bims-lypmec 2023-07-09 papers

bims-lypmec

Biomed News

on Lysosomal positioning and metabolism in cardiomyocytes

Issue of 2023–07–09
eight papers selected by
Satoru Kobayashi, New York Institute of Technology

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.
High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.
The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.
TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.
The role of redox-mediated lysosomal dysfunction and therapeutic strategies.
Ratiometric and discriminative visualization of autophagic processes with a novel dual-responded lysosome-specific fluorescent probe.
Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.
Current understanding of the contribution of lactate to the cardiovascular system and its therapeutic relevance.

Nat Commun. 2023 07 03. 14(1): 3911

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Alessia Calcagni', Leopoldo Staiano, Nicolina Zampelli, Nadia Minopoli, Niculin J Herz, Giuseppe Di Tullio, Tuong Huynh, Jlenia Monfregola, Alessandra Esposito, Carmine Cirillo, Aleksandar Bajic, Mahla Zahabiyon, Rachel Curnock, Elena Polishchuk, Luke Parkitny, Diego Luis Medina, Nunzia Pastore, Peter J Cullen, Giancarlo Parenti, Maria Antonietta De Matteis, Paolo Grumati, Andrea Ballabio.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

DOI: https://doi.org/10.1038/s41467-023-39643-7
Autophagy. 2023 Jul 05. 1-20

High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.

Gustav Godtliebsen, Kenneth Bowitz Larsen, Zambarlal Bhujabal, Ida S Opstad, Mireia Nager, Abhinanda R Punnakkal, Trine B Kalstad, Randi Olsen, Trine Lund, Dilip K Prasad, Krishna Agarwal, Truls Myrmel, Asa Birna Birgisdottir.

  Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

Keywords:  CLEM; SIM; deep learning; lysosomes; mitochondria; quality control

DOI:  https://doi.org/10.1080/15548627.2023.2230837
Front Mol Biosci. 2023 ;10 1168680

The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

Farzana Tuli, Patricia M Kane.

  Vacuolar H+-ATPases (V-ATPases) acidify several organelles in all eukaryotic cells and export protons across the plasma membrane in a subset of cell types. V-ATPases are multisubunit enzymes consisting of a peripheral subcomplex, V1, that is exposed to the cytosol and an integral membrane subcomplex, Vo, that contains the proton pore. The Vo a-subunit is the largest membrane subunit and consists of two domains. The N-terminal domain of the a-subunit (aNT) interacts with several V1 and Vo subunits and serves to bridge the V1 and Vo subcomplexes, while the C-terminal domain contains eight transmembrane helices, two of which are directly involved in proton transport. Although there can be multiple isoforms of several V-ATPase subunits, the a-subunit is encoded by the largest number of isoforms in most organisms. For example, the human genome encodes four a-subunit isoforms that exhibit a tissue- and organelle-specific distribution. In the yeast S. cerevisiae, the two a-subunit isoforms, Golgi-enriched Stv1 and vacuolar Vph1, are the only V-ATPase subunit isoforms. Current structural information indicates that a-subunit isoforms adopt a similar backbone structure but sequence variations allow for specific interactions during trafficking and in response to cellular signals. V-ATPases are subject to several types of environmental regulation that serve to tune their activity to their cellular location and environmental demands. The position of the aNT domain in the complex makes it an ideal target for modulating V1-Vo interactions and regulating enzyme activity. The yeast a-subunit isoforms have served as a paradigm for dissecting interactions of regulatory inputs with subunit isoforms. Importantly, structures of yeast V-ATPases containing each a-subunit isoform are available. Chimeric a-subunits combining elements of Stv1NT and Vph1NT have provided insights into how regulatory inputs can be integrated to allow V-ATPases to support cell growth under different stress conditions. Although the function and distribution of the four mammalian a-subunit isoforms present additional complexity, it is clear that the aNT domains of these isoforms are also subject to multiple regulatory interactions. Regulatory mechanisms that target mammalian a-subunit isoforms, and specifically the aNT domains, will be described. Altered V-ATPase function is associated with multiple diseases in humans. The possibility of regulating V-ATPase subpopulations via their isoform-specific regulatory interactions are discussed.

Keywords:  V-ATPase; a-subunit; acidification; isoform; organelle; regulation; reversible disassembly

DOI:  https://doi.org/10.3389/fmolb.2023.1168680
EMBO J. 2023 Jul 06. e113105

TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.

Namrita Kaur, Laura Rodriguez de la Ballina, Håvard Styrkestad Haukaas, Maria Lyngaas Torgersen, Maja Radulovic, Michael J Munson, Alan Sabirsh, Harald Stenmark, Anne Simonsen, Sven R Carlsson, Alf Håkon Lystad.

  Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

Keywords:  CASM; DysF; membrane damage; noncanonical autophagy; sphingomyelin

DOI:  https://doi.org/10.15252/embj.2022113105
Biomed Pharmacother. 2023 Jul 05. pii: S0753-3322(23)00912-5. [Epub ahead of print]165 115121

The role of redox-mediated lysosomal dysfunction and therapeutic strategies.

Xiaomeng He, Xuening Li, Wei Tian, Chenyu Li, Pengfei Li, Jingyuan Zhao, Shilei Yang, Shuai Li.

  Redox homeostasis refers to the dynamic equilibrium between oxidant and reducing agent in the body which plays a crucial role in maintaining normal physiological activities of the body. The imbalance of redox homeostasis can lead to the development of various human diseases. Lysosomes regulate the degradation of cellular proteins and play an important role in influencing cell function and fate, and lysosomal dysfunction is closely associated with the development of various diseases. In addition, several studies have shown that redox homeostasis plays a direct or indirect role in regulating lysosomes. Therefore, this paper systematically reviews the role and mechanisms of redox homeostasis in the regulation of lysosomal function. Therapeutic strategies based on the regulation of redox exerted to disrupt or restore lysosomal function are further discussed. Uncovering the role of redox in the regulation of lysosomes helps to point new directions for the treatment of many human diseases.

