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Metformin reduces prion infection in neuronal cells by enhancing autophagy
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Metformin reduces prion infection in neuronal cells by enhancing autophagy Dalia H. Abdelaziz a, b, c, d, 1, Simrika Thapa a, b, d, 1, Basant Abdulrahman a, b, c, d, Lauren Vankuppeveld a, d, Hermann M. Schatzl a, b, d, * a
Calgary Prion Research Unit, University of Calgary, Calgary, Alberta, Canada Hotchkiss Brain Institute (HBI), University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Cairo, Egypt d Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2019 Accepted 16 December 2019 Available online xxx
Prion diseases are fatal infectious neurodegenerative disorders in human and animals that are caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. No effective treatment is available for prion diseases. Metformin is a first-line medication for treatment of type 2 diabetes which is known to activate AMPK and induce autophagy through the inhibition of mammalian target of rapamycin (mTOR1) signaling. Metformin was reported to be beneficial in various protein misfolding and neurodegenerative diseases like Alzheimer’s and Huntington’s diseases. In this study we investigated the anti-prion effect of metformin in persistently prion-infected neuronal cells. Our data showed that metformin significantly decreased the PrPSc load in the treated cells, as shown by less PK resistant PrP in Western blots and reduced prion conversion activity in Real-Time Quaking-Induced Conversion (RTQuIC) assay in both 22L-ScN2a and RML-ScCAD5 cells. Additionally, metformin induced autophagy as shown by higher levels of LC3-II in treated cells compared with control cells. On the other hand, our mouse bioassay showed that oral metformin at a dose of 2 mg/ml in drinking water had no effect on the survival of prion-infected mice. In conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. This effect can be explained at least partially by the autophagy inducing activity of metformin. This study sheds light on metformin as an anti-prion candidate for the combination therapy of prion diseases. © 2019 Elsevier Inc. All rights reserved.
Keywords: Prion Prion disease Prion therapy Metformin Autophagy
1. Introduction Prion diseases are group of invariably fatal transmissible neurodegenerative diseases in human and animals characterized by vacuolation, neuronal loss and astrogliosis in the brain. These diseases are caused by aggregation of the misfolded and pathological isoform (PrPSc) of the cellular prion protein (PrPC) [1,2]. Over the past three decades many therapeutic agents have been tested for treatment of prion diseases with very limited success. Yet, there
Abbreviations: PrPC, Cellular prion protein; PrPSc, misfolded prion protein; mTOR1, mammalian target of rapamycin; RT-QuIC, Real-Time Quaking-Induced Conversion; AMPK, 50 adenosine monophosphate-activated protein kinase. * Corresponding author. Faculty of Veterinary Medicine, University of Calgary, TRW 2D10, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada . E-mail address: [email protected] (H.M. Schatzl). 1 authors equally contributed to the work.
are no preventive or therapeutic measures available against prion diseases [3,4]. Autophagy is a highly regulated basic cellular process for degradation and recycling of organelles and cytoplasmic proteins including misfolded proteins [5]. Autophagic vacuoles were described in neurons of prion-infected mice and hamsters [6,7], and in prion-infected neuronal cell lines [8]. Autophagy stimulants like rapamycin, trehalose and Imatinib mesylate were effective in decreasing the prion load in both prion infected cultured neuronal cells and mice [3]. Metformin is a widely used antidiabetic drug prescribed as first line medication for type 2 diabetes [9]. It provides efficient glycemic control with a low risk of hypoglycemia [10]. Metformin has potential neuroprotective effects due to its ability to counteract hyperphosphorylation of proteins and decrease oxidative stress [11]. Additionally, metformin is known to induce autophagy [12].
https://doi.org/10.1016/j.bbrc.2019.12.074 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Great efforts have been invested in studying the role of metformin in treatment of neurodegenerative diseases [13]. Metformin was found to protect against amyloid b (Ab) accumulation and hyperphosphorylation of tau protein in neuronal cell lines submitted to prolonged hyperinsulinemia [14]. Additionally, it mitigates the Abinduced apoptosis in cultured hippocampal neurons [15]. A recent clinical study revealed that metformin treatment improved cognitive function of patients with Huntington’s disease [16]. Long term usage of metformin was linked with lower risk of cognitive decline in diabetic patients [17]. Despite metformin has been tested on many neurodegenerative disease models, its effect on prion disease is unexplored. Thus, the aim of our study is to investigate the anti-prion effect of metformin in vitro using prion infected neuronal cell lines and in vivo using prion infected mouse models.
10 min. Cleared aliquots of lysates were incubated with 20 mg/ml PK for 30 min at 37 C, PK digestion was stopped by addition of 0.5 mM Pefabloc followed by methanol precipitation. Samples were run on 12.5% SDS-PAGE, electro-blotted on Amersham Hybond P 0.45 PVDF and analysed in immunoblot, using Luminata Western Chemiluminescent HRP Substrates. 2.6. Real-Time Quaking-Induced Conversion (RT-QuIC)
For the animals used in this study, we strictly followed the guidelines of the Canadian Council for Animal Care (CCAC). The animal protocol was approved by the institutional Health Sciences Animal Care Committee (HSACC), under protocol number AC170142.
The recombinant prion protein was prepared as previously described [22]. Ninety-eight microliters of the master mix (10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/mL rPrP, 10 mM ThT, and 1 mM of EDTA) were loaded into each well of a blackwalled 96-well plate with a clear bottom (Nunc, 165305) and reactions were seeded with 2 mL of the test dilution. Reactions were incubated at 42 C and shaken every other minute at 700 rpm. Serial dilutions of each sample were prepared. Plates were incubated in a FLUOstar Omega plate reader (BMG Labtech, USA) for 30 h. ThT fluorescence (450 nm excitation and 480 nm emission) were monitored every 15 min. The RT-QuIC data was averaged from four replicates, the average values were plotted against the reaction time and reactivity in 2 out of 4 wells was considered positive, Cut-off value was calculated as average þ 5 SD of the negative control.
2.2. Bioassay
2.7. Statistical analysis
Female FVB mice (4e6 weeks old) were used in this study. Mice were intracerebrally (i.c) inoculated with 22L prions (20 mL of 1% (w/v) brain homogenate (BH)). Metformin treated group (n ¼ 10) received metformin (2 mg/ml) in the drinking water [18], the water was changed weekly with fresh drug. The treatment started from the day of the inoculation for 110 days post inoculation (dpi). The control group (n ¼ 10) received water only. Two researchers were involved in monitoring animals for clinical signs of prion disease and making decision for euthanasia.
