bims-lysosi 2021-10-24 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒10‒24
forty-two papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement.
AKAP13 couples GPCR signaling to mTORC1 inhibition.
Pib2 as an Emerging Master Regulator of Yeast TORC1.
Role of the Transcriptional Repressor Zinc Finger with KRAB and SCAN Domains 3 (ZKSCAN3) in Retinal Pigment Epithelial Cells.
Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma.
Autophagy and the Lysosomal System in Cancer.
Impaired Bile Acid Metabolism and Gut Dysbiosis in Mice Lacking Lysosomal Acid Lipase.
Relationship between lysosomal dyshomeostasis and progression of diabetic kidney disease.
Activation of mTORC1 by Free Fatty Acids Suppresses LAMP2 and Autophagy Function via ER Stress in Alcohol-Related Liver Disease.
mTORC1 promotes malignant large cell/anaplastic histology and is a targetable vulnerability in SHH-TP53 mutant medulloblastomas.
mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia.
Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model.
Stem Cell Research Tools in Human Metabolic Disorders: An Overview.
The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.
His Domain Protein Tyrosine Phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation.
Sialidase neu4 deficiency is associated with neuroinflammation in mice.
Patient-Derived Induced Pluripotent Stem Cell Models for Phenotypic Screening in the Neuronal Ceroid Lipofuscinoses.
Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole.
Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases.
AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo.
The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.
Plasma Neurofilament Light (NfL) in Patients Affected by Niemann-Pick Type C Disease (NPCD).
RICTOR Affects Melanoma Tumorigenesis and Its Resistance to Targeted Therapy.
SEA and GATOR 10 Years Later.
Fission Yeast TORC2 Signaling Pathway Ensures Cell Proliferation under Glucose-Limited, Nitrogen-Replete Conditions.
The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.
Nuclear PHGDH protects cancer cells from nutrient stress.
Analysis of the early response to spinal cord injury identified a key role for mTORC1 signaling in the activation of neural stem progenitor cells.
The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress.
Ragulator-Rag and ROS TORment gasdermin D pore formation.
Role of PI3K-AKT-mTOR Pathway as a Pro-Survival Signaling and Resistance-Mediating Mechanism to Therapy of Prostate Cancer.
RIPK1 regulates starvation resistance by modulating aspartate catabolism.
Quantifying inter-organelle membrane contact sites using proximity ligation assay in fixed optic nerve sections.
A Novel Likely Pathogenic Variant in the BLOC1S5 Gene Associated with Hermansky-Pudlak Syndrome Type 11 and an Overview of Human BLOC-1 Deficiencies.
Renal Cell Carcinoma in Tuberous Sclerosis Complex.
Prenatal hydrops fetalis associated with infantile free sialic acid storage disease due to a novel homozygous deletion in the SLC17A5 gene.
Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice.
PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans.
Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain.
Comparative Transcriptome Analysis in Monocyte-Derived Macrophages of Asymptomatic GBA Mutation Carriers and Patients with GBA-Associated Parkinson's Disease.
Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.
MYC and TFEB Control DNA Methylation and Differentiation in AML.

Biomolecules. 2021 Sep 27. pii: 1412. [Epub ahead of print]11(10):

Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement.

Indranil Basak, Rachel A Hansen, Michael E Ward, Stephanie M Hughes.

  Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement-a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

Keywords:  Batten disease; CLN5; CRISPR; cathepsin B; iPSC-derived human neurons; lysosome acidity; lysosome enzyme activity; lysosome function; lysosome movement

DOI:  https://doi.org/10.3390/biom11101412
PLoS Genet. 2021 Oct 21. 17(10): e1009832

AKAP13 couples GPCR signaling to mTORC1 inhibition.

Shihai Zhang, Huanyu Wang, Chase H Melick, Mi-Hyeon Jeong, Adna Curukovic, Shweta Tiwary, Tshering D Lama-Sherpa, Delong Meng, Kelly A Servage, Nicholas G James, Jenna L Jewell.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.

DOI: https://doi.org/10.1371/journal.pgen.1009832
Biomolecules. 2021 Oct 09. pii: 1489. [Epub ahead of print]11(10):

Pib2 as an Emerging Master Regulator of Yeast TORC1.

Riko Hatakeyama.

