bims-lypmec 2023-06-18 papers

Biophys J. 2023 Jun 15. pii: S0006-3495(23)00375-2. [Epub ahead of print]

Lysosomal calcium loading promotes spontaneous calcium release by potentiating ryanodine receptors.

Zhaozheng Meng, Rebecca A Capel, Samuel J Bose, Erik Bosch, Sophia de Jong, Robert Planque, Antony Galione, Rebecca Ab Burton, Alfonso Bueno-Orovio.

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarisations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under β-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically-detailed mouse ventricular models including for the first time modelling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a pathway for fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum (SR) calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating RyR open probability. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and SERCA reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating RyR open probability, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action.

DOI: https://doi.org/10.1016/j.bpj.2023.06.007

PNAS Nexus. 2023 Jun;2(6): pgad174

Lysosomal Ca2+ flux modulates automaticity in ventricular cardiomyocytes and correlates with arrhythmic risk.

An Xie, Gyeoung-Jin Kang, Eun Ji Kim, Feng Feng, Sophie E Givens, Brenda M Ogle, Samuel C Dudley.

Automaticity involves Ca2+ handling at the cell membrane and sarcoplasmic reticulum (SR). Abnormal or acquired automaticity is thought to initiate ventricular arrhythmias associated with myocardial ischemia. Ca2+ flux from mitochondria can influence automaticity, and lysosomes also release Ca2+. Therefore, we tested whether lysosomal Ca2+ flux could influence automaticity. We studied ventricular human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), hiPSC 3D engineered heart tissues (EHTs), and ventricular cardiomyocytes isolated from infarcted mice. Preventing lysosomal Ca2+ cycling reduced automaticity in hiPSC-CMs. Consistent with a lysosomal role in automaticity, activating the transient receptor potential mucolipin channel (TRPML1) enhanced automaticity, and two channel antagonists reduced spontaneous activity. Activation or inhibition of lysosomal transcription factor EB (TFEB) increased or decreased total lysosomes and automaticity, respectively. In adult ischemic cardiomyocytes and hiPSC 3D EHTs, reducing lysosomal Ca2+ release also inhibited automaticity. Finally, TRPML1 was up-regulated in cardiomyopathic patients with ventricular tachycardia (VT) compared with those without VT. In summary, lysosomal Ca2+ handling modulates abnormal automaticity, and reducing lysosomal Ca2+ release may be a clinical strategy for preventing ventricular arrhythmias.

Keywords: automaticity; calcium; cardiomyocytes; human-induced pluripotent stem cell; lysosome

DOI: https://doi.org/10.1093/pnasnexus/pgad174

FEBS Lett. 2023 Jun 16.

Autophagy in a Nutshell.

Oren Shatz, Zvulun Elazar.

Autophagy is an intracellular catabolic process that eliminates cytoplasmic constituents selectively by tight engulfment in an isolation membrane or recycles bulk cytoplasm by nonselective sequestration. Completion of the isolation membrane forms a double membrane vesicle, termed autophagosome, that proceeds to fusion with the lysosome, where the inner membrane and its cytoplasmic content are degraded. Autophagosome biogenesis is unique in that the newly-formed membrane, termed phagophore, is elongated by direct lipid flow from a proximal ER-associated donor membrane. Recent years mark a tremendous advancement in delineating the direct regulation of this process by different lipid species and associated protein complexes. Here we schematically summarize the current view of autophagy and autophagosome biogenesis.

Keywords: PAS; autophagosome biogenesis; autophagy; lysosome; phagophore; vacuole

DOI: https://doi.org/10.1002/1873-3468.14679

Anal Chim Acta. 2023 Aug 29. pii: S0003-2670(23)00669-4. [Epub ahead of print]1271 341448

Ratiometric visualization of lysosomal pH fluctuations during autophagy by two-photon carbonized polymer dots-based probe.

Genxiu Yao, Junyong Sun, Shan Miao, Yue Wang, Feng Gao.

Monitoring the pH variation in lysosomes is very conducive to studying the autophagy process, and fluorescent ratiometric pH nanoprobes with inherent lysosome targeting ability are highly desirable. Here, a carbonized polymer dots-based pH probe (oAB-CPDs) was developed by self-condensation of o-aminobenzaldehyde and further carbonization at low temperature. The obtained oAB-CPDs display improved performance in pH sensing, including robust photostability, intrinsic lysosome-targeting ability, self-referenced ratiometric response, desirable two-photon-sensitized fluorescence property, and high selectivity. With the suitable pKa value of 5.89, the as-prepared nanoprobe was successfully applied to monitor the variation of lysosomal pH in HeLa cells. Moreover, the occurrence that lysosomal pH decreased during both starvation-induced and rapamycin-induced autophagy was observed by using oAB-CPDs as fluorescence probe. We believe that nanoprobe oAB-CPDs can work as a useful tool for visualizing autophagy in living cells.

Keywords: Autophagy; Carbonized polymer dots; Low-temperature synthesis; Ratiometric pH probe

DOI: https://doi.org/10.1016/j.aca.2023.341448

Nat Chem Biol. 2023 Jun 15.

Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.

Mijin Kim, Chen Chen, Zvi Yaari, Rune Frederiksen, Ewelina Randall, Jaina Wollowitz, Christian Cupo, Xiaojian Wu, Janki Shah, Daniel Worroll, Rachel E Lagenbacher, Dana Goerzen, Yue-Ming Li, Heeseon An, YuHuang Wang, Daniel A Heller.

Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.

DOI: https://doi.org/10.1038/s41589-023-01364-9

Brain Res. 2023 Jun 12. pii: S0006-8993(23)00233-0. [Epub ahead of print] 148462

Dimethyl fumarate ameliorates Parkinsonian pathology by modulating autophagy and apoptosis via Nrf2-TIGAR-LAMP2/Cathepsin D axis.

Mayuri Khot, Anika Sood, Kamatham Pushpa Tryphena, Poojitha Pinjala, Saurabh Srivastava, Shashi Bala Singh, Dharmendra Kumar Khatri.

Mounting evidence suggests a role for oxidative stress and accumulation of dysfunctional organelle and misfolded proteins in PD. Autophagosomes mediate the clearance of these cytoplasmic proteins via delivery to lysosomes to form autophagolysosomes, followed by degradation of the protein by lysosomal enzymes. In PD, autophagolysosome accumulation occurs initiating a plethora of events resulting in neuronal death by apoptosis. This study evaluated the effect of Dimethylfumarate (DMF), an Nrf2 activator in the rotenone-induced mouse PD model. In PD mice, there was decreased expression of LAMP2 and LC3, which resulted in inhibition of autophagic flux and increased expression of cathepsin D, which mediated apoptosis. The role of Nrf2 activation in alleviating oxidative stress is well known. Our study elucidated the novel mechanism underlying the neuroprotective effect of DMF. The loss of dopaminergic neurons induced by rotenone was lessened to a significant extent by pre-treatment with DMF. DMF promoted autophagosome formation and inhibited apoptosis by removing the inhibitory effect of p53 on TIGAR. TIGAR expression upregulated LAMP2 expression and downregulated Cathepsin D, promoting autophagy and inhibiting apoptosis. Thus, it was proved that DMF confers neuroprotection against rotenone-induced dopaminergic neurodegeneration and could be used as a potential therapeutic agent for PD and its progression.

Keywords: Dimethyl fumarate; Parkinson’s disease; apoptosis; autophagy; rotenone

DOI: https://doi.org/10.1016/j.brainres.2023.148462