Title: Beclin‐1‐mediated activation of autophagy improves proximal and distal urea cycle disorders
Abstract: Article28 December 2020Open Access Source DataTransparent process Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders Leandro R Soria Leandro R Soria orcid.org/0000-0002-7124-856X Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Sonam Gurung Sonam Gurung UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Giulia De Sabbata Giulia De Sabbata International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Dany P Perocheau Dany P Perocheau orcid.org/0000-0001-5450-8801 UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Angela De Angelis Angela De Angelis Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Gemma Bruno Gemma Bruno Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Debora Paris Debora Paris Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Paola Cuomo Paola Cuomo Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Andrea Motta Andrea Motta Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Michael Orford Michael Orford UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Youssef Khalil Youssef Khalil orcid.org/0000-0001-9025-3017 UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Simon Eaton Simon Eaton UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Philippa B Mills Philippa B Mills UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Simon N Waddington Simon N Waddington UCL Great Ormond Street Institute of Child Health, London, UK Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Search for more papers by this author Carmine Settembre Carmine Settembre orcid.org/0000-0002-5829-8589 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Andrés F Muro Andrés F Muro orcid.org/0000-0002-9628-0494 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Julien Baruteau Julien Baruteau orcid.org/0000-0003-0582-540X UCL Great Ormond Street Institute of Child Health, London, UK Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK Search for more papers by this author Nicola Brunetti-Pierri Corresponding Author Nicola Brunetti-Pierri [email protected] orcid.org/0000-0002-6895-8819 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medicine, Federico II University, Naples, Italy Search for more papers by this author Leandro R Soria Leandro R Soria orcid.org/0000-0002-7124-856X Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Sonam Gurung Sonam Gurung UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Giulia De Sabbata Giulia De Sabbata International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Dany P Perocheau Dany P Perocheau orcid.org/0000-0001-5450-8801 UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Angela De Angelis Angela De Angelis Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Gemma Bruno Gemma Bruno Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Debora Paris Debora Paris Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Paola Cuomo Paola Cuomo Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Andrea Motta Andrea Motta Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy Search for more papers by this author Michael Orford Michael Orford UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Youssef Khalil Youssef Khalil orcid.org/0000-0001-9025-3017 UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Simon Eaton Simon Eaton UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Philippa B Mills Philippa B Mills UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Simon N Waddington Simon N Waddington UCL Great Ormond Street Institute of Child Health, London, UK Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Search for more papers by this author Carmine Settembre Carmine Settembre orcid.org/0000-0002-5829-8589 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Andrés F Muro Andrés F Muro orcid.org/0000-0002-9628-0494 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Julien Baruteau Julien Baruteau orcid.org/0000-0003-0582-540X UCL Great Ormond Street Institute of Child Health, London, UK Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK Search for more papers by this author Nicola Brunetti-Pierri Corresponding Author