Statistical analysis was performed using GraphPad version 6.1. One-way ANOVA analysis with Dunnett’s multiple comparisons test was used. The percent survival was plotted in KaplaneMeier plot and Log-rank (Mantel-Cox) test was performed for statistical difference between groups. Significance was expressed as follows: ns: not significant, *: p < 0.05, **: p < 0.01, ***p < 0.001.
2. Materials and methods 2.1. Ethics statement
2.3. Reagents and antibodies Unless otherwise indicated, all the reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). Proteinase K (PK) was purchased from Roche (03115879001), Pefabloc inhibitor was from Roche (11286700). Sources of the antibodies were as follows; Anti-tubulin mAb Santa Cruz (sc-8035), anti-LC3 (Clone 2G6) NanoTools (0260-100); Anti-PrP monoclonal antibody (mAb) 4H11 has been previously described [19]. Peroxidase-conjugated immunoglobulins were from Jackson Immuno-research Lab (goat anti-mouse HRP). 2.4. Maintenance of cell culture The persistently prion-infected mouse neuroblastoma cell line 22L-ScN2a was cultured in Gibco OptiMEM Glutamax medium containing 10% fetal bovine serum, and penicillin/streptomycin in 5% CO2. CAD5 cells are a central nervous system catecholaminergic cell line [20]. RML-ScCAD5 cells were cultured in OptiMEM Glutamax medium containing 10% bovine serum and penicillin/streptomycin in a 5% CO2. Cells were plated and after 24 h they were treated with metformin at a dose of 0.5 or 1 mM. Every 48 h the media was changed, and metformin was added to fresh media. 2.5. Proteinase K (PK) digestion and Western blotting Immunoblot analysis was performed as previously described [21]. Briefly, confluent cells were lysed in cold cell lysis buffer for
3. Results Metformin reduced prion infection and induced autophagy in N2a cells persistently infected with 22L prions. Several studies have shown that metformin has beneficial effects in neurodegenerative diseases [13,14,23]. Here, we wanted to investigate whether metformin has anti-prion effects. So, we treated neuroblastoma cells persistently infected with 22L prions (22L-ScN2a) with two different concentrations of metformin (0.5 and 1 mM) for 7 days. We found significantly lower PrPSc signals in immunoblot in metformin-treated cells compared with vehicle (CT) treated cells. This effect was augmented by doubling the concentration of metformin (1 mM) (Fig. 1A and B). To assess the cytotoxicity we measured lactate dehydrogenase (LDH) levels in the cell culture media and we found no increase in LDH levels following metformin treatment, yet there was a significant decrease in the cytotoxicity which may be attributed to better viability of the cells (Fig. 1C). To confirm our findings, we used RT-QuIC assay to test the effect of metformin on the prion seeding activity in the lysate of 22L-ScN2a cells. The seeding activity in cells treated with metformin was markedly decreased when compared with the control, as shown by extended lag time and decreased maximum RFU (Fig. 1D). Metformin is known to induce autophagy in mTOR dependent manner in liver cells [12]. This directed us to test autophagy inducing capacity of metformin in N2a cells. We treated the cells with metformin for 30 min and 2 h, and cells were lysed and subjected to immunoblotting with anti-LC3 antibody. We found significantly increased LC3-II signals in metformin-treated cells compared with control (Fig. 1 E and F). Taken together, treating 22L-ScN2a cells with metformin for 7 days reduced the prion load as shown by immunoblot and RT-QuIC
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Fig. 1. Metformin treatment mitigates prion infection in persistently infected 22L-ScN2a cells. (A) Persistently 22L prion-infected neuroblastoma cells (22L-ScN2a) were treated for 7 days with 0.5 or 1 mM metformin, or not (CT). Cells were lysed and then the lysate was subjected to proteinase K digestion (þPK), or not (-PK), and submitted to immunoblot analysis. (B) Densitometric analysis of immunoblots. Data are represented as percentage of control (n ¼ 3). (C) Lactate dehydrogenase (LDH) cytotoxicity assay. Supernatant from cells treated with metformin or not (CT) for 7 days was tested to detect LDH levels. (D) RT-QuIC analysis using mouse recombinant PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of 22L-ScN2a cells treated with 0.5 or 1 mM of metformin, or vehicle (H2O), for 7 days. The uninfected N2a cell lysate served as negative control. Y-axis represents relative fluorescent units (RFU) and x-axis represents time in hours (h). (E) Uninfected N2a cells were treated or not (CT) with metformin (1 mM) for 30 min or 2 h. Then cells were lysed and subjected to immunoblotting with anti-LC3 antibody and tubulin was used as a loading control. (F) Densitometric analysis of LC3-II signals of 2 h metformin treatment immunoblots. Data are represented as percentage of control (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001.
