Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

Resource19 August 2021Open Access Source DataTransparent process Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease Sonja Blumenstock Sonja Blumenstock orcid.org/0000-0002-8124-3295 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany These authors contributed equally to this work Search for more papers by this author Elena Katharina Schulz-Trieglaff Elena Katharina Schulz-Trieglaff Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany These authors contributed equally to this work Search for more papers by this author Kerstin Voelkl Kerstin Voelkl orcid.org/0000-0002-4182-0764 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Anna-Lena Bolender Anna-Lena Bolender Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Paul Lapios Paul Lapios orcid.org/0000-0001-9251-1374 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Jana Lindner Jana Lindner Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Mark S Hipp Mark S Hipp orcid.org/0000-0002-0497-3016 Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Search for more papers by this author F Ulrich Hartl F Ulrich Hartl orcid.org/0000-0002-7941-135X Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Rüdiger Klein Rüdiger Klein orcid.org/0000-0002-3109-0163 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Irina Dudanova Corresponding Author Irina Dudanova [email protected] orcid.org/0000-0003-1052-8485 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Sonja Blumenstock Sonja Blumenstock orcid.org/0000-0002-8124-3295 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany These authors contributed equally to this work Search for more papers by this author Elena Katharina Schulz-Trieglaff Elena Katharina Schulz-Trieglaff Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany These authors contributed equally to this work Search for more papers by this author Kerstin Voelkl Kerstin Voelkl orcid.org/0000-0002-4182-0764 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Anna-Lena Bolender Anna-Lena Bolender Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Paul Lapios Paul Lapios orcid.org/0000-0001-9251-1374 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Jana Lindner Jana Lindner Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Mark S Hipp Mark S Hipp orcid.org/0000-0002-0497-3016 Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Search for more papers by this author F Ulrich Hartl F Ulrich Hartl orcid.org/0000-0002-7941-135X Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Rüdiger Klein Rüdiger Klein orcid.org/0000-0002-3109-0163 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Irina Dudanova Corresponding Author Irina Dudanova [email protected] orcid.org/0000-0003-1052-8485 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Author Information Sonja Blumenstock1,2, Elena Katharina Schulz-Trieglaff1, Kerstin Voelkl1,2, Anna-Lena Bolender1,2, Paul Lapios1,2, Jana Lindner1, Mark S Hipp3,4,5, F Ulrich Hartl3, Rüdiger Klein1 and Irina Dudanova *,1,2 1Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany 2Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, Martinsried, Germany 3Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany 4Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany *Corresponding author. Tel: +49 89 85783542; E-mail: [email protected] The EMBO Journal (2021)40:e107260https://doi.org/10.15252/embj.2020107260 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 cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases. SYNOPSIS This study describes a new reporter mouse line for monitoring neuronal proteostasis. The reporter reveals that protein aggregates associated with neurodegenerative diseases differ in their impact on the cellular protein quality control system. Fluc-EGFP reporter mouse allows studying neuronal proteostasis alterations in aging and disease. Fluc-EGFP reporter detects proteostasis impairments in tauopathy mice, but not in Huntington's disease mice. Mechanistic studies in cultured neurons show that different aggregating proteins cause distinct cellular compartment-specific defects of proteostasis. Introduction Maintaining the integrity of the cellular proteome is essential for survival. The cellular protein quality control system safeguards protein homeostasis (proteostasis) by ensuring correct folding of new proteins, detecting and refolding damaged proteins, and targeting terminally misfolded proteins for degradation (Balchin et al, 2016; Klaips et al, 2018). Age-dependent decline in protein quality control is believed to play a crucial role in neurodegenerative diseases, a group of brain disorders characterized by aggregation of misfolded proteins and