Nicola Brunetti-Pierri [email protected] orcid.org/0000-0002-6895-8819 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medicine, Federico II University, Naples, Italy Search for more papers by this author Author Information Leandro R Soria1, Sonam Gurung2, Giulia De Sabbata3, Dany P Perocheau2, Angela De Angelis1, Gemma Bruno1, Elena Polishchuk1, Debora Paris4, Paola Cuomo4, Andrea Motta4, Michael Orford2, Youssef Khalil2, Simon Eaton2, Philippa B Mills2, Simon N Waddington2,5, Carmine Settembre1, Andrés F Muro3, Julien Baruteau2,6 and Nicola Brunetti-Pierri *,1,7 1Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 2UCL Great Ormond Street Institute of Child Health, London, UK 3International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 4Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy 5Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa 6Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK 7Department of Translational Medicine, Federico II University, Naples, Italy *Corresponding author. Tel: +39 081 19230661; Fax: +39 081 5609877; E-mail: [email protected] EMBO Mol Med (2021)13:e13158https://doi.org/10.15252/emmm.202013158 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 Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell-penetrating autophagy-inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle. Synopsis Using mice with constitutive activation of autophagy and treating mice deficient for ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) with the autophagy inducing Tat-Beclin-1 (TB-1), this study shows that Beclin-1-dependent activation of autophagy improves the phenotypes of proximal and distal defects of the urea cycle. Beclin-1 is a central player in autophagy and a knock-in mouse model carrying a Becn1 mutation resulting in constitutively active autophagy shows enhanced ureagenesis and increased ammonia detoxification. TB-1 improves biochemical abnormalities and increases survival of mice with OTC deficiency (proximal urea cycle disorder). TB-1 improves biochemical abnormalities and survival of mice with ASL deficiency (distal urea cycle disorder). ASL deficient mice show cytoplasmic and nuclear glycogen accumulation that are both reduced by TB-1. The paper explained Problem Urea cycle disorders have high morbidity and mortality and require development of novel and more effective therapies. Ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies are the two most common urea cycle disorders. Results Mice carrying a Beclin-1 activating mutation have increased ammonia detoxification and treatment with the cell-penetrating autophagy-inducing Tat-Beclin-1 peptide improved phenotypic and biochemical abnormalities of mouse models of OTC and ASL deficiencies. Impact Drugs activating Beclin-1 have potential for therapy of UCD. Introduction Autophagy is highly active in liver. Proteins, glycogen and lipid droplets are degraded by autophagy in liver cells to release amino acids, glucose and free fatty acids that can be reused for synthesis of new proteins and macromolecules, or can enter the tricarboxylic acid (TCA) cycle to generate ATP (Kaur & Debnath, 2015). Liver autophagy was recently found to support ammonia detoxification by furnishing the urea cycle with intermediates and energy that increase urea cycle flux under conditions of excessive ammonia (Soria et al, 2018). Liver-specific deficiency of autophagy impaired ammonia detoxification whereas its enhancement resulted in increased urea synthesis and protection against hyperammonemia (Soria et al, 2018). Therefore, drugs enhancing autophagy have potential for treatment of urea cycle disorders (UCD) (Soria & Brunetti-Pierri, 2018, 2019). In a previous study (Soria et al, 2018), we showed that rapamycin reduces orotic acid in spf-ash mice, a mouse model of the ornithine transcarbamylase (OTC) deficiency that carries a single nucleotide mutation in the fourth exon of the Otc gene resulting in a splicing defect and 10% of residual enzyme activity (Hodges & Rosenberg, 1989). Although it has been efficiently used to promote autophagy, rapamycin does not completely inhibit its target, the mechanistic target of rapamycin