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Retrotransposon activation by distressed mitochondria in neurons
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Retrotransposon activation by distressed mitochondria in neurons Marius W. Baeken a, Bernd Moosmann a, Parvana Hajieva a, b, * a b
Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Department of Human Medicine, Medical School Hamburg, Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2020 Accepted 15 February 2020 Available online xxx
Retrotransposon activation occurs in a variety of neurological disorders including multiple sclerosis and Alzheimer’s Disease. While the origins of disease-related retrotransposon activation have remained mostly unidentified, this phenomenon may well contribute to disease progression by inducing inflammation, disrupting transcription and, potentially, genomic insertion. Here, we report that the inhibition of mitochondrial respiratory chain complex I by pharmacological agents widely used to model Parkinson’s disease leads to a significant increase in expression of the ORF1 protein of the long interspersed nucleotide element 1 (LINE1) retrotransposon in human dopaminergic LUHMES cells. These findings were recapitulated in midbrain lysates from accordingly treated wild-type mice that mimic Parkinson’s disease. Retrotransposon activation was paralleled by a loss of DNA cytosine methylation, providing a potential mechanism of retrotransposon mobilization. Loss of DNA methylation as well as retrotransposon activation were suppressed by the mitochondrial antioxidant phenothiazine, indicating that the well-established production of oxidants by inhibited complex I was causing these effects. Retrotransposon activation in some brain disorders may be less of a primary disease trigger rather than a consequence of mitochondrial distress, which is very common in neurodegenerative diseases. © 2020 Elsevier Inc. All rights reserved.
Keywords: Complex I inhibition DNA methylation Neurodegeneration ORF1p Parkinson’s disease Redox signalling
1. Introduction Mobilization of endogenous retroviruses has been described for a number of age-associated neurodegenerative diseases, particularly Alzheimer’s Disease, multiple sclerosis and amyotrophic lateral sclerosis [1e3]. Endogenous retroviruses could plausibly play a relevant role in these diseases since neurons appear to be more permissive to retrotransposition than other cell types [4,5], and they may also be preferably damaged by some of the reported toxic effector mechanisms of retrotransposon activation such as inflammation and microglial activation, toxic envelope protein expression and transcriptional dysregulation [1,3]. For example, the presence of LINE1 cDNA has been described to evoke persistent sterile inflammation in mice [6], the presence of the pHERV-W envelope protein in microglia stimulated these cells to attack myelinated axons in cell culture [7], and the evolutionary reintegration of Alu retrotransposons has been associated with transcriptional noise and mitochondrial dysfunction on theoretical
* Corresponding author. Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany. E-mail addresses: E-mail address: [email protected] (P. Hajieva).parvana. [email protected] (P. Hajieva).
grounds [8]. Even if retrotransposon insertion by LINE1 in patients has mostly been associated with tumor formation [9], it may also occur in syndromes belonging to the Parkinson’s disease spectrum [10]. In the majority of cases, the origins of retrotransposon activation in neurodegenerative disease have remained unknown. We here describe that mitochondrial distress involving increased radical production leads to a mobilization of LINE1 retrotransposons in human cells and in vivo. In view of the overwhelming evidence for mitochondrial distress as early common event in the majority of neurodegenerative disorders [11,12], our results provide a potential rationale for the strange mobilization of endogenous retroviruses in these diseases. 2. Materials and methods 2.1. Chemicals and cell culture media Cell culture media and reagents including Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F-12 Medium (F12), Dulbecco’s Phosphate-Buffered Saline (PBS), N2 Supplement and Hoechst 33258 were obtained from Invitrogen, Carlsbad, CA, USA. All other laboratory chemicals including phenothiazine were purchased at
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the highest available purity from Sigma-Aldrich, St. Louis, MO, USA unless otherwise stated. 2.2. Cell culture Lund human mesencephalic cells (LUHMES cells) were kindly provided by Dr. Jürgen Winkler (Division of Molecular Neurology, University of Erlangen, Germany) and cultivated as described [13]. In brief, the cells were grown in DMEM/F12 (1:1) medium containing 1x N2 supplement and 0.04 mg/ml bFGF (from R&D Systems, Minneapolis, MN, USA) at 37 C in a humidified atmosphere containing 5% CO2 on cell culture dishes that had been coated with 0.05 mg/ml poly-L-ornithine (MW range 30e70 kDa) and 0.001 mg/ml fibronectin (from bovine plasma). Differentiation into dopaminergic neurons was induced by adding 1 mM cAMP, 1 mg/ml tetracycline and 2 ng/ml GDNF (from R&D Systems) to the medium. After 4 days of differentiation in this medium, the medium was renewed and the cells were treated for 48 h with different small molecules in varying combinations: 1-methyl-4-phenylpyridinium iodide (MPPþ, 10 mM), phenothiazine (20 nM), rotenone (10 nM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 1 mM, from Tocris, Bristol, UK), paraquat (100 mM), 6-thioguanine (1 mM, from Tocris). 2.3. In vivo experiments In vivo experiments were performed by QPS Austria (Grambach, Austria), a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were cared for and housed in accordance with the animal welfare legislation of the Ministry of Science of the Austrian government. All procedures complied with the facility’s Animal Care and Welfare Committee. Male C57Bl/6J mice aged 10 ± 2 weeks were allocated to different groups: vehicle control group, MPTP group, MPTP þ phenothiazine group. Animals of the two groups receiving MPTP were treated with the drug on day 2 before tissue sampling (i.e. about 48 h before sacrifice). The MPTP treatment scheme on day 2 consisted of 4 ip injections, separated by 2 h, of 20 mg/kg body weight MPTP. The vehicle-treated group received equal amounts of saline at these points; the applied volume was 10 ml/g body weight. This protocol corresponded to the QPS standard protocol to induce parkinsonism in rodents. Phenothiazine was applied orally from day 5 to day 1 before tissue sampling. On each of these 5 days, the corresponding animals received two doses, separated by 4 h, of 10 mg/kg phenothiazine dissolved in vehicle (DMSO:corn oil 1:50). The two nonphenothiazine groups received equal amounts of vehicle at these points; the applied volume was 2.5 ml/g body weight. For tissue sampling, mice were deeply anesthetized, perfused with 0.9% saline, and the brains were prepared by mechanical dissection. The left hemisphere was divided into striatal tissue, midbrain (including the dopaminergic substantia nigra and ventral tegmental area), and residual brain. The samples were snap frozen and kept at 80 C until further analysis. 2.4. Immunocytochemistry LUHMES cells were seeded at a density of 7 104 cells/cm2 on glass cover slips with standard coating, differentiated for 4 days as above, before experimental treatment with different compounds for 48 h. At the end of each experiment, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by three washes with PBS for 5 min. Cells to be stained for DNA methylation were additionally incubated with 1.5 M HCl for
