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• W2895929116 abstract "Article22 October 2018free access Source DataTransparent process A novel lysosome-to-mitochondria signaling pathway disrupted by amyloid-β oligomers Andrés Norambuena Corresponding Author [email protected] orcid.org/0000-0003-2977-1709 Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Horst Wallrabe Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Rui Cao Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Dora Bigler Wang Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Antonia Silva Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Zdenek Svindrych Department of Biology, University of Virginia, Charlottesville, VA, USA W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Ammasi Periasamy Department of Biology, University of Virginia, Charlottesville, VA, USA W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Song Hu Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Rudolph E Tanzi Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Doo Yeon Kim Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author George S Bloom Corresponding Author [email protected] orcid.org/0000-0003-4781-7627 Department of Biology, University of Virginia, Charlottesville, VA, USA Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Department of Neuroscience, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Andrés Norambuena Corresponding Author [email protected] orcid.org/0000-0003-2977-1709 Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Horst Wallrabe Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Rui Cao Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Dora Bigler Wang Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Antonia Silva Department of Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Zdenek Svindrych Department of Biology, University of Virginia, Charlottesville, VA, USA W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Ammasi Periasamy Department of Biology, University of Virginia, Charlottesville, VA, USA W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Song Hu Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Rudolph E Tanzi Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Doo Yeon Kim Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author George S Bloom Corresponding Author [email protected] orcid.org/0000-0003-4781-7627 Department of Biology, University of Virginia, Charlottesville, VA, USA Department of Cell Biology, University of Virginia, Charlottesville, VA, USA Department of Neuroscience, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Author Information Andrés Norambuena *,1, Horst Wallrabe1, Rui Cao2, Dora Bigler Wang1, Antonia Silva1, Zdenek Svindrych1,3, Ammasi Periasamy1,3, Song Hu2, Rudolph E Tanzi4, Doo Yeon Kim4 and George S Bloom *,1,5,6 1Department of Biology, University of Virginia, Charlottesville, VA, USA 2Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA 3W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA 4Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA 5Department of Cell Biology, University of Virginia, Charlottesville, VA, USA 6Department of Neuroscience, University of Virginia, Charlottesville, VA, USA *Corresponding author. Tel: +1 434 982 5809; E-mail: [email protected] *Corresponding author. Tel: +1 434 243 3543; E-mail: [email protected] EMBO J (2018)37:e100241https://doi.org/10.15252/embj.2018100241 See also: JC Polanco & J Götz (November 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two-photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-β oligomers, which are precursors of amyloid plaques in Alzheimer's disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient-induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimer's disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles. Synopsis Nutrient-induced Mitochondrial Activation (NiMA) stimulates mitochondrial DNA synthesis through lysosomal mTORC1 and is blocked by amyloid-β oligomers in tau-dependent manner. This provides new insight into how impaired energy metabolism and mitochondrial activity contribute to the pathogenesis of Alzheimer's disease. Activation of lysosome-associated mTORC1 stimulates mitochondrial activity. NiMA is independent of mTORC1-mediated mRNA translation and mTOR association with mitochondria. Amyloid-β oligomers (AβOs) block NiMA in a soluble tau-dependent manner. AβO-mediated inhibition of NiMA is prevented by forcing mTORC1 localization or activity to lysosomes. NiMA is disrupted in fibroblasts from tuberous sclerosis patients. Introduction Mitochondria play a critical role in cellular physiology by producing ATP, and regulating lipid and calcium homeostasis, clearance of reactive oxygen species (ROS), cell death, and other processes (Nunnari & Suomalainen, 2012). These phenomena require coordinating cellular energy demands with nutrient availability, and with mitochondrial biogenesis and degradation. Not surprisingly, mitochondrial malfunctioning abounds in type 2 diabetes (De Felice & Ferreira, 2014), cancer (Vyas et al, 2016), and neurodegenerative disorders, like Alzheimer's disease (AD; DuBoff et al, 2013; Burté et al, 2014). While mitochondrial impairment is a well-known feature of AD neurons, studying mitochondrial activity in live cells and animals has been a challenge due to limitations in imaging dynamic metabolic processes in real time. There is evidence that amyloid-β oligomers (AβOs), which help initiate AD pathogenesis and are the soluble building blocks of the poorly soluble amyloid plaques that accumulate in AD brain, suppress mitochondrial fast axonal transport (Pigino et al, 2009), cause fragmentation of neuronal mitochondria (Wang et al, 2009), and directly affect mitochondrial dynamics at synapses by interacting with mitochondrial proteins (Manczak et al, 2006; Hansson Petersen et al, 2008). Together, these effects of AβOs lead to mitochondrial mislocalization, disrupted ATP production, impaired ROS clearance, excess cytoplasmic calcium, and oxidative stress. AβO-induced mitochondrial dysfunction thereby contributes to progressive loss of synaptic activity and neuron death, which together account for the behavioral deficits associated with AD (Wang et al, 2009; Calkins et al, 2011). mTORC1 is a multi-subunit protein complex that includes the serine–threonine protein kinase, mTOR, which also functions as the catalytic subunit of a related multi-protein complex, mTORC2. Together, mTORC1 and mTORC2 respond to cell surface receptors and transporters that detect insulin, growth factors, and nutrients, like amino acids (Saxton & Sabatini, 2017). The two mTOR complexes coordinately regulate fundamental cellular responses, such as protein synthesis, cell growth, cell cycle progression, and autophagy. mTOR kinase activity is elevated in AD brain (Oddo, 2012), and increasing or decreasing mTOR activity in AD model mice, respectively, worsens or ameliorates AD-like pathology and behavioral deficits (Caccamo et al, 2010; Caccamo et al, 2013). Although mTOR complexes and their regulators have been found in various subcellular compartments, including the plasma membrane (PM), nucleus, lysosomes, the Golgi complex, and peroxisomes (Betz & Hall, 2013; Arias et al, 2015; Ebner et al, 2017; Saxton & Sabatini, 2017), how the subcellular distribution of the mTOR complexes affects specific cellular responses is poorly understood. We recently identified an mTOR-dependent signaling network that leads to neuronal death in AD (Norambuena et al, 2017). We showed that AβOs activate mTORC1 at the PM in neurons, which leads to cell cycle re-entry (CCR), a frequent prelude to the death of neurons (Arendt et al, 2010; Norambuena et al, 2017), and that this effect of AβOs can be blocked by insulin and nutrients, like amino acids (Norambuena et al, 2017). These effects of AβOs on neuronal mTORC1 reinforce previous observations linking metabolic disorder to insulin resistance and AD pathogenesis (De Felice & Ferreira, 2014) and prompted us to test whether mTORC1 dysregulation contributes to mitochondrial dysfunction in AD. To that end, we adapted a two-photon fluorescence lifetime (2P-FLIM) assay for label-free imaging of mitochondrial activity in live cells by measuring fluorescence lifetimes of the fractions of free (a1%) and enzyme-bound (a2%) mitochondrial coenzymes, NADH and NADPH (Lakowicz, 2006; Alam et al, 2017; Wallrabe et al, 2018). Using this assay, along with multi-parametric photoacoustic microscopy (MP-PAM; Ning et al, 2015), we show that activation of lysosome-associated mTORC1 by insulin or amino acids in cultured neurons triggers rapid, Nutrient-induced Mitochondrial Activity (NiMA) and increases oxygen consumption in live mouse brain. NiMA involves neither mTORC1 association with mitochondria nor mTORC1-mediated regulation of mRNA translation or protein synthesis. Importantly, NiMA is strongly inhibited by AβO-induced activation of mTORC1 at the PM by a mechanism dependent on soluble tau, the precursor protein of the poorly soluble neurofibrillary tangles that form in AD brain. We also found NiMA to be defective in fibroblasts derived from patients with tuberous sclerosis, a genetic disorder that causes proliferation of benign tumors in brain and other organs because of dysregulated lysosomal mTORC1. Together, these results emphasize the critical importance of normal mTOR signaling and NiMA for neuronal health, and how their breakdown can serve as a seminal step in AD pathogenesis. Results Insulin and amino acids trigger mitochondrial activity in