neuronal cell death, such as Alzheimer's, Parkinson's, and Huntington's disease (HD; Soto & Pritzkow, 2018). Enhancing the capacity of the protein quality control system has therefore emerged as a promising therapeutic strategy for neurodegenerative proteinopathies (Smith et al, 2015; Klaips et al, 2018). However, our current knowledge about the proteostasis changes in vivo during disease progression is still scarce. While attempts to ameliorate aggregate toxicity by upregulating chaperones have been successful in cell culture, fly, and worm models (Carmichael et al, 2000; Auluck et al, 2002; Outeiro et al, 2006; Hageman et al, 2010; Vos et al, 2010; Wu et al, 2010; Kuo et al, 2013), they have produced less satisfactory results in mammalian models (Hansson et al, 2003; Liu et al, 2005; Zourlidou et al, 2007; Krishnan et al, 2008; Sharp et al, 2008; Shimshek et al, 2010; Labbadia et al, 2012; Xu et al, 2015). Reliable genetic reporters that allow monitoring the status of cellular proteostasis in vivo are essential for understanding disease mechanisms and for assessing the efficacy of potential treatments targeting the protein quality control system. Thus, transgenic mice expressing ubiquitin-proteasome system (UPS) reporters have been used successfully for investigating protein degradation in disease models (Lindsten et al, 2003; Kristiansen et al, 2007; Bett et al, 2009; Cheroni et al, 2009; Ortega et al, 2010; Myeku et al, 2016). However, tools for monitoring proteostasis in general are still lacking. Wild-type and mutated versions of the conformationally unstable firefly luciferase (Fluc) protein fused to EGFP are ideal for use as proteostasis sensors and have proven valuable in cell lines and in C. elegans (Gupta et al, 2011; Donnelly et al, 2014). These sensors depend on cellular chaperones for proper folding and enzymatic activity. Proteotoxic conditions that overload the protein quality control system lead to misfolding of Fluc-EGFP, which can be revealed by two readouts: decrease in bioluminescence due to decline in luciferase activity and formation of Fluc-EGFP foci in the cell as a result of decreased solubility of the misfolded sensor (Gupta et al, 2011). To gain a deeper understanding of proteostasis changes in aging and disease, we generated new reporter mice expressing Fluc-EGFP in the nervous system. Using these mice, we reveal unexpected differences in proteostasis alterations caused by different types of protein aggregates. Results Fluc-EGFP sensor reacts to proteostasis changes in primary neurons We first asked whether Fluc-EGFP variants can be used as reporters of proteotoxic stress in primary neurons. In the following experiments, we used two versions of Fluc, wild-type (FlucWT) and single mutant FlucR188Q (FlucSM). FlucSM is a conformationally destabilized mutant that was previously shown to have higher sensitivity to proteotoxic stress than FlucWT (Gupta et al, 2011), however, its expression levels in neurons were relatively low, possibly due to efficient degradation. Transfection of Fluc-EGFP constructs did not lead to toxicity in murine primary cortical cultures, as demonstrated by immunostaining against the apoptotic marker cleaved caspase-3 (Fig EV1A and B). Cultures transfected with Fluc-EGFP constructs were subjected to several treatments to induce proteotoxic stress. MG-132 was used to inhibit protein degradation by the proteasome, Bafilomycin A1 was used to inhibit autophagy, and 17-AAG was used to inhibit Hsp90, a major cytosolic chaperone (Appendix Fig S1A–C). All these treatments induced a change in the distribution of Fluc-EGFP, which typically formed several small compact foci in the perinuclear region, while the intensity of diffuse EGFP fluorescence in the rest of the cytoplasm decreased (Figs 1A and B, and Fig EV1C). In addition, cells were subjected to heat shock at 43°C, a treatment known to induce proteotoxic stress (Nishimura et al, 1991; Yang et al, 2008; Morimoto, 2011). Heat shock led to an even stronger Fluc-EGFP response, with many cells showing a complete loss of diffuse cytoplasmic EGFP fluorescence and formation of multiple Fluc-EGFP foci throughout the cytoplasm (Figs 1A and B, and Fig EV1C). Click here to expand this figure. Figure EV1. Additional experiments with Fluc-EGFP in primary neurons Examples of neurons expressing FlucSM-EGFP (green) that are negative (left) or positive (right) for cleaved caspase-3 (magenta). Nuclei are labeled with DAPI (blue). Quantification of the fraction of transfected neurons positive for cleaved caspase-3 at DIV 3 + 2. N = 3 independent experiments; one-way ANOVA. No significant differences were observed. Quantification of FlucWT-EGFP foci formation in transfected