30 min at this point. Unspecific epitopes were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 5 min at 4 C. The cells were then incubated with primary antibodies in PBS overnight at 4 C before incubation with secondary antibodies (Cy3-anti-rabbit and Cy2-anti-mouse from Dianova, Hamburg, Germany; dilution 1:400) for 2 h at room temperature. Hoechst 33258 (50 ng/ml) was used for nuclear counterstaining. The following primary antibodies were used: rabbit anti-ORF1 (#88701 from Cell Signaling, Cambridge, UK; dilution 1:200), mouse anti-MAP2 (#M4403 from Sigma Aldrich; dilution 1:200) and rabbit anti-5-methyl-cytosine (#28692 from Cell Signaling; dilution 1:200). The cells were analyzed and photographed using a confocal laser-scanning microscope (TCS SP5 from Leica Microsystems, Wetzlar, Germany). 2.5. Western blotting Brain tissue or LUHMES cells were homogenized in lysis buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 10% sucrose; 0.5 mM EDTA; 0.5 mM EGTA, 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail, both from Sigma-Aldrich) and briefly sonicated. Protein concentrations were determined with a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA), before loading 20 mg protein onto a denaturing 10% polyacrylamide gel for electrophoretic separation using standard protocols (10% SDS-PAGE). The separated proteins were blotted (by a second electrophoresis) onto nitrocellulose membranes, followed by blocking with 4% low-fat milk in PBST for 30 min. After three washes with PBST (30 min), the membranes were incubated with the following primary antibodies diluted in PBST: rabbit anti-ORF1 for human samples (#88701 from Cell Signaling; dilution 1:1000); rabbit anti-ORF1 for murine samples (#NBP2-66934 from Novus Biologicals, Littleton, CO, USA; dilution 1:1000); mouse anti-a-tubulin (#T9026 from SigmaAldrich; dilution 1:1000); mouse anti-histone H3 (#14269 from Cell Signaling; dilution 1:1000); mouse anti-ATP5A, mouse antiMTCO1 and mouse anti-UQCRC2 were applied as part of the Total OXPHOS Rodent WB Antibody Cocktail (#ab110413 from Abcam, Cambridge, UK; dilution 1:1000). Primary antibodies were detected by incubation with species-specific, horseradish peroxidaseconjugated anti-rabbit or anti-mouse secondary antibodies (from Dianova; dilution 1:10000), whose binding was visualized with luminescent substrates (Enhanced Chemiluminescence Plus from Amersham, Piscataway, NJ, USA), followed by densitometric quantification. 2.6. Statistical analysis All data are expressed as mean ± standard deviation (SD) of the indicated number of independent experiments. Statistically significant differences between treatment groups were identified by One-way ANOVA followed by a multiple comparisons test following the method of Benjamini and Hochberg. The p-values provided in the graphs derive from this post hoc test and are thus corrected for multiple testing (i.e., they represent q-values). 3. Results Lund human mesencephalic cells (LUHMES cells) were expanded and differentiated into dopaminergic neuronal cells as described [13]. In this differentiated state, LUHMES cells constitute a valuable model to study dopaminergic neurodegeneration, which underlies Parkinson’s disease [14]. LUHMES cells were treated with two mechanistically distinct respiratory chain complex I inhibitors widely used to model Parkinson’s disease, MPPþ and rotenone [15,16]. After 2 days of treatment with sublethal concentrations of these agents, a significant induction and nuclear accumulation of
Please cite this article as: M.W. Baeken et al., Retrotransposon activation by distressed mitochondria in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.106
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Fig. 1. Retrotransposon activation by complex I inhibitors in human neuronal LUHMES cells. (A) Images of differentiated LUHMES cells treated with 10 mM MPPþ, 10 nM rotenone, 20 nM phenothiazine (PHT), 1 mM FCCP, 100 mM paraquat or vehicle for 48 h (63x magnification, zoom factor 3). LINE1 ORF1p is visualized in red, the neuron-specific microtubule marker MAP2 in green and nuclear DNA in blue. (B,C) Quantification of ORF1p immunofluorescence from images taken at 40x magnification, normalized on the number of nuclei (70e100 cells were analyzed per n and treatment group in n ¼ 3 independent cultures). (D) Western blots showing ORF1p expression versus histone H3, the biological and loading control. Treatment conditions were identical as in A. (E,F) Quantification of ORF1p expression in n ¼ 3 Western blots. The indicated p-values derive from all pairwise Benjamini/ Hochberg post hoc tests conducted following One-way ANOVA; p-values in C and F are versus the control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the ORF1 protein encoded by LINE1 was observed by immunocytochemistry (Fig. 1AeC). FCCP, a mitochondrial uncoupler known to impair cellular ATP generation, did not recapitulate this effect. In contrast, a cytosolic and redox cycler and inducer of reactive oxygen species (ROS) formation, paraquat, also induced ORF1p expression (Fig. 1). Western blot analysis confirmed these findings and indicated that the increased immunoreactivity corresponded to a 42 kDa protein matching the size of full-length ORF1p (Fig. 1DeF). To further verify whether the well-established induction of ROS production by MPPþ was indeed causative to ORF1 induction, the radical scavenger phenothiazine was administered. Phenothiazine is a mitochondrial antioxidant that has been shown to be unusually effective in the suppression of oxidative damage from complex I ROS formation [16e18] while lacking any confounding neurotransmitter receptor-modulatory effects [17,19]. As shown in Fig. 1, nanomolar concentrations of phenothiazine entirely prevented the effect of complex I inhibition. Our findings correspond to a series of reports in urogenital cancer cells in which LINE1 ORF1p induction by exogenously added ROS and its suppression by antioxidants was described [20,21]. Despite their in vitro-differentiation into morphologically and biochemically mature dopaminergic-like neurons, human LUHMES cells may potentially be prone to retrotransposon mobilization because of their transformation with v-myc [13].