kinase complex 1 (mTORC1), and affects several biological processes besides autophagy (Li et al, 2014). Therefore, drugs targeting autophagy more specifically are attractive because they are expected to have less side effects. Tat-Beclin-1 (TB-1) is an engineered cell-permeable peptide that potently and specifically induces autophagy (Shoji-Kawata et al, 2013). TB-1 is formed by the HIV-1 Tat protein transduction domain attached via a diglycine linker to a peptide derived from Beclin-1 (Becn1), a key component of the autophagy induction machinery (Shoji-Kawata et al, 2013). In summary, TB-1 is an attractive therapeutic candidate for its specificity and at least in mice, it has shown great potential for treatment of various diseases, including several types of cancer, infections, cardiac dysfunction, skeletal disorders and axonal injuries (Cinque et al, 2015; He et al, 2016; Pietrocola et al, 2016; Bartolomeo et al, 2017; Song et al, 2018; Sun et al, 2018; Vega-Rubin-de-Celis et al, 2018). In the present study, we investigated the therapeutic potential of TB-1 for treatment of UCD. Results Constitutional hyperactivation of Beclin-1 enhances ammonia detoxification Beclin-1 is a central player in autophagy and regulates autophagosome formation and maturation (Liang et al, 2008). To investigate Becn1 functions in vivo, a knock-in mouse model carrying a Becn1 mutation (Becn1F121A) resulting in constitutively active autophagy has been recently generated (Rocchi et al, 2017). In these mice, Phe121 is mutated into alanine resulting in disruption of the BECN1-BCL2 binding and constitutive activation of BECN1 and autophagy in multiple tissues, including liver (Rocchi et al, 2017; Fernandez et al, 2018; Yamamoto et al, 2018). In these mice, we investigated ammonia detoxification by measurements of blood ammonia levels during acute hyperammonemia induced by an ammonia challenge. Despite no changes in blood ammonia at baseline, Becn1F121A mice showed 32% reduction in blood ammonia at 30 min after intraperitoneal (i.p.) injection of ammonium chloride compared to age-matched wild-type (WT) mice (Fig 1A). Accordingly, Becn1F121A mice showed enhanced ureagenesis compared to WT controls, as shown by increased blood levels of 15N-labelled urea from 15N-ammonium chloride (Fig EV1A). Improved ammonia clearance was not dependent on increased expression of urea cycle enzymes in Becn1F121A mice that showed similar enzyme levels by Western blotting compared to WT controls (Fig EV1B and C). Therefore, consistent with previous findings (Soria et al, 2018), gain-of-function mutation of the autophagy activator Becn1 protects against acute hyperammonemia in vivo, suggesting that Beclin-1 is a druggable candidate for therapy of hyperammonemia. Figure 1. Hyperactive Beclin-1 protects against hyperammonemia, and activation of hepatic autophagy improves the phenotype of OTC-deficient mice A. Blood ammonia in 8–9-week-old C57BL/6J wild-type (WT) mice (n = 8) and Becn1F121A mice with constitutive activation of autophagy (n = 8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **P < 0.01 (Unpaired t-test). ns: not statistically significant difference. B. Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n = 5 mice/group). *P < 0.05 (Two-way ANOVA). C, D. Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after 10 days of treatment with TB-1 or vehicle. β-actin was used as loading control. n = 5 mice/group. **P < 0.01, *P < 0.05 (Unpaired t-test). E. Survival curves of spf-ash mice fed with a high protein diet (HPD) for 10 days and treated with TB-1 alone or combined with scavenger drug (Na-benzoate) and l-Arginine, or treated with scavenger drug (Na-benzoate) and l-Arginine, or left untreated. WT control were included (n = 5/group). ***P < 0.001 (Log-rank Mantel–Cox test). F. Blood ammonia levels determined after 4 days under HPD (n = 5 mice/group); ***P < 0.001, **P < 0.01, *P < 0.05 (One-way ANOVA). Data information: Treatments in (E, F): Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and l-arginine (l-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± SEM. Exact P values are reported in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 