vitro and in vivo NADH and NADPH exhibit low-intensity, spectrally indistinguishable fluorescence that can be readily visualized in cells, including neurons, as small puncta that co-localize extensively with mitochondria (Fig 1A). The fluorescence lifetimes of these coenzymes are known to increase several-fold, from 0.3–0.8 ns to 1.0–6.5 ns, upon binding to enzyme partners, which signifies increased ATP production by NADH, and stimulation of various biosynthetic pathways and antioxidant activity for NADPH (Blacker et al, 2014). To analyze mitochondrial activity in live neurons, we used 2P-FLIM to monitor NAD(P)H lifetimes and their bound fraction in perikarya of wild-type (WT) mouse cortical neurons and human neurons differentiated from neuronal precursor cells (Fig 1A–D). Following serum starvation in Hank's balanced salt solution for 2 hours, individual fields of view were assayed by 2P-FLIM before and 30 min after stimulation of the cells with either insulin or a mixture of the amino acids, arginine and lysine (R + L). Both insulin and the amino acids induced an ~30% increase in enzyme-bound NAD(P)H (a2%), signifying a rise in mitochondrial activity (Fig 1C and D; left and middle panels). Figure 1. 2P-FLIM reveals nutrient control of neuronal mitochondrial activity A. The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by MitoTracker CMXRos). B. 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%). C, D. a2% values (starved, and after insulin or R + L addition) were recorded pixel by pixel for mouse cortical (C) and human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2–15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63 and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests. Download figure Download PowerPoint Although the excitation and emission of NADH and NADPH cannot be spectrally separated, their distinct fluorescence lifetimes enable them to be discriminated from each other by 2P-FLIM (Blacker et al, 2014). Accordingly, we determined the relative contributions of each coenzyme to total measured fluorescence lifetimes before and after insulin or R + L stimulation, and found that on average NADH accounted for 63% of the observed fluorescence lifetime signals, while the remaining 37% was due to NADPH. A slight, but significant decrease in the NADH/NADPH ratio was observed after addition of R + L, but not after insulin addition (Fig 1C and D; right panels). Thus, the NiMA response is dominated by NADH signals and predominantly reflects mitochondrial oxidative pathways. As insulin and R + L trigger lysosomal mTORC1 catalytic activity (Saxton & Sabatini, 2017), we challenged NiMA under conditions in which mTORC1 activity was inhibited by Torin1 (Liu et al, 2010) or by shRNA-mediated knockdown of Raptor, an essential mTORC1 subunit. Robust NiMA was observed 30 minutes after addition of insulin or R + L to starved mouse cortical or human neurons, and then Torin1 was added. Thirty minutes later, the enzyme-bound NAD(P)H (a2%) dropped to levels below those observed for starved cells prior to insulin or R + L addition (Fig 2A and B). Likewise, shRNA knockdown of Raptor in mouse cortical neurons blocked insulin-induced NiMA (Fig 2C). Together, these results indicate that NiMA is mTORC1-dependent. Figure 2. Nutrient-mediated regulation of mitochondrial activity in cultured neurons depends on mTORC1 A, B. Wild-type (WT) mouse cortical neurons cultured for 10 days in vitro (A) and human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 h. Next, the cells were imaged immediately (starved), after which either amino acids (R + L) or insulin was added. Thirty minutes later, the cells were imaged again. Finally, the mTOR inhibitor, Torin1, was added, and after an additional 30 min, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from five fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test. C. Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from four fields of view of a single replicate out of a total of three replicates. Western blots are representative of three independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test. Source data are available online for this figure. Source Data for Figure 2 [embj2018100241-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint To evaluate the NiMA-regulated, mitochondrial oxidative pathways in greater detail, we measured nutrient-induced changes in reactive oxygen species (ROS), ATP production, and oxygen consumption (Suomalainen & Battersby, 2018). The mitochondrial superoxide radical fluorescence sensor, MitoSox Red, indicated an ~20% increase in mitochondrial ROS 1 hour after insulin or R + L stimulation of cultured human (Fig 3A) and mouse (Fig EV1A) neurons. Surprisingly, total cellular ATP levels decreased by ~40% under these conditions (Figs 3A and EV1B), implying that ATP consumption exceeded production after nutrient stimulation. This change paralleled an ~10% increase in extracellular oxygen consumption (Fig 3A). No changes in the mitochondrial membrane potential were observed during the time-course of these experiments, as measured by tetramethylrhodamine methyl ester (TRME) fluorescence (Fig EV1C). Figure 3. Lysosomal mTORC1 regulates mitochondrial activity in cultured neurons and in brain Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 h. Next, amino acids (R + L), insulin, insulin + R + L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later, cellular reactive oxygen species (ROS), ATP levels, and oxygen consumption were measured. The data were collected from three independent assays in which each experimental condition contained six to eight replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test. MP-PAM imaging of wild-type (WT) mouse cerebral cortex through an open-skull window 1 h after topical application of amino acids (R + L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from three mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m. MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 min after suppression of mTORC1 activity by a single topical application of 1 μM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R + L was applied to the open-skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from four mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R + L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Nutrients control neuronal mitochondrial activity A, B. WT mouse cortical neurons were serum-starved in Hank's balanced salt solution for 2 h. Next, amino acids (R + L), insulin, or the oxidative phosphorylation inhibitor, NaN3, was added, and 1 h later, cellular ROS and ATP levels were measured. Error bars represent mean ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test and are representative of three independent assays. C. In otherwise identical experiments, the mitochondrial membrane potential indicator, tetramethylrhodamine ethyl ester (TMRE) was added 1 hour after the amino acids or insulin, or 20 min after the oxidative phosphorylation inhibitor, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added. Thirty minutes later, the cultures were washed to remove unbound TMRE, and cellular TMRE fluorescence was then imaged and quantified. Error bars represent mean ± s.e.m. Download figure Download PowerPoint We next sought evidence for NiMA in vivo by using MP-PAM (Cao et al, 2017) to measure oxygen metabolic responses in live mouse brain. MP-PAM imaging through a cranial window enables simultaneous quantification of hemoglobin concentration (CHb), oxygen saturation (sO2), and cerebral blood flow (CBF; Ning et al, 2015). This approach revealed significant decreases in both arterial and venous sO2, but no significant change in CBF in response to the R + L treatment (Fig 3B). These hemodynamic responses resulted in increased oxygen extraction fraction (OEF), but no significant rise in cerebral metabolic rate of oxygen (CMRO2; Fig 3B). Pre-application of the mTORC1 inhibitor, rapamycin, prevented all of the statistically significant effects of R+L on MP-PAM-measurable parameters (Fig 3C), indicating that mTORC1 controls NiMA not only in vitro, but in vivo as well. NiMA and mRNA translation are independently regulated by lysosomal mTORC1 Because mTORC1 protein kinase activity is associated with multiple subcellular compartments, most notably lysosomes and the PM (Saci et al, 2011; Betz & Hall, 2013; Menon et al, 2014; Saxton & Sabatini, 2017), we sought to determine where the mTORC1 that regulates NiMA is localized. Accordingly, we forced mTORC1 to accumulate preferentially on lysosomes or PM by lentiviral expression of Flag-tagged Raptor fused either to a lysosome-targeting region of Rheb (Flag-Raptor-Rheb15) or the PM-targeting region of H-Ras (Flag-Raptor-H-Ras25; Sancak et al, 2010). NiMA was stimulated by insulin by ~30% in non-transduced neurons and neurons expressing Flag-Raptor-Rheb15, but was insensitive to insulin in neurons expressing Flag-Raptor-H-Ras25 (Fig 4A). These observations implicate lysosomes as the mTORC1 activation site that leads to NiMA. Figure 4. Lysosomal, but not mitochondrial mTORC1 controls NiMA Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 h and then imaged by 2P-FLIM before and 30 min after addition of insulin. Each colored line refers to a single field of view containing 5–15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by Western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of three independent assays. Mouse cortical neurons starved in Hank's balanced salt solution for 2 h were treated with AβOs, insulin, or amino acids (R + L) for 30 min, and then were fixed and double-labeled with anti-mTOR, and either anti-LAMP1 or MitoTracker CMXRos. Error bars represent ± s.e.m. Data are representative of three independent assays. Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of three independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test. Source data are available online for this figure. Source Data for Figure 4 [embj2018100241-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint There is also evidence that mTOR controls mitochondrial activity by direct binding to the mitochondrial protein, Bcl-xL (Ramanathan & Schreiber, 2009), and by regulation of mitochondrial biogenesis and function through a translationally controlled mechanism (Morita et al, 2013). To test whether NiMA entails mTOR interactions with mitochondria in cultured mouse cortical neurons, we first analyzed nutrient-induced changes in the co-localization of mTOR with the mitochondrial marker, MitoTracker CMXRos, and with the lysosomal marker, LAMP1. In agreement with others (Menon et al, 2014) and our previous work (Norambuena et al, 2017), we found that insulin or R + L causes ~15–30% increases in the level of lysosomal mTOR. In contrast, nutrients did not induce recruitment of mTOR to mitochondria (Fig 4B). Next, we used 2P-FLIM to analyze NiMA in mouse neurons depleted of Bcl-xL by shRNA. As shown in Fig 4C, the mitochondrial responses to either insulin or R + L were unaffected by Bcl-xL knockdown. These findings imply that NiMA does not require any of the known direct interactions of mTORC1 with mitochondria. One of the main functions of mTORC1 is the regulation of mRNA translation. This process involves mTORC1-mediated control of two main downstream targets, the 70 kDa S6 ribosomal protein kinase (S6K) (Saxton & Sabatini, 2017) and the translation regulator, eukaryotic initiation factor 4E (eIF4E), whose activity is inhibited by its binding to 4EBPs, which in turn are regulated by mTORC1 phosphorylation (Ma & Blenis, 2009). As the 4EBP1-eIF4E interaction has been shown to control mitochondrial biogenesis (Morita et al, 2013), we sought to explore whether mRNA translational functions of mTORC1 are upstream of NiMA. We found that NiMA is unaffected by either antisense-mediated reduction of S6K or eIF4E (Fig EV2A), or pharmacological inhibition of S6K (Fig EV2B). Furthermore, exposure of neurons to AβOs, insulin, or R + L did not cause detectable changes in 35S-methionine incorporation into newly synthesized proteins (Fig EV2C). We thus conclude that NiMA and protein synthesis (Morita et al, 2013) are regulated independently by mTORC1. Click here to expand this figure. Figure EV2. NiMA occurs independently of mTOR-mediated regulation of transcription and translation Reducing expression in cultured mouse neurons of eIF4E (upper panel) or S6K (lower panel), which respectively regulate mRNA and protein synthesis, did not affect NiMA. Average data from three fields of view per condition are shown. Western blots are representative of three independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test. Pharmacological inhibition of S6K with PF-4708671 did not affect NiMA. Each colored line refers to a single field of view containing 2–15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test. NiMA does not obviously change protein synthesis in WT mouse cortical neurons (10 days in vitro), as judged by SDS gel autoradiography. The autoradiogram image is representative of two independent experiments. Source data are available online for this figure. Download figure Download PowerPoint NiMA regulates mitochondrial DNA replication Normal mitochondrial functioning depends on coordinated expression of > 1,000 nuclear-encoded genes and those encoded by mitochondrial DNA (mtDNA; Nunnari & Suomalainen, 2012; Suomalainen & Battersby, 2018). As defects in mtDNA maintenance have been linked to aging and neurodegeneration (Trifunovic et al, 2004; Manczak et al, 2011; Nunnari & Suomalainen, 2012), we investigated NiMA's role in mtDNA maintenance. To visualize nucleoids (Lewis et al, 2016), which correspond to mtDNA–protein complexes representing mitochondrial inheritance units (Garrido et al, 2003), we incubated live mouse neurons with 5-ethyl-2′-deoxyuridine (EdU) for 3 h in the absence or presence of a mixture of insulin and R + L (I/R/L). EdU is a thymidine analog that readily incorporates into DNA during active DNA synthesis. EdU can be detected by a copper-catalyzed reaction that covalently links (“clicks”) the alkyne group in EdU to the picolyl azide group in an Alex" @default.
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• W2895929116 title "A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers" @default.
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