Hence, the potential induction of LINE1 ORF1p was also probed in wild-type mice in vivo. These mice had been subjected to repeated intraperitoneal treatments with MPTP, a metabolic precursor of MPPþ, which in vivo selectively targets dopaminergic cells as occurring in the substantia nigra [22]; one group was orally administered with phenothiazine. Total midbrain lysates from MPTP-treated animals exhibited a significantly increased expression of ORF1p related to nuclear histone as well as cytosolic tubulin expression (Fig. 2AeC). For comparison, a number of respiratory chain complex subunits were examined, as they might have been induced in compensation of the complex I inhibition. However, none of these subunits were altered in the MPTP animals (Fig. 2DeG), indicating that LINE1 activation may be viewed to be a relatively early event upon respiratory chain inhibition. LINE1 retrotransposon mobilization has repeatedly been associated with epigenetic chromatin modifications like DNA demethylation [5,21,23]. Hence, we have analyzed whether global DNA cytosine methylation might also be influenced by our low doses of standard complex I inhibitors. The results in Fig. 3 provide evidence that this was indeed the case. Both MPPþ and rotenone treatment for 48 h induced a substantial loss of 5-methyl-cytosine immunoreactivity, which was prevented by phenothiazine co-treatment. Effect sizes were comparable to that of 6-thioguanine, an established inhibitor of DNA cytosine methylation [24].
Fig. 2. Retrotransposon mobilization in the midbrain of MPTP-treated mice. (A) Western blots showing ORF1p expression versus histone H3 and tubulin expression in three midbrain lysates from each of three treatment groups (Control; MPTP treatment; MPTP and phenothiazine (PHT) treatment). (B,C) Quantification of midbrain ORF1p expression in n ¼ 9 animals as determined by Western blot. Normalization was done versus histone H3 in B, and versus tubulin in C. (D) Western blots showing the expression (in four midbrain lysates per treatment group) of respiratory chain complex (RCC) subunit ATP5A of complex V, MTCO1 of complex IV, and UQCRC2 of complex III. (E,F,G) Quantification of midbrain RCC expression in n ¼ 9 animals per group. Normalization was done versus tubulin. The given p-values derive from Benjamini/Hochberg post hoc tests conducted following Oneway ANOVA.
Please cite this article as: M.W. Baeken et al., Retrotransposon activation by distressed mitochondria in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.106
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Fig. 3. Loss of DNA cytosine methylation in complex I inhibitor-treated LUHMES cells. Images and quantification of differentiated LUHMES cells treated with 10 mM MPPþ, 10 nM rotenone, 20 nM phenothiazine, 1 mM 6-thioguanine or vehicle for 48 h (63x magnification). Immunostained 5-methyl-cytosine is visualized in red. The quantification was conducted on images taken at 40x magnification and involved normalization on the number of nuclei; 70e100 cells were analyzed per n and treatment group in n ¼ 3 separate cultures. The indicated p-values refer to all pairwise Benjamini/Hochberg post hoc tests conducted following One-way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion The mobilization of LINE1 retrotransposons upon relatively mild pharmacologic complex I inhibition in LUHMES cells evidently depends on mitochondrion-to-nucleus redox signaling. We propose that it represents a side-effect of general mitochondrion-tonucleus signaling aimed at the facilitation of transcription of mitochondrial genes to restore mitochondrial function. During the evolution of the eukaryotes, the endosymbiotic precursors of the mitochondria have lost the vast majority of their genes, transferring many of them to the nucleus [25], such that modern mammalian mitochondria import ~1500 proteins that are nuclear encoded [26]. Clearly, mechanisms must have evolved by which mitochondria signal to the nucleus their protein demand [26,27]. About 80 nuclear genes encode for proteins of the respiratory electron transport chain [26], which may be viewed to represent just one single “mega-enzyme” performing a rather simple reaction, i.e., the reduction of oxygen to water with NADH (even if this mega-enzyme is exceptionally efficient in saving the released chemical energy by pumping protons across the inner mitochondrial membrane). Therefore, a certain degree of shared regulation of all nuclear genes encoding for respiratory chain subunits is plausible from an evolutionary as well as biochemical point of view [28]. Epigenetic mechanisms like regulatory DNA demethylation with their often broad spectrum of affected genes seem to be perfectly suited to accomplish this task. In fact, about 80% of nuclear genes encoding respiratory chain subunits have been shown to feature CpG islands in their promoter regions [28]. Which signal appears preferred for the purpose of a common (emergency) regulation of respiratory chain genes (and, as a side effect, of retrotransposons)? As a long linear redox chain, the mega-
enzyme may be inhibited at multiple sites by toxins, selective absence of certain protein subunits and cofactors as well as by protein oxidation, protein unfolding and other types of protein aging. A common consequence may be ATP loss, which has indeed been shown to induce compensatory responses including the transcription of nuclear genes that would act to counter ATP deficiency [29]. However, an identically common, but even more dangerous consequence of respiratory chain dysfunction is the generation of superoxide and other ROS at the highly reducing upstream sites of the chain, for example at the FMN and Q binding sites of complex I [27,30]. To regulate the transcription and translation of mitochondrially imported proteins, therefore, it appears quite reasonable to deploy the more sensitive redox signalling system [26,30] in addition to bioenergetic markers like ATP. This is because the latter might be influenced by many more events than just respiratory blockade, for example, by an acute high demand of ion pumps or motor proteins. In fact, AMP kinases sensing ATP deficiency primarily respond by rapidly shutting down energetic demand and by mobilizing catabolic substrates [29]. Regarding the mobilization of retrotransposons in the wake of respiratory inhibition, the following evidence points to an involvement of ROS redox signaling: the efficacy of both ROSproducing complex I inhibitors MPPþ and rotenone, the complete suppression of the MPPþ effect by the mitochondrial antioxidant phenothiazine, the inactivity of FCCP, a ROS-inactive mitochondrial uncoupler that only depletes ATP levels, and the efficacy of the cytosolic ROS-producing redox cycler paraquat (Fig. 1). After all, under the employed conditions of mild complex I inhibition in LUHMES cells with 10 mM MPPþ, cellular ATP levels were entirely unaltered (not shown), in agreement with the literature [14].