analysis. Induction of autophagy could be one of the mechanisms by which metformin mitigates prion infection in N2a cells. Long term treatment with metformin did not eradicate prion infection in 22L-ScN2a cells. We next tested whether metformin can completely cure the prion infection in vitro. We treated 22LScN2a cells with metformin (0.5 and 1 mM) for 14, 21 and 28 days, respectively (Fig. 2). Metformin strongly decreased the PrPSc signal and the seeding activity at all time points as shown by immunoblot and RT-QuIC analysis (Fig. 2), but no full clearance of PrPSc was observed, as the signal of prion infection were still detectable by Western blotting. Also, all the RT-QuIC curves were still positive indicating positive seeding activity (Fig. 2). Taken together, metformin markedly alleviates the prion load in 22L-ScN2a cells but cannot fully cure the cells from infection. Metformin decreased prion infection and induced autophagy in ScCAD5 cells persistently infected with RML prions. Prions consist of various strains with different conformational stability and biochemical properties [24]. It is therefore beneficial to test anti-prion effects on different prion strains and cell lines. This encouraged us to use CNS neuronal cells (CAD5) which are persistently infected with the RML prion strain. Metformin treatment of RML-ScCAD5 cells for 10 days showed decrease in the prion load as revealed by reduction in PrPSc signals in immunoblots compared with the untreated controls (Fig. 3A and B). Moreover, the seeding activity of the cells treated with metformin was markedly reduced compared to untreated cells as shown by
prolongation of the lag time of the RT-QuIC curves and decrease in the maximum RFU (Fig. 3D). Of note, treatment of the CAD5 cells with concentrations of 0.5 and 1 mM metformin for 10 days did not cause any cytotoxicity as shown by LDH assay (Fig. 3C). In addition, there was a significant increase of the LC3-II signals in metformintreated cells compared with the control (Fig. 3 E and F), indicating that metformin induces autophagy in the CAD5 neuronal cells. Together, treating RML-infected ScCAD5 cells with metformin for 10 days reduced prion infection which denotes that the in vitro anti-prion effect of metformin may be strain and cell type independent. Metformin treatment in vivo had no effect on the survival of prion-infected mice. To test the therapeutic effect of metformin in vivo, mice were intracerebrally infected with 22L prions. Then, they were treated from the day of inoculation with metformin in drinking water (2 mg/ml) for 110 days. The Kaplan-Meier curve showed no increase in survival time of the metformin-treated group compared with the control (Fig. 4). Notably, the mice receiving metformin did not show any hypoglycemia compared with the controls (data not shown). 4. Discussion Searching for therapeutic agents to treat prion diseases is an ongoing process that started more than 40 years ago. Unfortunately, no cure is available to date [4]. In this study, we showed that
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Fig. 2. Long-term treatment with metformin decreases prion load but does not fully cure the infection in persistently infected 22L-ScN2a cells. 22L-ScN2a cells were treated for 14 (A), 21 (B) or 28 days (C) with 0.5 and 1 mM metformin, or not treated (control, CT). Cells were lysed, subjected to proteinase K digestion (þ/-PK) and analysed in immunoblotting. (D-F) Densitometric analysis of immunoblots (14, 21, and 28 days, respectively). Data are represented as a percentage of control (n ¼ 3) (G-I) RT-QuIC analysis using recombinant mouse PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of cells treated with 0.5 and 1 mM of metformin, or vehicle treated (H2O) for 14 (G), 21 (H), and 28 days (I), respectively. The uninfected N2a cell lysate used as negative control. Y-axis represents relative fluorescent units (RFU) and xaxis is time in hours (h).
the antidiabetic drug metformin can reduce prion infection and induce autophagy in prion-infected neuronal cells. Metformin was reported to be a promising candidate for treatment of some neurodegenerative diseases [13]. A study showed that metformin prevented amyloid b (Ab) aggregation and tau protein hyperphosphorylation in N2a cells subjected to hyperinsulinemia [14]. Also, preconditioning with metformin provided neuroprotection against focal cerebral ischemia through induction of autophagy [25] and enhanced neurogenesis and spatial memory in wildtype mice [26]. In addition, metformin showed neuroprotective effect and improved the cognitive function in a Huntington’s disease mouse model [18] and prevented memory impairment and amyloid plaque deposition in APP/PS1 mice by enhancing neurogenesis and anti-inflammation [27]. Metformin is known to be an AMPK activator, which induces autophagy by inhibition of mTOR1 signaling in a dose dependent manner [12]. Our data showed that metformin induced autophagy as shown by higher LC3-II signals in treated cells. Interestingly, several autophagy inducers showed substantial effectiveness in treating prion infection both in vivo and in vitro [3]. Oral rapamycin treatment starting at 100 dpi prolonged the survival of 139A prion
infected mice [28]. Also, injection of imatinib mesylate (i.p.) for 30 days delayed neuro-invasion and prolonged survival of RMLinfected mice [29]. A recent data published from our lab showed that AR12, a celecoxib derivative which induces autophagy in neuronal cell lines, prolonged the survival of RML-infected mice [30]. In our study, the anti-prion effect achieved by metformin in vitro was not translated into an effect on the survival of prioninfected mice in vivo. One explanation could be that metformin was not crossing the blood brain barrier efficiently. However, studies in rodents showed that metformin can cross the blood brain barrier and accumulates in the CNS in considerable amounts [31], which may exclude the possibility of limited bioavailability of metformin in the CNS. The discrepancy between in vitro and in vivo effects of antiprion compounds has been reported before in many studies [3,4,32]. Treating of prion-infected N2a cells with branched polyamines, which act on prions in endosomes and lysosomes, resulted in eradication of PrPSc. However, none of these compounds had any anti-prion effect in vivo [33]. Another study reported that the immunosuppressant drug tacrolimus inhibited the replication of
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
D.H. Abdelaziz et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Fig. 3. Metformin treatment reduces prion infection in RML prion-infected ScCAD5 cells. (A) Persistently RML prion-infected ScCAD5 cells were treated for 10 days with 0.5 or 1 mM metformin, or vehicle only (CT). Cells were lysed, subjected to proteinase K digestion (þ/ PK), and then submitted to immunoblotting. Immunoblot was incubated with antiPrP monoclonal antibody 4H11 and anti-tubulin mAb as a loading control. (B) Densitometric analysis of immunoblots. Data are represented as a percentage of control (n ¼ 6), ***; P < 0.001. (C) Lactate dehydrogenase (LDH) cytotoxicity assay. Supernatant from cells treated with metformin or not (CT) for 10 days was tested to detect the level of LDH. (D) RTQuIC analysis using mouse recombinant PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of ScCAD5 cells treated with 0.5 and 1 mM of metformin, or vehicle only (H2O) for 10 days. The uninfected CAD5 cell lysate serve as negative control. Y-axis represents relative fluorescent units (RFU) and x-axis represents time in hours (h). (E) Uninfected CAD5 cells were treated or not (CT) with metformin (1 mM) for either 30 min or 2 h. Then cells were lysed and subjected to immunoblotting with anti-LC3 mAb and tubulin was used as a loading control. (F) Densitometric analysis of LC3-II signal in 2 h metformin treated cells. Data are represented as a percentage of control (n ¼ 3), *; P < 0.05.
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T im e (d p i) Fig. 4. Metformin does not affect the survival of prion-infected mice. Kaplan-Meier survival curves of metformin treated (n ¼ 10) and control group (n ¼ 10) of female FVB mice, inoculated i.c. with 1% 22L brain homogenate of a terminally prion sick mouse. The mice were treated with metformin (2 mg/ml) in drinking water from the day of inoculation consecutively for 110 days, dpi, days post inoculation.
22L and RML prions in the PK1 neuroblastoma cell line, but had no effect on the survival of RML-infected mice [34]. The reason for this inconsistency between in vitro and in vivo effects is unknown but could be explained at least partially by the existence of many
redundant pathways that control the prion life cycle in vivo [3]. So, it may be hard to clear prion infection by targeting only one pathway. Interestingly, metformin was shown to have gender-specific effects. In a study by Ma et al., metformin treatment in a Huntington’s disease mouse model prolonged the survival of only male mice but not the females [18]. In contrast, another study demonstrated that metformin treatment in a murine model of Alzheimer’s disease revealed increase in the memory dysfunction in males but was protective in female mice [35]. Here we used female mice only, so we could not conclude gender-dependent effects from the results of our study. In Conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. The in vitro anti-prion effect can be explained at least partially by the autophagy inducing activity of metformin. Yet, the anti-prion effect was not translated into in vivo efficacy, which requires further experiments using different dosing and different routes of administration. In the future, we are planning to combine metformin with other anti-prion candidate drugs to test if this will improve the in vivo anti-prion efficacy via working on different pathways.