1 [emmm202013158-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Ureagenesis in Becn1F121A mice 15N-labelled urea in blood at 5, 15 and 30 min after i.p. injection of 15NH4Cl tracer (10 mmol/kg) in 8–13-week-old wild-type (WT) and Becn1F121A mice. The number between parentheses indicates the number of mice per time-point. Two-way ANOVA analysis showed a significant difference between the two mice genotypes. Western blot analyses of urea cycle enzymes (NAGS, CPS1, OTC, ASS1, ASL, and ARG1) in livers of WT and Becn1F121A mice. β-actin was used as loading control: upper β-actin blot for CPS1, OTC and ASS1; lower β-actin blot for NAGS, ASL and ARG1. Densitometric quantifications (n = 4 mice/group) (Unpaired t-test). Data information: All values are shown as averages ± SEM. ns: not statistically significant difference. Source data are available online for this figure. Download figure Download PowerPoint Tat-Beclin-1 improves the phenotype of OTC-deficient mice To investigate the therapeutic efficacy of Becn1-mediated induction of autophagy in mouse models of UCD, we injected TB-1 i.p. in spf-ash mice (Hodges & Rosenberg, 1989), a model of OTC deficiency, the most common UCD. Body weights were unaffected by TB-1 (Fig EV2A). Although not normalized, in spf-ash mice the levels of the biochemical hallmark of OTC deficiency, urinary orotic acid, were significantly reduced by TB-1 (Fig 1B). Consistent with its autophagy enhancer activity, TB-1 increased the hepatic autophagic flux, as showed by reduced protein levels of the autophagosome marker LC3-II, and the two main autophagy cargo receptors, namely p62 and NBR1 (Fig 1C and D). Notably, OTC residual enzyme activity was unaffected by TB-1 (Fig EV2B), thus excluding reduction of urinary orotic acid as a consequence of increased residual OTC activity induced by TB-1. To further investigate the efficacy of TB-1-mediated increased liver autophagy for therapy of OTC deficiency, spf-ash mice were fed for 10 days with a high protein diet and were either treated with TB-1 or left untreated. Consistent with previous reports (Yang et al, 2016; Kurtz et al, 2019), spf-ash mice showed marked mortality under high protein diet compared to control WT mice (Fig 1E). An approximately 30% weight loss was observed in all spf-ash mice fed with the high protein diet, independently of TB-1 treatment (Fig EV2C). Although it did not significantly improved survival as single treatment, when combined with an ammonia scavenger drug (Na-benzoate) and l-arginine (l-Arg), TB-1 increased survival whereas ammonia scavengers and l-Arg did not affect survival of spf-ash mice under high protein diet (Fig 1E). Consistent with the increased survival, blood ammonia levels measured after 4 days with high protein diet (a time-point prior to mortality) were significantly lower in spf-ash mice treated with the combination of TB-1 and Na-benzoate and l-Arg compared to untreated spf-ash mice (Fig 1F). TB-1 alone decreased slightly but not significantly blood ammonia whereas Na-benzoate and l-Arg significantly reduced blood ammonia levels, consistent with the human data (Enns et al, 2007; Fig 1F). Notably, Na-benzoate and l-Arg treatment did not affect the levels of urinary orotic acid increased by the high protein diet whereas TB-1 either alone or in combination with Na-benzoate and l-Arg efficiently blunted the increase in urinary orotic acid induced by the high protein diet (Fig EV2D). Taken together, these results support the therapeutic potential of activation of liver autophagy by TB-1 in combination with conventional treatments, such as ammonia scavenger drugs and l-Arg (Enns et al, 2007; Haberle et al, 2019), for treatment of OTC deficiency, the most common UCD. Click here to expand this figure. Figure EV2. TB-1 treatment in spf-ash mice Body weights of spf-ash mice at baseline and up to day 10 of treatment with Tat-Beclin-1 (TB-1) (related to Fig 1B) (n = 5 mice/group). Liver ornithine transcarbamylase (OTC) catalytic activity in spf-ash mice injected with TB-1 or vehicle (n = 5 mice/group) compared to wild-type (WT) levels (n = 3) (Unpaired t-test). Body weights of WT and spf-ash mice fed with