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In summary, our data indicate that elevated ROS levels due to respiratory chain complex I blockade induce DNA demethylation [31] and potentially other epigenetic alterations to facilitate the broad-spectrum transcription of genes encoding for mitochondrially imported proteins. As an inadvertent side-effect, also retrotransposons are activated in consequence of the attained DNA demethylation. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments Support from the Corona foundation and the Manfred-undUrsula-Müller foundation, members of the “Stifterverband für die Deutsche Wissenschaft”, is gratefully acknowledged. Both entities were not involved in the study’s design, execution or evaluation. The authors thank the team from QPS Austria for their careful planning and excellent execution of the animal experiments. References [1] A. Saleh, A. Macia, A.R. Muotri, Transposable elements, inflammation, and neurological disease, Front. Neurol. 10 (2019) 894. [2] A.L. Savage, G.G. Schumann, G. Breen, V.J. Bubb, A. Al-Chalabi, J.P. Quinn, Retrotransposons in the development and progression of amyotrophic lateral sclerosis, J. Neurol. Neurosurg. Psychiatry 90 (2019) 284e293. [3] O.H. Tam, L.W. Ostrow, M. Gale Hammell, Diseases of the nERVous system: retrotransposon activity in neurodegenerative disease, Mobile DNA 10 (2019) 32. [4] J.L. Goodier, Restricting retrotransposons: a review, Mobile DNA 7 (2016) 16. [5] A. Macia, T.J. Widmann, S.R. Heras, et al., Engineered LINE-1 retrotransposition in nondividing human neurons, Genome Res. 27 (2017) 335e348. [6] M. Simon, M. Van Meter, J. Ablaeva, et al., LINE1 derepression in aged wildtype and SIRT6-deficient mice drives inflammation, Cell Metabol. 29 (2019) 871e885. [7] D. Kremer, J. Gruchot, V. Weyers, et al., pHERV-W envelope protein fuels microglial cell-dependent damage of myelinated axons in multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A. 116 (2019) 15216e15225. [8] P.A. Larsen, M.W. Lutz, K.E. Hunnicutt, M. Mihovilovic, A.M. Saunders, A.D. Yoder, A.D. Roses, The Alu neurodegeneration hypothesis: a primatespecific mechanism for neuronal transcription noise, mitochondrial dysfunction, and manifestation of neurodegenerative disease, Alzheim. Dement 13 (2017) 828e838. [9] D.C. Hancks, H.H. Kazazian Jr., Roles for retrotransposon insertions in human disease, Mobile DNA 7 (2016) 9. [10] S. Makino, R. Kaji, S. Ando, et al., Reduced neuron-specific expression of the TAF1 gene is associated with X-linked dystonia-parkinsonism, Am. J. Hum. Genet. 80 (2007) 393e406. [11] A. Johri, M.F. Beal, Mitochondrial dysfunction in neurodegenerative diseases, J. Pharmacol. Exp. Therapeut. 342 (2012) 619e630.
[12] J.V. Cabral-Costa, A.J. Kowaltowski, Neurological disorders and mitochondria, Mol. Aspect. Med. 100826 (2019). € ltl, A. Genewsky, M. Weng, T. Waldmann, S. Schildknecht, [13] D. Scholz, D. Po M. Leist, Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line, J. Neurochem. 119 (2011) 957e971. [14] X.M. Zhang, M. Yin, M.H. Zhang, Cell-based assays for Parkinson’s disease using differentiated human LUHMES cells, Acta Pharmacol. Sin. 35 (2014) 945e956. [15] J.R. Richardson, Y. Quan, T.B. Sherer, J.T. Greenamyre, G.W. Miller, Paraquat neurotoxicity is distinct from that of MPTP and rotenone, Toxicol. Sci. 88 (2005) 193e201. [16] P. Hajieva, J.B. Mocko, B. Moosmann, C. Behl, Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity, J. Neurochem. 110 (2009) 118e132. [17] J.B. Mocko, A. Kern, B. Moosmann, C. Behl, P. Hajieva, Phenothiazines interfere with dopaminergic neurodegeneration in Caenorhabditis elegans models of Parkinson’s disease, Neurobiol. Dis. 40 (2010) 120e129. [18] V. Tapias, J.L. McCoy, J.T. Greenamyre, Phenothiazine normalizes the NADH/ NADþ ratio, maintains mitochondrial integrity and protects the nigrostriatal dopamine system in a chronic rotenone model of Parkinson’s disease, Redox Biol. 24 (2019), 101164. [19] M.J. Ohlow, B. Moosmann, Phenothiazine: the seven lives of pharmacology’s first lead structure, Drug Discov. Today 16 (2011) 119e131. [20] P. Whongsiri, C. Pimratana, U. Wijitsettakul, D. Jindatip, A. Sanpavat, W.A. Schulz, M.J. Hoffmann, W. Goering, C. Boonla, LINE-1 ORF1 protein is upregulated by reactive oxygen species and associated with bladder urothelial carcinoma progression, Canc. Genom. Proteom. 