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Author contributions D.H.A, S.T, B.A and L.V conducted the experiments, D.H.A, S.T and H.M.S designed the experiments, D.H.A and H.M.S wrote the manuscript and HMS provided the financial support for the project. The manuscript has been revised and approved by all authors. Funding This work was performed within the framework of the Calgary Prion Research Unit, supported by an Alberta Innovates/Alberta Prion Research Institute grant (201600010). Declaration of competing interest There are no financial interests to be disclosed. Acknowledgements We really appreciate the great help provided by Dr. Lilian Oribhabor and Bukola Alli for animal care. D.H.A. was a University of Calgary Eyes High postdoctoral fellow; S.T. is a University of Calgary Eyes High, Killam doctoral and AIHS doctoral, B.A was an AIHS postdoctoral fellow. References [1] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13363e13383. http://www.ncbi.nlm.nih.gov/pubmed/9811807. accessed April 13, 2017. [2] S.B. Prusiner, Novel proteinaceous infectious particles cause scrapie, Science 216 (1982) 136e144. http://www.ncbi.nlm.nih.gov/pubmed/6801762. accessed June 26, 2018. [3] D.H. Abdelaziz, B.A. Abdulrahman, S. Gilch, H.M. Schatzl, Autophagy pathways in the treatment of prion diseases, Curr. Opin. Pharmacol. 44 (2019) 46e52, https://doi.org/10.1016/j.coph.2019.04.013. [4] K. Giles, S.H. Olson, S.B. Prusiner, Developing therapeutics for PrP prion diseases, Cold Spring Harb. Perspect. Med. 7 (2017), https://doi.org/10.1101/ cshperspect.a023747. [5] B. Levine, D.J. Klionsky, A. Arbor, Development By Self Consumption 6, 2004, pp. 463e477, https://doi.org/10.1016/S1534-5807(04)00099-1. [6] J.W. Boellaard, M. Kao, W. Schlote, H. Diringer, Neuronal autophagy in experimental scrapie, Acta Neuropathol. 82 (1991) 225e228, https://doi.org/ 10.1007/bf00294449. [7] J.W. Boellaard, W. Schlote, J. Tateishi, Neuronal autophagy in experimental Creutzfeldt-Jakob’s disease, Acta Neuropathol. 78 (1989) 410e418, https:// doi.org/10.1007/bf00688178. €tzl, L. Laszlo, D.M. Holtzman, J. Tatzelt, S.J. DeArmond, R.I. Weiner, [8] H.M. Scha W.C. Mobley, S.B. Prusiner, A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis, J. Virol. 71 (1997) 8821e8831. http://www.ncbi.nlm.nih.gov/pubmed/9343242. accessed February 11, 2019. [9] P.I. Moreira, Metformin in the diabetic brain: friend or foe? Ann. Transl. Med. 2 (2014) 54, https://doi.org/10.3978/j.issn.2305-5839.2014.06.10. [10] A.I. Adler, E.J. Shaw, T. Stokes, F. Ruiz, Guideline Development Group, Newer agents for blood glucose control in type 2 diabetes: summary of NICE guidance, BMJ 338 (2009) b1668, https://doi.org/10.1136/bmj.b1668. [11] C. Rotermund, G. Machetanz, J.C. Fitzgerald, The therapeutic potential of metformin in neurodegenerative diseases, Front. Endocrinol. 9 (2018) 400, https://doi.org/10.3389/fendo.2018.00400. [12] J.J. Howell, K. Hellberg, M. Turner, G. Talbott, M.J. Kolar, D.S. Ross, G. Hoxhaj, A. Saghatelian, R.J. Shaw, B.D. Manning, Metformin inhibits hepatic mTORC1 signaling via dose-dependent mechanisms involving AMPK and the TSC complex, Cell Metabol. 25 (2017) 463e471, https://doi.org/10.1016/ j.cmet.2016.12.009. , E. Mikiciuk[13] M. Markowicz-Piasecka, J. Sikora, A. Szydłowska, A. Skupien Olasik, K.M. Huttunen, Metformin e a future therapy for neurodegenerative diseases, Pharm. Res. 34 (2017) 2614e2627, https://doi.org/10.1007/s11095017-2199-y. [14] A. Gupta, B. Bisht, C.S. Dey, Peripheral insulin-sensitizer drug metformin ameliorates neuronal insulin resistance and Alzheimer’s-like changes, Neuropharmacology 60 (2011) 910e920, https://doi.org/10.1016/ j.neuropharm.2011.01.033. [15] B. Chen, Y. Teng, X. Zhang, X. Lv, Y. Yin, Metformin alleviated A b -induced apoptosis via the suppression of JNK MAPK signaling pathway in cultured hippocampal neurons, BioMed Res. Int. (2016) 1e8, https://doi.org/10.1155/ 2016/1421430, 2016.