  Cell growth is dynamically regulated in response to external cues such as nutrient availability, growth factor signals, and stresses. Central to this adaptation process is the Target of Rapamycin Complex 1 (TORC1), an evolutionarily conserved kinase complex that fine-tunes an enormous number of cellular events. How upstream signals are sensed and transmitted to TORC1 has been intensively studied in major model organisms including the budding yeast Saccharomyces cerevisiae. This field recently saw a breakthrough: the identification of yeast phosphatidylInositol(3)-phosphate binding protein 2 (Pib2) protein as a critical regulator of TORC1. Although the study of Pib2 is still in its early days, multiple groups have provided important mechanistic insights on how Pib2 relays nutrient signals to TORC1. There remain, on the other hand, significant gaps in our knowledge and mysteries that warrant further investigations. This is the first dedicated review on Pib2 that summarizes major findings and outstanding questions around this emerging key player in cell growth regulation.

Keywords:  Pib2; TOR; TORC1; mTOR; mTORC1; yeast

DOI:  https://doi.org/10.3390/biom11101489
Cells. 2021 Sep 22. pii: 2504. [Epub ahead of print]10(10):

Role of the Transcriptional Repressor Zinc Finger with KRAB and SCAN Domains 3 (ZKSCAN3) in Retinal Pigment Epithelial Cells.

Hsuan-Yeh Pan, Mallika Valapala.

  Lysosomes are important for proper functioning of the retinal pigment epithelial (RPE) cells. RPE cells have a daily burden of phagocytosis of photoreceptor outer segments (POS) and also degrade cellular waste by autophagy. Here, we identified the role of Zinc-finger protein with KRAB and SCAN domains 3 (ZKSCAN3) in co-ordinate regulation of lysosomal function and autophagy in the RPE. Our studies show that in the RPE, ZKSCAN3 is predominantly nuclear in healthy cells and its nuclear expression is reduced upon nutrient deprivation. siRNA-mediated knockdown of ZKSCAN3 results in de-repression of some of the ZKSCAN3 target genes. Knockdown of ZKSCAN3 also resulted in an induction in autophagy flux, increase in the number of functional lysosomes and accompanied activation of lysosomal cathepsin B activity in ARPE-19 cells. We also demonstrated that inhibition of P38 mitogen-activated protein kinase (MAPK) retains ZKSCAN3 in the nucleus in nutrient-deprived cells. In summary, our studies elucidated the role of ZKSCAN3 as a transcriptional repressor of autophagy and lysosomal function in the RPE.

Keywords:  autophagy; lysosomal function; retinal pigment epithelium (RPE); zinc finger with KRAB and SCAN domains 3 (ZKSCAN3)

DOI:  https://doi.org/10.3390/cells10102504
Cell Rep. 2021 Oct 19. pii: S2211-1247(21)01312-7. [Epub ahead of print]37(3): 109848

Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma.

Wanlu Du, Mingxue Gu, Meiqin Hu, Prateeksunder Pinchi, Wei Chen, Michael Ryan, Timothy Nold, Ahmed Bannaga, Haoxing Xu.

  During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.

Keywords:  ML-SAs; ML-SIs; TRPML1; Zn(2+); cell death; lysosome; metastatic melanoma; mitochondria; small molecule

DOI:  https://doi.org/10.1016/j.celrep.2021.109848
Cells. 2021 Oct 14. pii: 2752. [Epub ahead of print]10(10):

Autophagy and the Lysosomal System in Cancer.

Suresh Kumar, Miguel Sánchez-Álvarez, Fidel-Nicolás Lolo, Flavia Trionfetti, Raffaele Strippoli, Marco Cordani.

  Autophagy and the lysosomal system, together referred to as the autophagolysosomal system, is a cellular quality control network which maintains cellular health and homeostasis by removing cellular waste including protein aggregates, damaged organelles, and invading pathogens. As such, the autophagolysosomal system has roles in a variety of pathophysiological disorders, including cancer, neurological disorders, immune- and inflammation-related diseases, and metabolic alterations, among others. The autophagolysosomal system is controlled by TFEB, a master transcriptional regulator driving the expression of multiple genes, including autophagoly sosomal components. Importantly, Reactive Oxygen Species (ROS) production and control are key aspects of the physiopathological roles of the autophagolysosomal system, and may hold a key for synergistic therapeutic interventions. In this study, we reviewed our current knowledge on the biology and physiopathology of the autophagolysosomal system, and its potential for therapeutic intervention in cancer.

Keywords:  AMPK; TFEB; autophagy; cancer; lysosomes; mTOR; nanoparticles

DOI:  https://doi.org/10.3390/cells10102752
Cells. 2021 Oct 01. pii: 2619. [Epub ahead of print]10(10):

Impaired Bile Acid Metabolism and Gut Dysbiosis in Mice Lacking Lysosomal Acid Lipase.