15 (2018) 143e151. [21] P. Whongsiri, C. Pimratana, U. Wijitsettakul, A. Sanpavat, D. Jindatip, M.J. Hoffmann, W. Goering, W.A. Schulz, C. Boonla, Oxidative stress and LINE1 reactivation in bladder cancer are epigenetically linked through active chromatin formation, Free Radic. Biol. Med. 134 (2019) 419e428. [22] J.R. Cannon, J.T. Greenamyre, The role of environmental exposures in neurodegeneration and neurodegenerative diseases, Toxicol. Sci. 124 (2011) 225e250. [23] E.M. Wolff, H.M. Byun, H.F. Han, S. Sharma, P.W. Nichols, K.D. Siegmund, A.S. Yang, P.A. Jones, G. Liang, Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer, PLoS Genet. 6 (2010), e1000917. [24] K. Agrawal, V. Das, P. Vyas, M. Hajdúch, Nucleosidic DNA demethylating epigenetic drugs - a comprehensive review from discovery to clinic, Pharmacol. Ther. 188 (2018) 45e79. [25] J.N. Timmis, M.A. Ayliffe, C.Y. Huang, W. Martin, Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes, Nat. Rev. Genet. 5 (2004) 123e135. [26] D.C. Wallace, Mitochondria and cancer, Nat. Rev. Canc. 12 (2012) 685e698. [27] M.D. Brand, Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling, Free Radic. Biol. Med. 100 (2016) 14e31. [28] C. van Waveren, C.T. Moraes, Transcriptional co-expression and co-regulation of genes coding for components of the oxidative phosphorylation system, BMC Genom. 9 (2008) 18. [29] M.M. Mihaylova, R.J. Shaw, The AMPK signalling pathway coordinates cell growth, autophagy and metabolism, Nat. Cell Biol. 13 (2011) 1016e1023. [30] E.B. Tahara, F.D. Navarete, A.J. Kowaltowski, Tissue-, substrate-, and sitespecific characteristics of mitochondrial reactive oxygen species generation, Free Radic. Biol. Med. 46 (2009) 1283e1297. [31] Q. Wu, X. Ni, ROS-mediated DNA methylation pattern alterations in carcinogenesis, Curr. Drug Targets 16 (2015) 13e19.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Retrotransposon activation by distressed mitochondria in neurons Marius W. Baeken a, Bernd Moosmann a, Parvana Hajieva a, b, * a b
Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Department of Human Medicine, Medical School Hamburg, Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2020 Accepted 15 February 2020 Available online xxx
Retrotransposon activation occurs in a variety of neurological disorders including multiple sclerosis and Alzheimer’s Disease. While the origins of disease-related retrotransposon activation have remained mostly unidentified, this phenomenon may well contribute to disease progression by inducing inflammation, disrupting transcription and, potentially, genomic insertion. Here, we report that the inhibition of mitochondrial respiratory chain complex I by pharmacological agents widely used to model Parkinson’s disease leads to a significant increase in expression of the ORF1 protein of the long interspersed nucleotide element 1 (LINE1) retrotransposon in human dopaminergic LUHMES cells. These findings were recapitulated in midbrain lysates from accordingly treated wild-type mice that mimic Parkinson’s disease. Retrotransposon activation was paralleled by a loss of DNA cytosine methylation, providing a potential mechanism of retrotransposon mobilization. Loss of DNA methylation as well as retrotransposon activation were suppressed by the mitochondrial antioxidant phenothiazine, indicating that the well-established production of oxidants by inhibited complex I was causing these effects. Retrotransposon activation in some brain disorders may be less of a primary disease trigger rather than a consequence of mitochondrial distress, which is very common in neurodegenerative diseases. © 2020 Elsevier Inc. All rights reserved.
Keywords: Complex I inhibition DNA methylation Neurodegeneration ORF1p Parkinson’s disease Redox signalling
1. Introduction Mobilization of endogenous retroviruses has been described for a number of age-associated neurodegenerative diseases, particularly Alzheimer’s Disease, multiple sclerosis and amyotrophic lateral sclerosis [1e3]. Endogenous retroviruses could plausibly play a relevant role in these diseases since neurons appear to be more permissive to retrotransposition than other cell types [4,5], and they may also be preferably damaged by some of the reported toxic effector mechanisms of retrotransposon activation such as inflammation and microglial activation, toxic envelope protein expression and transcriptional dysregulation [1,3]. For example, the presence of LINE1 cDNA has been described to evoke persistent sterile inflammation in mice [6], the presence of the pHERV-W envelope protein in microglia stimulated these cells to attack myelinated axons in cell culture [7], and the evolutionary reintegration of Alu retrotransposons has been associated with transcriptional noise and mitochondrial dysfunction on theoretical
* Corresponding author. Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany. E-mail addresses: E-mail address: [email protected] (P. Hajieva).parvana. [email protected] (P. Hajieva).