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He, G.M. Keller, P.W. Frankland, D.R. Kaplan, F.D. Miller, Metformin activates an atypical PKCCBP pathway to promote neurogenesis and enhance spatial memory formation, Cell Stem Cell 11 (2012) 23e35, https://doi.org/10.1016/ j.stem.2012.03.016. [27] Z. Ou, X. Kong, X. Sun, X. He, L. Zhang, Z. Gong, J. Huang, B. Xu, D. Long, J. Li, Q. Li, L. Xu, A. Xuan, Metformin treatment prevents amyloid plaque deposition and memory impairment in APP/PS1 mice, Brain Behav. Immun. 69 (2018) 351e363, https://doi.org/10.1016/j.bbi.2017.12.009. €tzl, Lithium induces [28] A. Heiseke, Y. Aguib, C. Riemer, M. Baier, H.M. Scha clearance of protease resistant prion protein in prion-infected cells by induction of autophagy, J. Neurochem. 109 (2009) 25e34, https://doi.org/ 10.1111/j.1471-4159.2009.05906.x. [29] S.-W. Yun, A. Ertmer, E. Flechsig, S. Gilch, P. Riederer, M. Gerlach, H.M. Sch€ atzl, M.A. Klein, The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery, J. Neurovirol. 13 (2007) 328e337, https://doi.org/10.1080/13550280701361516. [30] B.A. Abdulrahman, W. Tahir, K. Doh-Ura, S. Gilch, H.M. Schatzl, Combining autophagy stimulators and cellulose ethers for therapy against prion disease, Prion 13 (2019) 185e196, https://doi.org/10.1080/19336896.2019.1670928. , Quantifi[31] K. Łabuzek, D. Suchy, B. Gabryel, A. Bielecka, S. Liber, B. Okopien cation of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide, Pharmacol. Rep. 62 (2010) 956e965. http://www.ncbi.nlm.nih.gov/pubmed/21098880. accessed July 9, 2019. [32] A. Aguzzi, A.K.K. Lakkaraju, K. Frontzek, Toward therapy of human prion diseases, Annu. Rev. Pharmacol. Toxicol. 58 (2018) 331e351, https://doi.org/ 10.1146/annurev-pharmtox-010617-052745. [33] S. Supattapone, H.O.B. 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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Metformin reduces prion infection in neuronal cells by enhancing autophagy Dalia H. Abdelaziz a, b, c, d, 1, Simrika Thapa a, b, d, 1, Basant Abdulrahman a, b, c, d, Lauren Vankuppeveld a, d, Hermann M. Schatzl a, b, d, * a
Calgary Prion Research Unit, University of Calgary, Calgary, Alberta, Canada Hotchkiss Brain Institute (HBI), University of Calgary, Calgary, Alberta, Canada Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Cairo, Egypt d Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2019 Accepted 16 December 2019 Available online xxx
Prion diseases are fatal infectious neurodegenerative disorders in human and animals that are caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. No effective treatment is available for prion diseases. Metformin is a first-line medication for treatment of type 2 diabetes which is known to activate AMPK and induce autophagy through the inhibition of mammalian target of rapamycin (mTOR1) signaling. Metformin was reported to be beneficial in various protein misfolding and neurodegenerative diseases like Alzheimer’s and Huntington’s diseases. In this study we investigated the anti-prion effect of metformin in persistently prion-infected neuronal cells. Our data showed that metformin significantly decreased the PrPSc load in the treated cells, as shown by less PK resistant PrP in Western blots and reduced prion conversion activity in Real-Time Quaking-Induced Conversion (RTQuIC) assay in both 22L-ScN2a and RML-ScCAD5 cells. Additionally, metformin induced autophagy as shown by higher levels of LC3-II in treated cells compared with control cells. On the other hand, our mouse bioassay showed that oral metformin at a dose of 2 mg/ml in drinking water had no effect on the survival of prion-infected mice. In conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. This effect can be explained at least partially by the autophagy inducing activity of metformin. This study sheds light on metformin as an anti-prion candidate for the combination therapy of prion diseases. © 2019 Elsevier Inc. All rights reserved.
Keywords: Prion Prion disease Prion therapy Metformin Autophagy
1. Introduction Prion diseases are group of invariably fatal transmissible neurodegenerative diseases in human and animals characterized by vacuolation, neuronal loss and astrogliosis in the brain. These diseases are caused by aggregation of the misfolded and pathological isoform (PrPSc) of the cellular prion protein (PrPC) [1,2]. Over the past three decades many therapeutic agents have been tested for treatment of prion diseases with very limited success. Yet, there
Abbreviations: PrPC, Cellular prion protein; PrPSc, misfolded prion protein; mTOR1, mammalian target of rapamycin; RT-QuIC, Real-Time Quaking-Induced Conversion; AMPK, 50 adenosine monophosphate-activated protein kinase. * Corresponding author. Faculty of Veterinary Medicine, University of Calgary, TRW 2D10, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada . E-mail address: [email protected] (H.M. Schatzl). 1 authors equally contributed to the work.
are no preventive or therapeutic measures available against prion diseases [3,4]. Autophagy is a highly regulated basic cellular process for degradation and recycling of organelles and cytoplasmic proteins including misfolded proteins [5]. Autophagic vacuoles were described in neurons of prion-infected mice and hamsters [6,7], and in prion-infected neuronal cell lines [8]. Autophagy stimulants like rapamycin, trehalose and Imatinib mesylate were effective in decreasing the prion load in both prion infected cultured neuronal cells and mice [3]. Metformin is a widely used antidiabetic drug prescribed as first line medication for type 2 diabetes [9]. It provides efficient glycemic control with a low risk of hypoglycemia [10]. Metformin has potential neuroprotective effects due to its ability to counteract hyperphosphorylation of proteins and decrease oxidative stress [11]. Additionally, metformin is known to induce autophagy [12].
https://doi.org/10.1016/j.bbrc.2019.12.074 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Great efforts have been invested in studying the role of metformin in treatment of neurodegenerative diseases [13]. Metformin was found to protect against amyloid b (Ab) accumulation and hyperphosphorylation of tau protein in neuronal cell lines submitted to prolonged hyperinsulinemia [14]. Additionally, it mitigates the Abinduced apoptosis in cultured hippocampal neurons [15]. A recent clinical study revealed that metformin treatment improved cognitive function of patients with Huntington’s disease [16]. Long term usage of metformin was linked with lower risk of cognitive decline in diabetic patients [17]. Despite metformin has been tested on many neurodegenerative disease models, its effect on prion disease is unexplored. Thus, the aim of our study is to investigate the anti-prion effect of metformin in vitro using prion infected neuronal cell lines and in vivo using prion infected mouse models.
10 min. Cleared aliquots of lysates were incubated with 20 mg/ml PK for 30 min at 37 C, PK digestion was stopped by addition of 0.5 mM Pefabloc followed by methanol precipitation. Samples were run on 12.5% SDS-PAGE, electro-blotted on Amersham Hybond P 0.45 PVDF and analysed in immunoblot, using Luminata Western Chemiluminescent HRP Substrates. 2.6. Real-Time Quaking-Induced Conversion (RT-QuIC)
For the animals used in this study, we strictly followed the guidelines of the Canadian Council for Animal Care (CCAC). The animal protocol was approved by the institutional Health Sciences Animal Care Committee (HSACC), under protocol number AC170142.