neurons upon indicated treatments. N = 3–4 independent experiments; two-tailed t-test. DIV 3 + 2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q25-mCherry (magenta) and treated with 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cultured neurons were stained for MAP2 (gray) as a neuronal marker, and nuclei were labeled with DAPI (blue). Data information: Error bars represent SD. Significance: *P < 0.05; ***P < 0.001. Scale bars: A, 20 µm; D, 5 µm. Download figure Download PowerPoint Figure 1. Fluc-EGFP reacts to proteostasis changes in primary neurons Representative images of DIV 3 + 2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 h; 10 nM Bafilomycin A1 (BafA1) for 24 h; 0.5 µM 17-AAG for 4 h; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes. Quantification of FlucSM-EGFP foci formation in transfected neurons. N = 4 biological replicates for BafA1 and corresponding control group, 3 biological replicates for all the other conditions; two-tailed t-test. Left, quantification of specific luciferase activity of FlucSM-EGFP upon indicated treatments, normalized to respective vehicle-treated controls. N = 3 biological replicates; one-sample t-test. Right, representative Western blot of neuronal lysates for the indicated conditions. Tubulin was used as a loading control. DIV 3 + 2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (gray) as a neuronal marker, and nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig EV1D. Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N = 4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **P = 0.0046; 4-PBA, n.s.; HTT × 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. Data information: Error bars represent SD. Significance: *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: A and D, 5 µm; insets in A, 2 µm. Source data are available online for this figure. Source Data for Figure 1 [embj2020107260-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint We used the luciferase assay to evaluate the enzymatic activity of Fluc-EGFP as an additional readout of proteostasis alterations. Throughout the study, luciferase activity measurements were normalized to Fluc-EGFP protein quantity determined by Western blot to obtain specific activity values. As expected, proteasome inhibition with MG-132 and Hsp90 inhibition with 17-AAG both resulted in a significant decrease in specific luciferase activity of FlucSM-EGFP by ∼65% and ∼85%, respectively (Fig 1C). Overall, the response of Fluc-EGFP to heat shock and small-molecule inhibitors in primary neurons appeared stronger than the response observed in non-neuronal cell lines (Gupta et al, 2011), probably due to the high sensitivity of neurons to proteotoxic stress. We next asked whether Fluc-EGFP reacts to the proteostasis dysbalance caused by an aggregating protein. To this end, we co-transfected primary neurons with Fluc-EGFP and the pathologically expanded form of mutant Huntingtin (mHTT)-exon1 (HTT-Q97-mCherry). mHTT-exon1 is a key pathogenic version of the protein that is sufficient to recapitulate HD phenotypes (Mangiarini et al, 1996; Sathasivam et al, 2013; Yang et al, 2020). Co-transfection with HTT-Q97-mCherry resulted in a significant increase in Fluc-EGFP foci compared to control cells co-transfected with HTT-Q25-Cherry (Figs 1D and E, and Fig EV1D). To ensure that the changes in Fluc-EGFP solubility were proteostasis-dependent, we treated the cultures with the chemical chaperone 4-phenylbutyrate (4-PBA). 4-PBA has been shown to counteract protein misfolding and aggregation in several proteinopathy models (Yam et al, 2007; Wiley et al, 2011; Winter et al, 2014; Hirata et al, 2020). The frequency of Fluc-EGFP foci in HTT-Q97-mCherry cells treated with 4-PBA was not significantly different from that in control HTT-Q25-mCherry cells (Fig 1D and E), suggesting that formation of Fluc-EGFP foci is indeed due to protein misfolding. Taken together, these results demonstrate that Fluc-EGFP can be reliably used to detect proteostasis disturbances in primary neurons. Fluc-EGFP reporter mouse for in vivo analysis of proteostasis For in vivo studies of the protein quality control system in mouse models, we generated transgenic mouse lines expressing FlucWT-EGFP or FlucSM-EGFP under the control of the prion protein (PrP) promoter (Fig 2A). In line with our observations in primary neurons, FlucSM-EGFP mouse lines showed rather low expression of the sensor. For further experiments, we selected the FlucWT-EGFP line 1,214 (from here on, Fluc-EGFP mice), which had a broad expression of the transgene throughout the brain, including regions affected in neurodegenerative proteinopathies. In particular, stronger expression was detected in the neocortex