grounds [8]. Even if retrotransposon insertion by LINE1 in patients has mostly been associated with tumor formation [9], it may also occur in syndromes belonging to the Parkinson’s disease spectrum [10]. In the majority of cases, the origins of retrotransposon activation in neurodegenerative disease have remained unknown. We here describe that mitochondrial distress involving increased radical production leads to a mobilization of LINE1 retrotransposons in human cells and in vivo. In view of the overwhelming evidence for mitochondrial distress as early common event in the majority of neurodegenerative disorders [11,12], our results provide a potential rationale for the strange mobilization of endogenous retroviruses in these diseases. 2. Materials and methods 2.1. Chemicals and cell culture media Cell culture media and reagents including Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F-12 Medium (F12), Dulbecco’s Phosphate-Buffered Saline (PBS), N2 Supplement and Hoechst 33258 were obtained from Invitrogen, Carlsbad, CA, USA. All other laboratory chemicals including phenothiazine were purchased at
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the highest available purity from Sigma-Aldrich, St. Louis, MO, USA unless otherwise stated. 2.2. Cell culture Lund human mesencephalic cells (LUHMES cells) were kindly provided by Dr. Jürgen Winkler (Division of Molecular Neurology, University of Erlangen, Germany) and cultivated as described [13]. In brief, the cells were grown in DMEM/F12 (1:1) medium containing 1x N2 supplement and 0.04 mg/ml bFGF (from R&D Systems, Minneapolis, MN, USA) at 37 C in a humidified atmosphere containing 5% CO2 on cell culture dishes that had been coated with 0.05 mg/ml poly-L-ornithine (MW range 30e70 kDa) and 0.001 mg/ml fibronectin (from bovine plasma). Differentiation into dopaminergic neurons was induced by adding 1 mM cAMP, 1 mg/ml tetracycline and 2 ng/ml GDNF (from R&D Systems) to the medium. After 4 days of differentiation in this medium, the medium was renewed and the cells were treated for 48 h with different small molecules in varying combinations: 1-methyl-4-phenylpyridinium iodide (MPPþ, 10 mM), phenothiazine (20 nM), rotenone (10 nM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 1 mM, from Tocris, Bristol, UK), paraquat (100 mM), 6-thioguanine (1 mM, from Tocris). 2.3. In vivo experiments In vivo experiments were performed by QPS Austria (Grambach, Austria), a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were cared for and housed in accordance with the animal welfare legislation of the Ministry of Science of the Austrian government. All procedures complied with the facility’s Animal Care and Welfare Committee. Male C57Bl/6J mice aged 10 ± 2 weeks were allocated to different groups: vehicle control group, MPTP group, MPTP þ phenothiazine group. Animals of the two groups receiving MPTP were treated with the drug on day 2 before tissue sampling (i.e. about 48 h before sacrifice). The MPTP treatment scheme on day 2 consisted of 4 ip injections, separated by 2 h, of 20 mg/kg body weight MPTP. The vehicle-treated group received equal amounts of saline at these points; the applied volume was 10 ml/g body weight. This protocol corresponded to the QPS standard protocol to induce parkinsonism in rodents. Phenothiazine was applied orally from day 5 to day 1 before tissue sampling. On each of these 5 days, the corresponding animals received two doses, separated by 4 h, of 10 mg/kg phenothiazine dissolved in vehicle (DMSO:corn oil 1:50). The two nonphenothiazine groups received equal amounts of vehicle at these points; the applied volume was 2.5 ml/g body weight. For tissue sampling, mice were deeply anesthetized, perfused with 0.9% saline, and the brains were prepared by mechanical dissection. The left hemisphere was divided into striatal tissue, midbrain (including the dopaminergic substantia nigra and ventral tegmental area), and residual brain. The samples were snap frozen and kept at 80 C until further analysis. 2.4. Immunocytochemistry LUHMES cells were seeded at a density of 7 104 cells/cm2 on glass cover slips with standard coating, differentiated for 4 days as above, before experimental treatment with different compounds for 48 h. At the end of each experiment, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by three washes with PBS for 5 min. Cells to be stained for DNA methylation were additionally incubated with 1.5 M HCl for
30 min at this point. Unspecific epitopes were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 5 min at 4 C. The cells were then incubated with primary antibodies in PBS overnight at 4 C before incubation with secondary antibodies (Cy3-anti-rabbit and Cy2-anti-mouse from Dianova, Hamburg, Germany; dilution 1:400) for 2 h at room temperature. Hoechst 33258 (50 ng/ml) was used for nuclear counterstaining. The following primary antibodies were used: rabbit anti-ORF1 (#88701 from Cell Signaling, Cambridge, UK; dilution 1:200), mouse anti-MAP2 (#M4403 from Sigma Aldrich; dilution 1:200) and rabbit anti-5-methyl-cytosine (#28692 from Cell Signaling; dilution 1:200). The cells were analyzed and photographed using a confocal laser-scanning microscope (TCS SP5 from Leica Microsystems, Wetzlar, Germany). 2.5. Western blotting Brain tissue or LUHMES cells were homogenized in lysis buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 10% sucrose; 0.5 mM EDTA; 0.5 mM EGTA, 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail, both from Sigma-Aldrich) and briefly sonicated. Protein concentrations were determined with a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA), before loading 20 mg protein onto a denaturing 10% polyacrylamide gel for electrophoretic separation using standard protocols (10% SDS-PAGE). The separated proteins were blotted (by a second electrophoresis) onto nitrocellulose membranes, followed by blocking with 4% low-fat milk in PBST for 30 min. After three washes with PBST (30 min), the membranes were incubated with the following primary antibodies diluted in PBST: rabbit anti-ORF1 for human samples (#88701 from Cell Signaling; dilution 1:1000); rabbit anti-ORF1 for murine samples (#NBP2-66934 from Novus Biologicals, Littleton, CO, USA; dilution 1:1000); mouse anti-a-tubulin (#T9026 from SigmaAldrich; dilution 1:1000); mouse anti-histone H3 (#14269 from Cell Signaling; dilution 1:1000); mouse anti-ATP5A, mouse antiMTCO1 and mouse anti-UQCRC2 were applied as part of the Total OXPHOS Rodent WB Antibody Cocktail (#ab110413 from Abcam, Cambridge, UK; dilution 1:1000). Primary antibodies were detected by incubation with species-specific, horseradish peroxidaseconjugated anti-rabbit or anti-mouse secondary antibodies (from Dianova; dilution 1:10000), whose binding was visualized with luminescent substrates (Enhanced Chemiluminescence Plus from Amersham, Piscataway, NJ, USA), followed by densitometric quantification. 