Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy

Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8

Prognostic factors for multi-organ dysfunction in pediatric oncology patients admitted to the pediatric intensive care unit

Background: Pediatric oncology patients who require admission to the pediatric intensive care unit (PICU) have worse outcomes compared to their non-cancer peers. Although multi-organ dysfunction (MOD) plays a pivotal role in PICU mortality and morbidity, risk factors for MOD have not yet been identified. We aimed to identify risk factors at PICU admission for new or progressive MOD (NPMOD) during the first week of PICU stay.Methods: This retrospective cohort study included all pediatric oncology patients aged 0 to 18 years admitted to the PICU between June 2018 and June 2021. We used the recently published PODIUM criteria for defining multi-organ dysfunction and estimated the association between covariates at PICU baseline and the outcome NPMOD using a multivariable logistic regression model, with PICU admission as unit of study. To study the predictive performance, the model was internally validated by using bootstrap.Results: A total of 761 PICU admissions of 571 patients were included. NPMOD was present in 154 PICU admissions (20%). Patients with NPMOD had a high mortality compared to patients without NPMOD, 14% and 1.0% respectively. Hemato-oncological diagnosis, number of failing organs and unplanned admission were independent risk factors for NPMOD. The prognostic model had an overall good discrimination and calibration.Conclusion: The risk factors at PICU admission for NPMOD may help to identify patients who may benefit from closer monitoring and early interventions. When applying the PODIUM criteria, we found some opportunities for fine-tuning these criteria for pediatric oncology patients, that need to be validated in future studies.</p

Prognostic factors for multi-organ dysfunction in pediatric oncology patients admitted to the pediatric intensive care unit

Background: Pediatric oncology patients who require admission to the pediatric intensive care unit (PICU) have worse outcomes compared to their non-cancer peers. Although multi-organ dysfunction (MOD) plays a pivotal role in PICU mortality and morbidity, risk factors for MOD have not yet been identified. We aimed to identify risk factors at PICU admission for new or progressive MOD (NPMOD) during the first week of PICU stay.Methods: This retrospective cohort study included all pediatric oncology patients aged 0 to 18 years admitted to the PICU between June 2018 and June 2021. We used the recently published PODIUM criteria for defining multi-organ dysfunction and estimated the association between covariates at PICU baseline and the outcome NPMOD using a multivariable logistic regression model, with PICU admission as unit of study. To study the predictive performance, the model was internally validated by using bootstrap.Results: A total of 761 PICU admissions of 571 patients were included. NPMOD was present in 154 PICU admissions (20%). Patients with NPMOD had a high mortality compared to patients without NPMOD, 14% and 1.0% respectively. Hemato-oncological diagnosis, number of failing organs and unplanned admission were independent risk factors for NPMOD. The prognostic model had an overall good discrimination and calibration.Conclusion: The risk factors at PICU admission for NPMOD may help to identify patients who may benefit from closer monitoring and early interventions. When applying the PODIUM criteria, we found some opportunities for fine-tuning these criteria for pediatric oncology patients, that need to be validated in future studies.</p

Prognostic factors for multi-organ dysfunction in pediatric oncology patients admitted to the pediatric intensive care unit

Background: Pediatric oncology patients who require admission to the pediatric intensive care unit (PICU) have worse outcomes compared to their non-cancer peers. Although multi-organ dysfunction (MOD) plays a pivotal role in PICU mortality and morbidity, risk factors for MOD have not yet been identified. We aimed to identify risk factors at PICU admission for new or progressive MOD (NPMOD) during the first week of PICU stay.Methods: This retrospective cohort study included all pediatric oncology patients aged 0 to 18 years admitted to the PICU between June 2018 and June 2021. We used the recently published PODIUM criteria for defining multi-organ dysfunction and estimated the association between covariates at PICU baseline and the outcome NPMOD using a multivariable logistic regression model, with PICU admission as unit of study. To study the predictive performance, the model was internally validated by using bootstrap.Results: A total of 761 PICU admissions of 571 patients were included. NPMOD was present in 154 PICU admissions (20%). Patients with NPMOD had a high mortality compared to patients without NPMOD, 14% and 1.0% respectively. Hemato-oncological diagnosis, number of failing organs and unplanned admission were independent risk factors for NPMOD. The prognostic model had an overall good discrimination and calibration.Conclusion: The risk factors at PICU admission for NPMOD may help to identify patients who may benefit from closer monitoring and early interventions. When applying the PODIUM criteria, we found some opportunities for fine-tuning these criteria for pediatric oncology patients, that need to be validated in future studies.</p

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBPRegulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRING(KO) cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRING(KO) cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling

Autophagosomes in the larval muscle are filled with glycogen.

<p>(A) Sectioned third instar <i>OreR</i> larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body). (B) Glycogen was also detected in muscle from <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i> larvae, immunostained with an antiglycogen monoclonal antibody. (C) GFP–Atg8 vesicles colocalized with glycogen in <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i> larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red). (D) HRP–Lamp1 vesicles show less colocalization with glycogen in UAS–HRP–Lamp1;Dmef2–Gal4 larvae starved and treated with CQ. (E) Quantification of GFP–Atg8 or HRP–Lamp1 vesicles with glycogen. (F–G) EM from <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>whitei</i> larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>whitei</i> larva starved on low-nutrient food for 6 h. Arrow points to double membrane.</p

Interaction and colocalization of Glycogen synthase with Atg8 is disrupted in R593A and W609A mutants.