and hippocampus, while lower levels were observed in the basal ganglia and cerebellum (Fig 2A and B). Co-staining with cell type markers demonstrated that Fluc-EGFP was present in Neurotrace+ neurons, while it was not detectable in GFAP+ astrocytes, APC+ oligodendrocytes, or Iba+ microglia (Fig EV2A and Appendix Fig S2A). In neurons, Fluc-EGFP showed cytoplasmic localization in the soma and dendrites (Figs 2B and EV2A, and Appendix Fig S2A). Figure 2. Fluc-EGFP reporter reveals proteostasis impairment in aging mice Scheme of the transgenic construct (top) and sagittal brain section of a Fluc-EGFP mouse from line 1,214 at 3 months of age immunostained for EGFP (bottom). Representative images of the indicated brain regions of a Fluc-EGFP mouse, stained for EGFP (green) and the neuronal marker NeuN (magenta). Insets show higher magnification of the areas indicated by the boxes. Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N = 3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test). Representative Western blot of hippocampal lysates from Fluc-EGFP mice at the indicated ages. Short bracket with an arrow indicates the high molecular weight species observed in older mice. Long bracket indicates the part of the lane that was used for Fluc protein quantification (see Materials and Methods). Several lanes on the blot between 6-month-old and 12-month-old samples were digitally removed. Fluc-EGFP specific luciferase activity measured in the indicated brain regions of Fluc-EGFP mice at the indicated ages. Values are normalized to the 3-month-old group. N = 5 Fluc-EGFP mice for each age group. One-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Hippocampus, *P = 0.0205; Cortex, **P = 0.0077; Cerebellum, n.s. Significant pairwise comparisons to the 3-month-old group are indicated on the graphs. Fluc-EGFP protein quantity measured in the indicated brain regions of Fluc-EGFP mice at the indicated ages. Values are normalized to the 3-month-old group. N = 5 Fluc-EGFP mice for each age group. One-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Hippocampus, *P = 0.0278; Cortex, n.s; Cerebellum, **P = 0.0058. Significant pairwise comparisons to the 3-month-old group are indicated on the graphs. Protein quantity normalized to total protein instead of tubulin is shown in Appendix Fig S2C. Data information: Error bars represent SD. Significance: *P < 0.05; **P < 0.01, ***P < 0.001. Scale bars: A, 2 mm; B, 20 µm. Source data are available online for this figure. Source Data for Figure 2 [embj2020107260-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Characterization of Fluc-EGFP reporter mice A. Analysis of Fluc-EGFP expression in neurons and glia. Cortical sections from 3 to 4-month-old Fluc-EGFP mice were stained against GFAP (magenta), APC (yellow), and Iba1 (white). Fluc-EGFP was detected by EGFP fluorescence (green), and neurons were labeled with Neurotrace (blue). Arrowheads point to Fluc-EGFP-positive cells. Dashed lines indicate borders of cortical layers. The experiment was repeated in N = 5 animals with similar results. Similar analysis in the hippocampus is shown in Appendix Fig S2A. B–D. Linearity of luciferase activity in tissue samples. Cortical lysates from 5 Fluc-EGFP mice (shown in shades of gray) were used in different dilutions (25, 50, 75, 100, 125 and 150 μg input protein quantity). All values were normalized to the 75 μg dilution. (B) Measured luciferase activity of the samples. (C) Measured protein quantity. (D) Linear regression of input protein quantity to specific luciferase activity (measured luciferase activity normalized to measured protein quantity) for individual mice (gray lines) and mean regression of five mice (black line); red line shows the corrected relation where 100% input protein quantity corresponds to 100% specific activity. E. Quantification of luciferase activity in Fluc-EGFP mice and non-transgenic littermates. N = 3 mice per genotype. F. Western blots of acute brain slices of indicated genotype treated with 5 µM MG-132 or vehicle (control) for 4 h. The part of the lane used for quantification of ubiquitin is indicated with a bracket (left). Total protein load (right, stain-free blot) was used for normalization. G. Quantification of increase in ubiquitinated proteins upon MG-132 treatment in brain slices from Fluc-EGFP mice and control littermates. N = 5 mice of each genotype. Green and gray asterisks indicate comparisons to the corresponding vehicle-treated control slices by one-sample t-test. Comparison between MG-132 treated slices of different genotypes was performed by two-tailed t-test. Data information: Error bars represent SD. Significance: *P < 0.05; **P < 0.01; n.s.