a high protein diet (HPD) (related to Fig 1E) (n = 4–6 mice/group). Urinary orotic acid levels at baseline (D0) and after 4 days of HPD (D4) (related to Fig 1E) (n ≥ 4 mice/group); ***P < 0.001, **P < 0.01 (One-way ANOVA). Data information: All values are shown as averages ± SEM. ns: not statistically significant difference. Exact P values are reported in Appendix Table S1. Source data are available online for this figure. Download figure Download PowerPoint Tat-Beclin-1 enhances ureagenesis and corrects metabolic abnormalities of argininosuccinic aciduria To investigate the efficacy of autophagy enhancement for therapy of argininosuccinic aciduria (ASA), the second most frequent UCD (Baruteau et al, 2019a), we investigated TB-1 treatment in the hypomorphic murine model of argininosuccinate lyase (ASL) deficiency (AslNeo/Neo) that expresses approximately 16% of residual enzyme activity and recapitulates the main biochemical and clinical abnormalities of ASA patients (Erez et al, 2011; Nagamani et al, 2012; Baruteau et al, 2018; Burrage et al, 2020). Besides impaired urea synthesis and ammonia detoxification, systemic manifestations of ASA, such as reduced body weight, increased blood pressure, and reduced survival are also associated with nitric oxide (NO)-deficiency (Erez et al, 2011; Nagamani et al, 2012; Baruteau et al, 2018; Kho et al, 2018). AslNeo/Neo mice treated with TB-1 but without any additional treatment showed increased survival compared to vehicle-treated controls that started dying by 10 days of age (Fig 2A). Weight gain was unaffected by TB-1 (Fig EV3A). Consistent with our previous work (Soria et al, 2018), TB-1-mediated activation of autophagy in AslNeo/Neo mice was associated with increased incorporation of 15N into urea (+88%, P < 0.05) indicating enhanced ureagenesis (Fig 2B). Consistent with the increased ureagenesis, blood ammonia levels were lowered by TB-1 in AslNeo/Neo mice (Fig EV3B). As expected, autophagic flux was enhanced in livers of AslNeo/Neo mice injected with TB-1, as shown by reduced LC3-II and decreased autophagy substrates (p62 and NBR1) in livers (Fig 2C and D), whereas residual ASL enzyme activity was unaffected (Fig EV3C). Argininosuccinic acid levels were reduced in dried blood spots (Fig 2E) in TB-1-treated AslNeo/Neo mice. Consistent with this reduction, hepatic content of 15N-labelled argininosuccinic acid was also reduced in mice treated with TB-1 (Fig 2F). Metabolomic analysis by 1H-NMR spectroscopy (Soria et al, 2018) showed that the whole-liver metabolome of vehicle-treated AslNeo/Neo mice was well separated from healthy WT controls but it was shifted towards non-diseased WT controls in AslNeo/Neo mice injected with TB-1 (Fig 2G and Appendix Fig S1), suggesting that TB-1 corrects at least partially the liver metabolic deregulation caused by ASL deficiency. Notably, NMR confirmed that liver content of argininosuccinate, along with its two precursors citrulline and aspartate, was reduced (Fig EV4). Moreover, levels of key compounds of the TCA cycle (fumarate and succinate) and glucose were rescued by TB-1 (Fig EV4). In summary, TB-1 improved several biochemical alterations of ASA, confirming the efficacy of autophagy enhancer molecules for therapy of UCD. Figure 2. Enhancement of liver autophagy improves survival, increases ureagenesis, and corrects metabolic defects of ASL-deficient mice Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 h starting at day 10 of age) or vehicle. WT + Vehicle n = 8; WT + TB-1 n = 8; AslNeo/Neo + Vehicle n = 20; AslNeo/Neo + TB-1 n = 16. *P < 0.05 (Log-rank Mantel–Cox test). Isotopic enrichment of 15N-labelled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n = 15) or vehicle (n = 9). *P < 0.05 (Unpaired t-test). Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. Densitometric quantifications. AslNeo/Neo + Vehicle n = 5; AslNeo/Neo + TB-1 n = 9. **P < 0.01, *P < 0.05 (Unpaired t-test). Argininosuccinate in dried blood spots of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n = 4–14 mice/group). ***P < 0.001, *P < 0.05 (One-way ANOVA). Isotopic enrichment of 15N-labelled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n = 11) or vehicle (n = 5). *P < 0.05 (Unpaired t-test). Orthogonal Projection to Latent Structure-Discriminant Analysis (OPLS-DA) score plot obtained from high-resolution 1H-NMR spectroscopy performed on livers of vehicle-treated AslNeo/Neo mice (n = 6), WT controls (n = 4) and AslNeo/Neo mice injected with TB-1 (n = 6). A statistical model with R2 = 0.78 (goodness of fit), Q2 = 0.57 (power in prediction) and P = 0.0056 was obtained. See also Appendix Fig S1. Data information: All values are shown as averages ± SEM. ns: not statistically significant difference. Exact P values are reported in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 2 [emmm202013158-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Short-term treatment of TB-1 in ASL-deficient mice Body weights of wild-type (WT) and ASL-deficient (AslNeo/Neo) mice injected with Tat-Beclin-1 (TB-1) or vehicle (n = 8–16 mice/group). Blood ammonia in WT and AslNeo/Neo mice injected with TB-1 or vehicle (n = 4–11 mice/group). *P < 0.05 (One-way ANOVA with Dunnett's post-test compared to WT). ns: not statistically significant difference. Liver arginosuccinate lyase (ASL) catalytic activity in WT and AslNeo/Neo mice injected with TB-1 or vehicle (n = 4–7 mice/group). ASL residual activity is unaffected in TB-1-treated mice. **P < 0.01 (One-way ANOVA). Data information: All values are shown as averages ± SEM. Exact P values are reported in Appendix Table S1. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Enhancement of liver autophagy corrects the metabolic defects of AslNeo/Neo mice Liver metabolites acquired and quantified through 1H-NMR analysis with statistically significant differences between control wild-type (WT) compared to ASL-deficient (AslNeo/Neo) mice treated with Tat-Beclin-1 (TB-1) or vehicle (n ≥ 4 mice/group). See also Appendix Fig S1. The values are shown as averages ± SEM. **P < 0.01, *P < 0.05 (One-way ANOVA). ASA: argininosuccinic acid; ns: not statistically significant difference. Exact P values are reported in Appendix Table S1. Source data are available online for this figure. Download figure Download PowerPoint Tat-Beclin-1 reduces injury and abnormal glycogen deposition in livers with ASL deficiency Chronic hepatocellular injury is a common complication in patients with ASL deficiency (Mori et al, 2002; Yaplito-Lee et al, 2013; Baruteau et al, 2017; Ranucci et al, 2019). Despite the underlying mechanism triggering the liver disease remains unclear, evidence in human and mouse suggests that it is related to massive accumulation of cytoplasmic glycogen (Badizadegan & Perez-Atayde, 1997; Bigot et al, 2017; Burrage et al, 2020). Moreover, because activation of autophagy was found to be effective in clearance of glycogen storage in glycogen storage diseases (Ashe et al, 2010; Spampanato et al, 2013; Martina et al, 2014; Farah et al, 2016), we investigated whether TB-1 promotes glycogen clearance in ASA livers. To this end, AslNeo/Neo mice received protein-restricted diet and daily administration of Na-benzoate and L-Arg in combination with either TB-1 or vehicle, started on day 10 of life and lasting for 3 weeks. Consistent with previous data (Erez et al, 2011; Ashley et al, 2018; Baruteau et al, 2018; Burrage et al, 2020), vehicle-treated AslNeo/Neo mice showed vacuolated hepatocyte cytoplasm by haematoxylin and eosin (H&E) staining in contrast to WT mice, whereas TB-1 treatment markedly improved the microscopic changes of liver architecture (Fig 3A). Although body weight was unaffected (Fig EV5A), TB-1 treatment resulted in a trend of reduction in hepatomegaly (Fig EV5B) and a mild decrease in serum alanine aminotransferase (ALT) levels in AslNeo/Neo mice (Fig EV5C). Moreover, AslNeo/Neo mice treated with TB-1 showed partial reduction of liver glycogen storage by periodic acid Schiff (PAS) staining (Fig 3A and B) and glycogen quantification (Fig 3C) compared to controls. Notably, glycogen accumulation was not observed in livers of spf-ash mice (Fig EV5D). Glycogen in hepatocytes is catabolized either in cytosol by the coordinated action of enzymes involved in glycogenolysis or in the lysosome by the acid glucosidase (Prats