Keywords:  Autophagy; LMP; Lysosomal; Redox homeostasis; Targeting ROS

DOI:  https://doi.org/10.1016/j.biopha.2023.115121
Biomater Res. 2023 Jul 06. 27(1): 66

Ratiometric and discriminative visualization of autophagic processes with a novel dual-responded lysosome-specific fluorescent probe.

Fan Zheng, Yeshuo Ma, Jipeng Ding, Shuai Huang, Shengwang Zhang, Xueyan Huang, Bin Feng, Hongliang Zeng, Fei Chen, Wenbin Zeng.

   BACKGROUND: Autophagy is a critical self-eating pathway involved in numerous physiological and pathological processes. Lysosomal degradation of dysfunctional organelles and invading microorganisms is central to the autophagy mechanism and essential for combating disease-related conditions. Therefore, monitoring fluctuations in the lysosomal microenvironment is vital for tracking the dynamic process of autophagy. Although much effort has been put into designing probes for measuring lysosomal viscosity or pH separately, there is a need to validate the concurrent imaging of the two elements to enhance the understanding of the dynamic progression of autophagy.
METHODS: Probe HFI was synthesized in three steps and was developed to visualize changes in viscosity and pH within lysosomes for real-time autophagy tracking. Then, the spectrometric determination was carried out. Next, the probe was applied to image autophagy in cells under nutrient-deprivation or external stress. Additionally, the performance of HFI to monitor autophagy was employed to evaluate acetaminophen-induced liver injury.
RESULTS: We constructed a ratiometric dual-responsive probe, HFI, with a large Stokes shift over 200 nm, dual-wavelength emission, and small background interference. The ratiometric fluorescent signal (R = I 610/I 460) of HFI had an excellent correlation with both viscosity and pH. More importantly, high viscosity and low pH had a synergistic promotion effect on the emission intensity of HFI, which enabled it to specially lit lysosomes without disturbing the inherent microenvironment. We then successfully used HFI to monitor intracellular autophagy induced by starvation or drugs in real-time. Interestingly, HFI also enabled us to visualize the occurrence of autophagy in the liver tissue of a DILI model, as well as the reversible effect of hepatoprotective drugs on this event.
CONCLUSIONS: In this study, we developed the first ratiometric dual-responsive fluorescent probe, HFI, for real-time revealing autophagic details. It could image lysosomes with minimal perturbation to their inherent pH, allowing us to track changes in lysosomal viscosity and pH in living cells. Ultimately, HFI has great potential to serve as a useful indicator for autophagic changes in viscosity and pH in complex biological samples and can also be used to assess drug safety.

Keywords:  Acetaminophen-induced liver injury; Autophagy visualization; Dual-responsive; Fluorescent probe; Lysosome-specific; Ratiometric imaging; Viscosity; pH

DOI:  https://doi.org/10.1186/s40824-023-00409-3
Dis Model Mech. 2023 Jul 04. pii: dmm.050066. [Epub ahead of print]

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Kevin R Zhang, Connor S R Jankowski, Rayna Marshall, Rohini Nair, Néstor Más Gómez, Ahab Alnemri, Yingrui Liu, Elizabeth Erler, Julia Ferrante, Ying Song, Brent A Bell, Bailey H Baumann, Jacob Sterling, Brandon Anderson, Sierra Foshe, Jennifer Roof, Hossein Fazelinia, Lynn A Spruce, Jen-Zen Chuang, Ching-Hwa Sung, Anuradha Dhingra, Kathleen Boesze-Battaglia, Venkata R M Chavali, Joshua D Rabinowitz, Claire H Mitchell, Joshua L Dunaief.

  Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly, with retinal pigment epithelial (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured iPS-RPE cells increased lysosomal abundance, impaired proteolysis, and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase and acid sphingomyelinase. In a murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy, and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes, and ceramides. The proteolytic enzyme cathepsin D had impaired maturation. A large proportion of lysosomes were galectin-3 positive, suggesting cytotoxic lysosomal membrane permeabilization (LMP). Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Keywords:  Age-related macular degeneration; Aging; Lysosome; Oxidative stress; Retina

DOI:  https://doi.org/10.1242/dmm.050066
Front Endocrinol (Lausanne). 2023 ;14 1205442

Current understanding of the contribution of lactate to the cardiovascular system and its therapeutic relevance.

Panyun Wu, Tengteng Zhu, Yiyuan Huang, Zhenfei Fang, Fei Luo.

  Research during the past decades has yielded numerous insights into the presence and function of lactate in the body. Lactate is primarily produced via glycolysis and plays special roles in the regulation of tissues and organs, particularly in the cardiovascular system. In addition to being a net consumer of lactate, the heart is also the organ in the body with the greatest lactate consumption. Furthermore, lactate maintains cardiovascular homeostasis through energy supply and signal regulation under physiological conditions. Lactate also affects the occurrence, development, and prognosis of various cardiovascular diseases. We will highlight how lactate regulates the cardiovascular system under physiological and pathological conditions based on evidence from recent studies. We aim to provide a better understanding of the relationship between lactate and cardiovascular health and provide new ideas for preventing and treating cardiovascular diseases. Additionally, we will summarize current developments in treatments targeting lactate metabolism, transport, and signaling, including their role in cardiovascular diseases.

Keywords:  angiogenesis; cardiovascular disease; lactate; pathophysiology; signal transduction

DOI:  https://doi.org/10.3389/fendo.2023.1205442