The recombinant prion protein was prepared as previously described [22]. Ninety-eight microliters of the master mix (10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/mL rPrP, 10 mM ThT, and 1 mM of EDTA) were loaded into each well of a blackwalled 96-well plate with a clear bottom (Nunc, 165305) and reactions were seeded with 2 mL of the test dilution. Reactions were incubated at 42 C and shaken every other minute at 700 rpm. Serial dilutions of each sample were prepared. Plates were incubated in a FLUOstar Omega plate reader (BMG Labtech, USA) for 30 h. ThT fluorescence (450 nm excitation and 480 nm emission) were monitored every 15 min. The RT-QuIC data was averaged from four replicates, the average values were plotted against the reaction time and reactivity in 2 out of 4 wells was considered positive, Cut-off value was calculated as average þ 5 SD of the negative control.
2.2. Bioassay
2.7. Statistical analysis
Female FVB mice (4e6 weeks old) were used in this study. Mice were intracerebrally (i.c) inoculated with 22L prions (20 mL of 1% (w/v) brain homogenate (BH)). Metformin treated group (n ¼ 10) received metformin (2 mg/ml) in the drinking water [18], the water was changed weekly with fresh drug. The treatment started from the day of the inoculation for 110 days post inoculation (dpi). The control group (n ¼ 10) received water only. Two researchers were involved in monitoring animals for clinical signs of prion disease and making decision for euthanasia.
Statistical analysis was performed using GraphPad version 6.1. One-way ANOVA analysis with Dunnett’s multiple comparisons test was used. The percent survival was plotted in KaplaneMeier plot and Log-rank (Mantel-Cox) test was performed for statistical difference between groups. Significance was expressed as follows: ns: not significant, *: p < 0.05, **: p < 0.01, ***p < 0.001.
2. Materials and methods 2.1. Ethics statement
2.3. Reagents and antibodies Unless otherwise indicated, all the reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). Proteinase K (PK) was purchased from Roche (03115879001), Pefabloc inhibitor was from Roche (11286700). Sources of the antibodies were as follows; Anti-tubulin mAb Santa Cruz (sc-8035), anti-LC3 (Clone 2G6) NanoTools (0260-100); Anti-PrP monoclonal antibody (mAb) 4H11 has been previously described [19]. Peroxidase-conjugated immunoglobulins were from Jackson Immuno-research Lab (goat anti-mouse HRP). 2.4. Maintenance of cell culture The persistently prion-infected mouse neuroblastoma cell line 22L-ScN2a was cultured in Gibco OptiMEM Glutamax medium containing 10% fetal bovine serum, and penicillin/streptomycin in 5% CO2. CAD5 cells are a central nervous system catecholaminergic cell line [20]. RML-ScCAD5 cells were cultured in OptiMEM Glutamax medium containing 10% bovine serum and penicillin/streptomycin in a 5% CO2. Cells were plated and after 24 h they were treated with metformin at a dose of 0.5 or 1 mM. Every 48 h the media was changed, and metformin was added to fresh media. 2.5. Proteinase K (PK) digestion and Western blotting Immunoblot analysis was performed as previously described [21]. Briefly, confluent cells were lysed in cold cell lysis buffer for
3. Results Metformin reduced prion infection and induced autophagy in N2a cells persistently infected with 22L prions. Several studies have shown that metformin has beneficial effects in neurodegenerative diseases [13,14,23]. Here, we wanted to investigate whether metformin has anti-prion effects. So, we treated neuroblastoma cells persistently infected with 22L prions (22L-ScN2a) with two different concentrations of metformin (0.5 and 1 mM) for 7 days. We found significantly lower PrPSc signals in immunoblot in metformin-treated cells compared with vehicle (CT) treated cells. This effect was augmented by doubling the concentration of metformin (1 mM) (Fig. 1A and B). To assess the cytotoxicity we measured lactate dehydrogenase (LDH) levels in the cell culture media and we found no increase in LDH levels following metformin treatment, yet there was a significant decrease in the cytotoxicity which may be attributed to better viability of the cells (Fig. 1C). To confirm our findings, we used RT-QuIC assay to test the effect of metformin on the prion seeding activity in the lysate of 22L-ScN2a cells. The seeding activity in cells treated with metformin was markedly decreased when compared with the control, as shown by extended lag time and decreased maximum RFU (Fig. 1D). Metformin is known to induce autophagy in mTOR dependent manner in liver cells [12]. This directed us to test autophagy inducing capacity of metformin in N2a cells. We treated the cells with metformin for 30 min and 2 h, and cells were lysed and subjected to immunoblotting with anti-LC3 antibody. We found significantly increased LC3-II signals in metformin-treated cells compared with control (Fig. 1 E and F). Taken together, treating 22L-ScN2a cells with metformin for 7 days reduced the prion load as shown by immunoblot and RT-QuIC
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Fig. 1. Metformin treatment mitigates prion infection in persistently infected 22L-ScN2a cells. (A) Persistently 22L prion-infected neuroblastoma cells (22L-ScN2a) were treated for 7 days with 0.5 or 1 mM metformin, or not (CT). Cells were lysed and then the lysate was subjected to proteinase K digestion (þPK), or not (-PK), and submitted to immunoblot analysis. (B) Densitometric analysis of immunoblots. Data are represented as percentage of control (n ¼ 3). (C) Lactate dehydrogenase (LDH) cytotoxicity assay. Supernatant from cells treated with metformin or not (CT) for 7 days was tested to detect LDH levels. (D) RT-QuIC analysis using mouse recombinant PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of 22L-ScN2a cells treated with 0.5 or 1 mM of metformin, or vehicle (H2O), for 7 days. The uninfected N2a cell lysate served as negative control. Y-axis represents relative fluorescent units (RFU) and x-axis represents time in hours (h). (E) Uninfected N2a cells were treated or not (CT) with metformin (1 mM) for 30 min or 2 h. Then cells were lysed and subjected to immunoblotting with anti-LC3 antibody and tubulin was used as a loading control. (F) Densitometric analysis of LC3-II signals of 2 h metformin treatment immunoblots. Data are represented as percentage of control (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001.