2.6. Statistical analysis All data are expressed as mean ± standard deviation (SD) of the indicated number of independent experiments. Statistically significant differences between treatment groups were identified by One-way ANOVA followed by a multiple comparisons test following the method of Benjamini and Hochberg. The p-values provided in the graphs derive from this post hoc test and are thus corrected for multiple testing (i.e., they represent q-values). 3. Results Lund human mesencephalic cells (LUHMES cells) were expanded and differentiated into dopaminergic neuronal cells as described [13]. In this differentiated state, LUHMES cells constitute a valuable model to study dopaminergic neurodegeneration, which underlies Parkinson’s disease [14]. LUHMES cells were treated with two mechanistically distinct respiratory chain complex I inhibitors widely used to model Parkinson’s disease, MPPþ and rotenone [15,16]. After 2 days of treatment with sublethal concentrations of these agents, a significant induction and nuclear accumulation of
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Fig. 1. Retrotransposon activation by complex I inhibitors in human neuronal LUHMES cells. (A) Images of differentiated LUHMES cells treated with 10 mM MPPþ, 10 nM rotenone, 20 nM phenothiazine (PHT), 1 mM FCCP, 100 mM paraquat or vehicle for 48 h (63x magnification, zoom factor 3). LINE1 ORF1p is visualized in red, the neuron-specific microtubule marker MAP2 in green and nuclear DNA in blue. (B,C) Quantification of ORF1p immunofluorescence from images taken at 40x magnification, normalized on the number of nuclei (70e100 cells were analyzed per n and treatment group in n ¼ 3 independent cultures). (D) Western blots showing ORF1p expression versus histone H3, the biological and loading control. Treatment conditions were identical as in A. (E,F) Quantification of ORF1p expression in n ¼ 3 Western blots. The indicated p-values derive from all pairwise Benjamini/ Hochberg post hoc tests conducted following One-way ANOVA; p-values in C and F are versus the control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the ORF1 protein encoded by LINE1 was observed by immunocytochemistry (Fig. 1AeC). FCCP, a mitochondrial uncoupler known to impair cellular ATP generation, did not recapitulate this effect. In contrast, a cytosolic and redox cycler and inducer of reactive oxygen species (ROS) formation, paraquat, also induced ORF1p expression (Fig. 1). Western blot analysis confirmed these findings and indicated that the increased immunoreactivity corresponded to a 42 kDa protein matching the size of full-length ORF1p (Fig. 1DeF). To further verify whether the well-established induction of ROS production by MPPþ was indeed causative to ORF1 induction, the radical scavenger phenothiazine was administered. Phenothiazine is a mitochondrial antioxidant that has been shown to be unusually effective in the suppression of oxidative damage from complex I ROS formation [16e18] while lacking any confounding neurotransmitter receptor-modulatory effects [17,19]. As shown in Fig. 1, nanomolar concentrations of phenothiazine entirely prevented the effect of complex I inhibition. Our findings correspond to a series of reports in urogenital cancer cells in which LINE1 ORF1p induction by exogenously added ROS and its suppression by antioxidants was described [20,21]. Despite their in vitro-differentiation into morphologically and biochemically mature dopaminergic-like neurons, human LUHMES cells may potentially be prone to retrotransposon mobilization because of their transformation with v-myc [13].
Hence, the potential induction of LINE1 ORF1p was also probed in wild-type mice in vivo. These mice had been subjected to repeated intraperitoneal treatments with MPTP, a metabolic precursor of MPPþ, which in vivo selectively targets dopaminergic cells as occurring in the substantia nigra [22]; one group was orally administered with phenothiazine. Total midbrain lysates from MPTP-treated animals exhibited a significantly increased expression of ORF1p related to nuclear histone as well as cytosolic tubulin expression (Fig. 2AeC). For comparison, a number of respiratory chain complex subunits were examined, as they might have been induced in compensation of the complex I inhibition. However, none of these subunits were altered in the MPTP animals (Fig. 2DeG), indicating that LINE1 activation may be viewed to be a relatively early event upon respiratory chain inhibition. LINE1 retrotransposon mobilization has repeatedly been associated with epigenetic chromatin modifications like DNA demethylation [5,21,23]. Hence, we have analyzed whether global DNA cytosine methylation might also be influenced by our low doses of standard complex I inhibitors. The results in Fig. 3 provide evidence that this was indeed the case. Both MPPþ and rotenone treatment for 48 h induced a substantial loss of 5-methyl-cytosine immunoreactivity, which was prevented by phenothiazine co-treatment. Effect sizes were comparable to that of 6-thioguanine, an established inhibitor of DNA cytosine methylation [24].
Fig. 2. Retrotransposon mobilization in the midbrain of MPTP-treated mice. (A) Western blots showing ORF1p expression versus histone H3 and tubulin expression in three midbrain lysates from each of three treatment groups (Control; MPTP treatment; MPTP and phenothiazine (PHT) treatment). (B,C) Quantification of midbrain ORF1p expression in n ¼ 9 animals as determined by Western blot. Normalization was done versus histone H3 in B, and versus tubulin in C. (D) Western blots showing the expression (in four midbrain lysates per treatment group) of respiratory chain complex (RCC) subunit ATP5A of complex V, MTCO1 of complex IV, and UQCRC2 of complex III. (E,F,G) Quantification of midbrain RCC expression in n ¼ 9 animals per group. Normalization was done versus tubulin. The given p-values derive from Benjamini/Hochberg post hoc tests conducted following Oneway ANOVA.