<p>(A) Protein sequence alignment of the C-terminal region of <i>D. melanogaster</i>, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined. (B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag–Atg8 binds to a Venus–GlyS or Venus–GlyS (S651A) protein complex in response to starvation. Flag–Atg8 is unable to co-IP with either Venus–GlyS (R593A) or Venus–GlyS (W609A). Venus–GlyS and Venus–GlyS mutants were co-IP'd from muscle lysate from <i>Dmef2</i>–<i>Gal4/UAS</i>–<i>Flag</i>–<i>Atg8</i> or <i>UAS</i>–<i>Venus</i>–<i>GlyS(WT or mutant)/+;Dmef2</i>–<i>Gal4/UAS</i>–<i>Flag</i>–<i>Atg8</i> third instar larvae. These were fed on high-nutrient food for 18 h, and then transferred to fresh high-nutrient food or low-nutrient food for 6 h. (C–F) <i>UAS</i>–<i>Venus</i>–<i>GlyS (WT or mutant)/+;Dmef2</i>–<i>Gal4/UAS</i>–<i>Flag</i>–<i>Atg8</i> larvae were treated with starved 6 h in low-nutrient food +2.5 mg/ml CQ (Venus, green; α-Flag, red). Purple arrows mark examples of the presence or absence of colocalization. (C) Venus–GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus–GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus–GlyS (S651A) was localized to the autophagosomes. (F) Venus–GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.</p

Degradation of glycogen via the autophagy lysosome system is regulated by nutrients and the Tor pathway.

<p>(A–D) Time course of autophagy induction in <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i> muscles, accompanied by quantification of GFP–Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0–8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP–Atg8-labeled vesicles began to appear. (C–D) At 6 and 8 h of starvation, the majority of GFP–Atg8-labeled vesicles colocalized with glycogen. (E) Time course of glycogen levels in <i>Dmef2</i>–<i>Gal4</i> carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0–24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for <i>n</i> = 5–8 samples (*<i>p</i><.05, **<i>p</i><.01). (F–G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control <i>Dmef2</i>–<i>Gal4/UAS</i>–<i>whitei</i> larvae. Muscles from (G) <i>UAS-Rheb/+; Dmef2-Gal4/+</i>, (H) <i>Dmef2</i>–<i>Gal4/UAS</i>–<i>Tsc1i</i>, and (I) <i>Dmef2</i>–<i>Gal4/UAS</i>–<i>gigi</i> all failed to induce autophagy.</p

Chloroquine (CQ) treatment blocks autophagosome–lysosome fusion and induces myopathy in the larva.

<p>(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green). (B–D) GFP–Atg8, overexpressed using the Dmef2–Gal4 driver, labels autophagosomes. <i>Dmef2–Gal4</i>, <i>UAS–GFP–Atg8</i> animals were fed on high-nutrient food (B), starved on low-nutrient food for 6 h (C), or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (D). GFP–Atg8-labeled vesicles appeared only in the starved animals (C–D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP–Atg8-labeled vesicles. (E–G) <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/UAS</i>–<i>HRP</i>–<i>Lamp1</i> animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP–Atg8 and HRP–Lamp1-labeled vesicles. (F) Colocalization of GFP–Atg8 and HRP–Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP–Atg8 and HRP–Lamp-labeled vesicles, but they failed to colocalize. (H) Quantification of the number of GFP–Atg8, HRP–Lamp1, or GFP–Atg8+HRP–Lamp1 vesicles in starved or starved +CQ muscles. (I–M) The core <i>Atg</i> genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/UAS</i>–<i>Atg1</i> larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that <i>Atg1</i> knockdown completely abolished the formation of GFP–Atg8-labeled autophagosomes (compare I–J to C–D). (K–L) <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i>, <i>Atg1^Δ3d</i> larvae failed to form GFP–Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to <i>Atg</i> gene knockdown in <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i> larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 <i>UAS</i>–<i>Atg</i> RNAi transgenes tested caused a highly significant decrease (<i>p</i><.01) in the total area of GFP–Atg8 vesicles. SEM is indicated, with <i>n</i> = 5 ventral longitudinal muscles from individual animals. (N–O) EM of muscles from <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>whitei</i> larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N). (P) CQ treatment increased the larval crawling time of <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>whitei</i> larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>whitei</i> larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for <i>n</i> = 10 larvae (*<i>p</i><.05, **<i>p</i><.01).</p

Autophagy and glycogenolysis compensate for each other, but both systems are required for maximal glycogen catabolism.

<p>(A–B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) <i>UAS</i>–<i>GlyPi/+; Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/+</i> larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP–Atg8 and glycogen. (B) Higher magnification of region outlined in (A). (C–F) <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8</i> larvae with <i>GlyP</i> and/or <i>Atg1</i> knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) <i>UAS</i>–<i>GlyPi/+; Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/+</i> larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation <i>UAS</i>–<i>GlyPi/+; Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/+</i> muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation <i>Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/UAS</i>–<i>Atg1i</i> muscles contained no glycogen. (F) Double-mutant larvae <i>UAS</i>–<i>GlyPi/+; Dmef2</i>–<i>Gal4</i>, <i>UAS</i>–<i>GFP</i>–<i>Atg8/UAS</i>–<i>Atg1i</i> larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen. (G) Time course of glycogen levels in <i>Dmef2</i>–<i>Gal4</i> carcasses (muscle+body wall) with expression of UAS–RNAi transgenes targeting <i>white</i>, <i>GlyP</i>, <i>Atg1</i>, or <i>GlyP+Atg1</i>. Simultaneous knockdown of <i>GlyP</i> and <i>Atg1</i>, but not either gene alone, significantly reduced glycogen degradation compared to the <i>white</i> control after 24 h of starvation, consistent with immunostaining (C–F). Between 6 and 12 h of starvation, individual knockdown of <i>GlyP</i> or <i>Atg1</i> caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for <i>n</i> = 5–8 samples. The <i>p</i> values were calculated relative to white RNAi control at each time point (*<i>p</i><.05, **<i>p</i><.01).</p