glucose metabolic crosstalk and regulation in brain function and diseases. Prog Neurobiol 204:102089 Epub 2021/06/13. 10.1016/j.pneurobio.2021.102089
Moon JH, Park SY (2020) Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux. Cell Commun Signal 18(1):109 Epub 2020/07/12. 10.1186/s12964-020-00590-1
Fan XY, Tian C, Wang H, Xu Y, Ren K, Zhang BY et al (2015) Activation of the AMPK-ULK1 pathway plays an important role in autophagy during prion infection. Sci Rep 5:14728. 10.1038/srep14728PubMed PMID: 26423766; PubMed Central PMCID: PMC4589734
Traxler L, Herdy JR, Stefanoni D, Eichhorner S, Pelucchi S, Szücs A et al (2022) Warburg-like metabolic transformation underlies neuronal degeneration in sporadic Alzheimer's disease. Cell Metab 34(9):1248–1263 PubMed PMID: 35987203; PubMed Central PMCID: PMCPMC9458870
Cheng J, Zhang R, Xu Z, Ke Y, Sun R, Yang H et al (2021) Early glycolytic reprogramming controls microglial inflammatory activation. J Neuroinflammation 18(1):129 Epub 2021/06/11. 10.1186/s12974-021-02187-y
Liberti MV, Locasale JW (2016) The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci 41(3):211–218 Epub 2016/01/19. 10.1016/j.tibs.2015.12.001
Vaupel P, Multhoff G (2021) Revisiting the Warburg effect: historical dogma versus current understanding. The Journal of physiology. ;599(6):1745-57. Epub 2020/12/22. doi: 10.1113/jp278810. PubMed PMID: 33347611
Zong WX, Rabinowitz JD, White E (2016) Mitochondria and Cancer. Mol Cell 61(5):667–676 Epub 2016/03/05. 10.1016/j.molcel.2016.02.011
Lu J, Tan M, Cai Q (2015) The Warburg effect in tumor progression: mitochondrial oxidative metabolism as an anti-metastasis mechanism. Cancer Lett. ;356(2 Pt A):156 – 64. Epub 2014/04/16. doi: 10.1016/j.canlet.2014.04.001. PubMed PMID: 24732809; PubMed Central PMCID: PMCPMC4195816
Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 11(5):325–337 Epub 2011/04/22. 10.1038/nrc3038
Min J, Zeng T, Roux M, Lazar D, Chen L, Tudzarova S. The Role of HIF1α-PFKFB3 Pathway in Diabetic Retinopathy. The Journal of clinical endocrinology and metabolism. 2021;106(9):2505-19. Epub 2021/05/22. doi: 10.1210/clinem/dgab362. PubMed PMID: 34019671; PubMed Central PMCID: PMCPMC8372643.

Download PDF

Journal Publication

published 20 Sep, 2023

Read the published version in Molecular Neurobiology →

Editorial decision: Major Revisions Needed
13 Jul, 2023
Reviewers agreed at journal
22 May, 2023
Reviewers invited by journal
13 Apr, 2023
Editor assigned by journal
20 Feb, 2023
First submitted to journal
19 Feb, 2023

You are reading this latest preprint version

Accumulation of prion triggers the enhanced glycolysis via activation of AMKP pathway in prion infected rodent and cell models

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Ethics Statement

Antibodies and reagents

Scrapie infected rodent models

Preparation of brain homogenates

Cell culture and cell lysates

Drug treatment upon cultured cells

Mitochondrial isolation

Western blot

Immunohistochemical assay (IHC)

Immunofluorescent assay (IFA)

Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

Statistical analysis

Results

Increases of HK2, PFKFB3, PKM2 in the brains of scrapie infected rodents at terminal stage

Colocalization of the increased PFKFB3 and HK2 with different types of cells in brains

Increases of HK2, PFKFB3, PKM2 in the prion infected cell line SMB-S15

Downregulation of mitochondrial complexes in prion infected cells and experimental hamsters

Increase of p-AMPKa in the scrapie infected rodents and cultured cells

Removal of prion replication in SMB-S15 cells decreases the levels of glycolysis

Discussion

Declarations

References

Status:

Journal Publication

Version 1