analysis. Induction of autophagy could be one of the mechanisms by which metformin mitigates prion infection in N2a cells. Long term treatment with metformin did not eradicate prion infection in 22L-ScN2a cells. We next tested whether metformin can completely cure the prion infection in vitro. We treated 22LScN2a cells with metformin (0.5 and 1 mM) for 14, 21 and 28 days, respectively (Fig. 2). Metformin strongly decreased the PrPSc signal and the seeding activity at all time points as shown by immunoblot and RT-QuIC analysis (Fig. 2), but no full clearance of PrPSc was observed, as the signal of prion infection were still detectable by Western blotting. Also, all the RT-QuIC curves were still positive indicating positive seeding activity (Fig. 2). Taken together, metformin markedly alleviates the prion load in 22L-ScN2a cells but cannot fully cure the cells from infection. Metformin decreased prion infection and induced autophagy in ScCAD5 cells persistently infected with RML prions. Prions consist of various strains with different conformational stability and biochemical properties [24]. It is therefore beneficial to test anti-prion effects on different prion strains and cell lines. This encouraged us to use CNS neuronal cells (CAD5) which are persistently infected with the RML prion strain. Metformin treatment of RML-ScCAD5 cells for 10 days showed decrease in the prion load as revealed by reduction in PrPSc signals in immunoblots compared with the untreated controls (Fig. 3A and B). Moreover, the seeding activity of the cells treated with metformin was markedly reduced compared to untreated cells as shown by
prolongation of the lag time of the RT-QuIC curves and decrease in the maximum RFU (Fig. 3D). Of note, treatment of the CAD5 cells with concentrations of 0.5 and 1 mM metformin for 10 days did not cause any cytotoxicity as shown by LDH assay (Fig. 3C). In addition, there was a significant increase of the LC3-II signals in metformintreated cells compared with the control (Fig. 3 E and F), indicating that metformin induces autophagy in the CAD5 neuronal cells. Together, treating RML-infected ScCAD5 cells with metformin for 10 days reduced prion infection which denotes that the in vitro anti-prion effect of metformin may be strain and cell type independent. Metformin treatment in vivo had no effect on the survival of prion-infected mice. To test the therapeutic effect of metformin in vivo, mice were intracerebrally infected with 22L prions. Then, they were treated from the day of inoculation with metformin in drinking water (2 mg/ml) for 110 days. The Kaplan-Meier curve showed no increase in survival time of the metformin-treated group compared with the control (Fig. 4). Notably, the mice receiving metformin did not show any hypoglycemia compared with the controls (data not shown). 4. Discussion Searching for therapeutic agents to treat prion diseases is an ongoing process that started more than 40 years ago. Unfortunately, no cure is available to date [4]. In this study, we showed that
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Fig. 2. Long-term treatment with metformin decreases prion load but does not fully cure the infection in persistently infected 22L-ScN2a cells. 22L-ScN2a cells were treated for 14 (A), 21 (B) or 28 days (C) with 0.5 and 1 mM metformin, or not treated (control, CT). Cells were lysed, subjected to proteinase K digestion (þ/-PK) and analysed in immunoblotting. (D-F) Densitometric analysis of immunoblots (14, 21, and 28 days, respectively). Data are represented as a percentage of control (n ¼ 3) (G-I) RT-QuIC analysis using recombinant mouse PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of cells treated with 0.5 and 1 mM of metformin, or vehicle treated (H2O) for 14 (G), 21 (H), and 28 days (I), respectively. The uninfected N2a cell lysate used as negative control. Y-axis represents relative fluorescent units (RFU) and xaxis is time in hours (h).
the antidiabetic drug metformin can reduce prion infection and induce autophagy in prion-infected neuronal cells. Metformin was reported to be a promising candidate for treatment of some neurodegenerative diseases [13]. A study showed that metformin prevented amyloid b (Ab) aggregation and tau protein hyperphosphorylation in N2a cells subjected to hyperinsulinemia [14]. Also, preconditioning with metformin provided neuroprotection against focal cerebral ischemia through induction of autophagy [25] and enhanced neurogenesis and spatial memory in wildtype mice [26]. In addition, metformin showed neuroprotective effect and improved the cognitive function in a Huntington’s disease mouse model [18] and prevented memory impairment and amyloid plaque deposition in APP/PS1 mice by enhancing neurogenesis and anti-inflammation [27]. Metformin is known to be an AMPK activator, which induces autophagy by inhibition of mTOR1 signaling in a dose dependent manner [12]. Our data showed that metformin induced autophagy as shown by higher LC3-II signals in treated cells. Interestingly, several autophagy inducers showed substantial effectiveness in treating prion infection both in vivo and in vitro [3]. Oral rapamycin treatment starting at 100 dpi prolonged the survival of 139A prion
infected mice [28]. Also, injection of imatinib mesylate (i.p.) for 30 days delayed neuro-invasion and prolonged survival of RMLinfected mice [29]. A recent data published from our lab showed that AR12, a celecoxib derivative which induces autophagy in neuronal cell lines, prolonged the survival of RML-infected mice [30]. In our study, the anti-prion effect achieved by metformin in vitro was not translated into an effect on the survival of prioninfected mice in vivo. One explanation could be that metformin was not crossing the blood brain barrier efficiently. However, studies in rodents showed that metformin can cross the blood brain barrier and accumulates in the CNS in considerable amounts [31], which may exclude the possibility of limited bioavailability of metformin in the CNS. The discrepancy between in vitro and in vivo effects of antiprion compounds has been reported before in many studies [3,4,32]. Treating of prion-infected N2a cells with branched polyamines, which act on prions in endosomes and lysosomes, resulted in eradication of PrPSc. However, none of these compounds had any anti-prion effect in vivo [33]. Another study reported that the immunosuppressant drug tacrolimus inhibited the replication of
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Fig. 3. Metformin treatment reduces prion infection in RML prion-infected ScCAD5 cells. (A) Persistently RML prion-infected ScCAD5 cells were treated for 10 days with 0.5 or 1 mM metformin, or vehicle only (CT). Cells were lysed, subjected to proteinase K digestion (þ/ PK), and then submitted to immunoblotting. Immunoblot was incubated with antiPrP monoclonal antibody 4H11 and anti-tubulin mAb as a loading control. (B) Densitometric analysis of immunoblots. Data are represented as a percentage of control (n ¼ 6), ***; P < 0.001. (C) Lactate dehydrogenase (LDH) cytotoxicity assay. Supernatant from cells treated with metformin or not (CT) for 10 days was tested to detect the level of LDH. (D) RTQuIC analysis using mouse recombinant PrP as substrate. Each quadruplicate RT-QuIC reaction was seeded with 2 ml cell lysate (at dilution 102) of ScCAD5 cells treated with 0.5 and 1 mM of metformin, or vehicle only (H2O) for 10 days. The uninfected CAD5 cell lysate serve as negative control. Y-axis represents relative fluorescent units (RFU) and x-axis represents time in hours (h). (E) Uninfected CAD5 cells were treated or not (CT) with metformin (1 mM) for either 30 min or 2 h. Then cells were lysed and subjected to immunoblotting with anti-LC3 mAb and tubulin was used as a loading control. (F) Densitometric analysis of LC3-II signal in 2 h metformin treated cells. Data are represented as a percentage of control (n ¼ 3), *; P < 0.05.