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Fig. 3. Loss of DNA cytosine methylation in complex I inhibitor-treated LUHMES cells. Images and quantification of differentiated LUHMES cells treated with 10 mM MPPþ, 10 nM rotenone, 20 nM phenothiazine, 1 mM 6-thioguanine or vehicle for 48 h (63x magnification). Immunostained 5-methyl-cytosine is visualized in red. The quantification was conducted on images taken at 40x magnification and involved normalization on the number of nuclei; 70e100 cells were analyzed per n and treatment group in n ¼ 3 separate cultures. The indicated p-values refer to all pairwise Benjamini/Hochberg post hoc tests conducted following One-way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion The mobilization of LINE1 retrotransposons upon relatively mild pharmacologic complex I inhibition in LUHMES cells evidently depends on mitochondrion-to-nucleus redox signaling. We propose that it represents a side-effect of general mitochondrion-tonucleus signaling aimed at the facilitation of transcription of mitochondrial genes to restore mitochondrial function. During the evolution of the eukaryotes, the endosymbiotic precursors of the mitochondria have lost the vast majority of their genes, transferring many of them to the nucleus [25], such that modern mammalian mitochondria import ~1500 proteins that are nuclear encoded [26]. Clearly, mechanisms must have evolved by which mitochondria signal to the nucleus their protein demand [26,27]. About 80 nuclear genes encode for proteins of the respiratory electron transport chain [26], which may be viewed to represent just one single “mega-enzyme” performing a rather simple reaction, i.e., the reduction of oxygen to water with NADH (even if this mega-enzyme is exceptionally efficient in saving the released chemical energy by pumping protons across the inner mitochondrial membrane). Therefore, a certain degree of shared regulation of all nuclear genes encoding for respiratory chain subunits is plausible from an evolutionary as well as biochemical point of view [28]. Epigenetic mechanisms like regulatory DNA demethylation with their often broad spectrum of affected genes seem to be perfectly suited to accomplish this task. In fact, about 80% of nuclear genes encoding respiratory chain subunits have been shown to feature CpG islands in their promoter regions [28]. Which signal appears preferred for the purpose of a common (emergency) regulation of respiratory chain genes (and, as a side effect, of retrotransposons)? As a long linear redox chain, the mega-
enzyme may be inhibited at multiple sites by toxins, selective absence of certain protein subunits and cofactors as well as by protein oxidation, protein unfolding and other types of protein aging. A common consequence may be ATP loss, which has indeed been shown to induce compensatory responses including the transcription of nuclear genes that would act to counter ATP deficiency [29]. However, an identically common, but even more dangerous consequence of respiratory chain dysfunction is the generation of superoxide and other ROS at the highly reducing upstream sites of the chain, for example at the FMN and Q binding sites of complex I [27,30]. To regulate the transcription and translation of mitochondrially imported proteins, therefore, it appears quite reasonable to deploy the more sensitive redox signalling system [26,30] in addition to bioenergetic markers like ATP. This is because the latter might be influenced by many more events than just respiratory blockade, for example, by an acute high demand of ion pumps or motor proteins. In fact, AMP kinases sensing ATP deficiency primarily respond by rapidly shutting down energetic demand and by mobilizing catabolic substrates [29]. Regarding the mobilization of retrotransposons in the wake of respiratory inhibition, the following evidence points to an involvement of ROS redox signaling: the efficacy of both ROSproducing complex I inhibitors MPPþ and rotenone, the complete suppression of the MPPþ effect by the mitochondrial antioxidant phenothiazine, the inactivity of FCCP, a ROS-inactive mitochondrial uncoupler that only depletes ATP levels, and the efficacy of the cytosolic ROS-producing redox cycler paraquat (Fig. 1). After all, under the employed conditions of mild complex I inhibition in LUHMES cells with 10 mM MPPþ, cellular ATP levels were entirely unaltered (not shown), in agreement with the literature [14].
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In summary, our data indicate that elevated ROS levels due to respiratory chain complex I blockade induce DNA demethylation [31] and potentially other epigenetic alterations to facilitate the broad-spectrum transcription of genes encoding for mitochondrially imported proteins. As an inadvertent side-effect, also retrotransposons are activated in consequence of the attained DNA demethylation. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments Support from the Corona foundation and the Manfred-undUrsula-Müller foundation, members of the “Stifterverband für die Deutsche Wissenschaft”, is gratefully acknowledged. Both entities were not involved in the study’s design, execution or evaluation. The authors thank the team from QPS Austria for their careful planning and excellent execution of the animal experiments. References [1] A. Saleh, A. Macia, A.R. Muotri, Transposable elements, inflammation, and neurological disease, Front. Neurol. 10 (2019) 894. [2] A.L. Savage, G.G. Schumann, G. Breen, V.J. Bubb, A. Al-Chalabi, J.P. Quinn, Retrotransposons in the development and progression of amyotrophic lateral sclerosis, J. Neurol. Neurosurg. Psychiatry 90 (2019) 284e293. [3] O.H. Tam, L.W. Ostrow, M. Gale Hammell, Diseases of the nERVous system: retrotransposon activity in neurodegenerative disease, Mobile DNA 10 (2019) 32. [4] J.L. Goodier, Restricting retrotransposons: a review, Mobile DNA 7 (2016) 16. [5] A. Macia, T.J. Widmann, S.R. Heras, et al., Engineered LINE-1 retrotransposition in nondividing human neurons, Genome Res. 27 (2017) 335e348. [6] M. Simon, M. Van Meter, J. Ablaeva, et al., LINE1 derepression in aged wildtype and SIRT6-deficient mice drives inflammation, Cell Metabol. 29 (2019) 871e885. [7] D. Kremer, J. Gruchot, V. Weyers, et al., pHERV-W envelope protein fuels microglial cell-dependent damage of myelinated axons in multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A. 116 (2019) 15216e15225. [8] P.A. Larsen, M.W. Lutz, K.E. Hunnicutt, M. Mihovilovic, A.M. Saunders, A.D. Yoder, A.D. Roses, The Alu neurodegeneration hypothesis: a primatespecific mechanism for neuronal transcription noise, mitochondrial dysfunction, and manifestation of neurodegenerative disease, Alzheim. Dement 13 (2017) 828e838. [9] D.C. Hancks, H.H. Kazazian Jr., Roles for retrotransposon insertions in human disease, Mobile DNA 7 (2016) 9. [10] S. Makino, R. Kaji, S. Ando, et al., Reduced neuron-specific expression of the TAF1 gene is associated with X-linked dystonia-parkinsonism, Am. J. Hum. Genet. 80 (2007) 393e406. [11] A. Johri, M.F. Beal, Mitochondrial dysfunction in neurodegenerative diseases, J. Pharmacol. Exp. Therapeut. 342 (2012) 619e630.
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