—not significant. Scale bars in A, 30 µm. Source data are available online for this figure. Download figure Download PowerPoint Unlike in cell culture conditions, the quantity of the Fluc-EGFP protein might vary considerably between tissue samples, which could lead to a bias when using bioluminescence measurements in vivo. We therefore tested whether luciferase activity changes linearly to the Fluc-EGFP protein concentration using serial dilutions of 5 cortical tissue samples from 2-month-old Fluc-EGFP mice. We found that higher levels of Fluc-EGFP resulted in lower bioluminescence values than expected (Fig EV2B and C). The relationship between Fluc-EGFP protein quantity in the sample (x) and expected specific luciferase activity of the sample (y) was described by the formula: y = −0.45488x + 145.488 (Fig EV2D). All measurements of specific activity in tissue samples were therefore corrected accordingly. No luciferase activity was detected in non-transgenic littermate controls (Fig EV2E). As prolonged expression of an unstable protein such as Fluc might cause adaptive changes in the proteostasis system, we compared response to proteotoxic stress in Fluc-EGFP mice and wild-type littermates. Acute brain slices were treated with the proteasome inhibitor MG-132 for 4 h, and levels of ubiquitinated proteins were determined in the slice lysates by Western blot (Fig EV2F and G). Of note, we observed time-dependent decrease in Fluc-EGFP protein levels in brain slices (Appendix Fig S2B), which was more prominent in heat shock conditions (Fig 2C and Appendix Fig S2B). All experiments with slices were therefore performed keeping incubation times as short as possible, and not exceeding 4 h. The increase in ubiquitinated proteins induced by MG-132 was not different between Fluc-EGFP and control mice (Fig EV2F and G). These data suggest that Fluc-EGFP expression does not lead to a major change in the cellular proteostasis capacity in our transgenic mouse model. To ensure that Fluc-EGFP responds to proteostasis alterations also in brain tissue, we performed a proteostasis rescue experiment. Acute brain slices from Fluc-EGFP mice were pre-incubated with 4-PBA or vehicle control and then subjected to heat shock for 15 min at 43°C. In control slices, heat shock resulted in a marked reduction in Fluc-EGFP-specific luciferase activity. Importantly, 4-PBA treatment led to a significant twofold increase in specific luciferase activity of heat-shocked slices without a respective change in Fluc-EGFP protein quantity (Fig 2C), indicating that Fluc-EGFP response in brain tissue indeed reflects changes in proteostasis. In summary, we have generated a reporter mouse that can be used for monitoring neuronal proteostasis in the brain. Fluc-EGFP reporter reveals proteostasis impairment in aging mice Using Fluc-EGFP mice, we first asked whether the reporter reacts to the in vivo changes in proteostasis capacity that are believed to occur in normal aging (Brehme et al, 2014; Labbadia & Morimoto, 2015; Klaips et al, 2018). To this end, we analyzed several brain regions of Fluc-EGFP mice at different ages (3, 6, 12, and 24 months). We did not detect major changes in total Fluc-EGFP protein levels between age groups (Fig 2D and F and Appendix Fig S2C). In 12- and 24-month-old mice, a high molecular weight smear was visible on the Western blot, likely representing aggregated Fluc-EGFP species, and there was a corresponding reduction in the monomeric form (Fig 2D). Interestingly, we observed a significant age-dependent decline in specific luciferase activity in the hippocampus and cortex, but not in the cerebellum (Fig 2E). These results show that Fluc-EGFP mice can be used to monitor in vivo alterations in proteostasis and highlight intrinsic differences in the protein quality control of different brain regions. Fluc-EGFP sensor reacts to proteostasis defects in tauopathy mice To investigate how proteostasis changes in disease, we crossed Fluc-EGFP mice to the rTg4510 line, a double-transgenic tauopathy model where expression of the responder transgene containing human four-repeat tau with the familial P301L mutation (tetO-tauP301L) is controlled by the CaMKIIα-tTA activator transgene. rTg4510 mice develop neurofibrillary tangle-like pathology and memory defects at 4 months, while neuronal loss and brain atrophy do not occur until 5.5 months of age (Santacruz et al, 2005). Consistent with previous reports (Santacruz et al, 2005; Myeku et al, 2016), immunostaining for phosphorylated tau (p-tau) revealed tau pathology in the cortex and hippocampus of 4-month-old rTg4510:Fluc-EGFP mice (Figs 3A and Fig EV3A). At this time point, multiple Fluc-EGFP foci were observed in both cortex and hippocampus. While foci were