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T im e (d p i) Fig. 4. Metformin does not affect the survival of prion-infected mice. Kaplan-Meier survival curves of metformin treated (n ¼ 10) and control group (n ¼ 10) of female FVB mice, inoculated i.c. with 1% 22L brain homogenate of a terminally prion sick mouse. The mice were treated with metformin (2 mg/ml) in drinking water from the day of inoculation consecutively for 110 days, dpi, days post inoculation.
22L and RML prions in the PK1 neuroblastoma cell line, but had no effect on the survival of RML-infected mice [34]. The reason for this inconsistency between in vitro and in vivo effects is unknown but could be explained at least partially by the existence of many
redundant pathways that control the prion life cycle in vivo [3]. So, it may be hard to clear prion infection by targeting only one pathway. Interestingly, metformin was shown to have gender-specific effects. In a study by Ma et al., metformin treatment in a Huntington’s disease mouse model prolonged the survival of only male mice but not the females [18]. In contrast, another study demonstrated that metformin treatment in a murine model of Alzheimer’s disease revealed increase in the memory dysfunction in males but was protective in female mice [35]. Here we used female mice only, so we could not conclude gender-dependent effects from the results of our study. In Conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. The in vitro anti-prion effect can be explained at least partially by the autophagy inducing activity of metformin. Yet, the anti-prion effect was not translated into in vivo efficacy, which requires further experiments using different dosing and different routes of administration. In the future, we are planning to combine metformin with other anti-prion candidate drugs to test if this will improve the in vivo anti-prion efficacy via working on different pathways.
Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074
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Author contributions D.H.A, S.T, B.A and L.V conducted the experiments, D.H.A, S.T and H.M.S designed the experiments, D.H.A and H.M.S wrote the manuscript and HMS provided the financial support for the project. The manuscript has been revised and approved by all authors. Funding This work was performed within the framework of the Calgary Prion Research Unit, supported by an Alberta Innovates/Alberta Prion Research Institute grant (201600010). Declaration of competing interest There are no financial interests to be disclosed. Acknowledgements We really appreciate the great help provided by Dr. Lilian Oribhabor and Bukola Alli for animal care. D.H.A. was a University of Calgary Eyes High postdoctoral fellow; S.T. is a University of Calgary Eyes High, Killam doctoral and AIHS doctoral, B.A was an AIHS postdoctoral fellow. References [1] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13363e13383. http://www.ncbi.nlm.nih.gov/pubmed/9811807. accessed April 13, 2017. [2] S.B. Prusiner, Novel proteinaceous infectious particles cause scrapie, Science 216 (1982) 136e144. http://www.ncbi.nlm.nih.gov/pubmed/6801762. accessed June 26, 2018. [3] D.H. Abdelaziz, B.A. Abdulrahman, S. Gilch, H.M. Schatzl, Autophagy pathways in the treatment of prion diseases, Curr. Opin. Pharmacol. 44 (2019) 46e52, https://doi.org/10.1016/j.coph.2019.04.013. [4] K. Giles, S.H. Olson, S.B. Prusiner, Developing therapeutics for PrP prion diseases, Cold Spring Harb. Perspect. Med. 7 (2017), https://doi.org/10.1101/ cshperspect.a023747. [5] B. Levine, D.J. Klionsky, A. Arbor, Development By Self Consumption 6, 2004, pp. 463e477, https://doi.org/10.1016/S1534-5807(04)00099-1. [6] J.W. Boellaard, M. Kao, W. Schlote, H. Diringer, Neuronal autophagy in experimental scrapie, Acta Neuropathol. 82 (1991) 225e228, https://doi.org/ 10.1007/bf00294449. [7] J.W. Boellaard, W. Schlote, J. Tateishi, Neuronal autophagy in experimental Creutzfeldt-Jakob’s disease, Acta Neuropathol. 78 (1989) 410e418, https:// doi.org/10.1007/bf00688178. €tzl, L. Laszlo, D.M. Holtzman, J. Tatzelt, S.J. DeArmond, R.I. Weiner, [8] H.M. Scha W.C. Mobley, S.B. Prusiner, A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis, J. Virol. 71 (1997) 8821e8831. http://www.ncbi.nlm.nih.gov/pubmed/9343242. accessed February 11, 2019. [9] P.I. Moreira, Metformin in the diabetic brain: friend or foe? Ann. Transl. Med. 2 (2014) 54, https://doi.org/10.3978/j.issn.2305-5839.2014.06.10. [10] A.I. Adler, E.J. Shaw, T. Stokes, F. Ruiz, Guideline Development Group, Newer agents for blood glucose control in type 2 diabetes: summary of NICE guidance, BMJ 338 (2009) b1668, https://doi.org/10.1136/bmj.b1668. [11] C. Rotermund, G. Machetanz, J.C. Fitzgerald, The therapeutic potential of metformin in neurodegenerative diseases, Front. Endocrinol. 9 (2018) 400, https://doi.org/10.3389/fendo.2018.00400. [12] J.J. Howell, K. Hellberg, M. Turner, G. Talbott, M.J. Kolar, D.S. Ross, G. Hoxhaj, A. Saghatelian, R.J. Shaw, B.D. Manning, Metformin inhibits hepatic mTORC1 signaling via dose-dependent mechanisms involving AMPK and the TSC complex, Cell Metabol. 25 (2017) 463e471, https://doi.org/10.1016/ j.cmet.2016.12.009. , E. Mikiciuk[13] M. Markowicz-Piasecka, J. Sikora, A. Szydłowska, A. Skupien Olasik, K.M. Huttunen, Metformin e a future therapy for neurodegenerative diseases, Pharm. Res. 34 (2017) 2614e2627, https://doi.org/10.1007/s11095017-2199-y. [14] A. Gupta, B. Bisht, C.S. Dey, Peripheral insulin-sensitizer drug metformin ameliorates neuronal insulin resistance and Alzheimer’s-like changes, Neuropharmacology 60 (2011) 910e920, https://doi.org/10.1016/ j.neuropharm.2011.01.033. [15] B. Chen, Y. Teng, X. Zhang, X. Lv, Y. Yin, Metformin alleviated A b -induced apoptosis via the suppression of JNK MAPK signaling pathway in cultured hippocampal neurons, BioMed Res. Int. (2016) 1e8, https://doi.org/10.1155/ 2016/1421430, 2016.
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Please cite this article as: D.H. Abdelaziz et al., Metformin reduces prion infection in neuronal cells by enhancing autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.074