Vinay Sachdev, Madalina Duta-Mare, Melanie Korbelius, Nemanja Vujić, Christina Leopold, Jan Freark de Boer, Silvia Rainer, Peter Fickert, Dagmar Kolb, Folkert Kuipers, Branislav Radovic, Gregor Gorkiewicz, Dagmar Kratky.

  Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mice to diet-induced obesity. Our results indicate that LAL-derived lipolytic products might be important metabolic effectors in the maintenance of whole-body lipid homeostasis.

Keywords:  FGF15; Western-type diet; cholesterol absorption; gut microbiota; lysosomal acid lipase

DOI:  https://doi.org/10.3390/cells10102619
Cell Death Dis. 2021 Oct 18. 12(11): 958

Relationship between lysosomal dyshomeostasis and progression of diabetic kidney disease.

Man Wu, Minjie Zhang, Yaozhi Zhang, Zixian Li, Xingyu Li, Zejian Liu, Huafeng Liu, Xiaoyu Li.

Lysosomes are organelles involved in cell metabolism, waste degradation, and cellular material circulation. They play a key role in the maintenance of cellular physiological homeostasis. Compared with the lysosomal content of other organs, that of the kidney is abundant, and lysosomal abnormalities are associated with the occurrence and development of certain renal diseases. Lysosomal structure and function in intrinsic renal cells are impaired in diabetic kidney disease (DKD). Promoting lysosomal biosynthesis and/or restoring lysosomal function can repair damaged podocytes and proximal tubular epithelial cells, and delay the progression of DKD. Lysosomal homeostasis maintenance may be advantageous in alleviating DKD. Here, we systematically reviewed the latest advances in the relationship between lysosomal dyshomeostasis and progression of DKD based on recent literature to further elucidate the mechanism of renal injury in diabetes mellitus and to highlight the application potential of lysosomal homeostasis maintenance as a new prevention and treatment strategy for DKD. However, research on screening effective interventions for lysosomal dyshomeostasis is still in its infancy, and thus should be the focus of future research studies. The screening out of cell-specific lysosomal function regulation targets according to the different stages of DKD, so as to realize the controllable targeted regulation of cell lysosomal function during DKD, is the key to the successful clinical development of this therapeutic strategy.

DOI: https://doi.org/10.1038/s41419-021-04271-w
Cells. 2021 Oct 13. pii: 2730. [Epub ahead of print]10(10):

Activation of mTORC1 by Free Fatty Acids Suppresses LAMP2 and Autophagy Function via ER Stress in Alcohol-Related Liver Disease.

Wei Guo, Wei Zhong, Liuyi Hao, Xinguo Sun, Zhanxiang Zhou.

  Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.

Keywords:  ER stress; LAMP2; alcohol-related liver disease; free fatty acid; inflammation; mTORC1

DOI:  https://doi.org/10.3390/cells10102730
JCI Insight. 2021 Oct 21. pii: e153462. [Epub ahead of print]

mTORC1 promotes malignant large cell/anaplastic histology and is a targetable vulnerability in SHH-TP53 mutant medulloblastomas.

Valentina Conti, Manuela Cominelli, Valentina Pieri, Alberto L Gallotti, Ilaria Pagano, Matteo Zanella, Stefania Mazzoleni, Flavia Pivetta, Monica Patanè, Giulia M Scotti, Ignazio S Piras, Bianca Pollo, Andrea Falini, Alessio Zippo, Antonella Castellano, Roberta Maestro, Pietro L Poliani, Rossella Galli.

  Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog (SHH)-activated MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MBs developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mechanistic Target Of Rapamycin Complex 1 (mTORC1) hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MBs and CSC-derived MBs resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patients' samples and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.

Keywords:  Brain cancer; Mouse models; Neuroscience; Oncology

DOI:  https://doi.org/10.1172/jci.insight.153462
Elife. 2021 Oct 22. pii: e70079. [Epub ahead of print]10

mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia.

Soyeon Lim, You-Joung Kim, Sooyeon Park, Ji-Heon Choi, Younghoon Sung, Katsuhiko Nishimori, Zybmek Kozmik, Han-Woong Lee, Jin Woo Kim.

  Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-a (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.

Keywords:  developmental biology; mouse

DOI:  https://doi.org/10.7554/eLife.70079
Sci Rep. 2021 Oct 22. 11(1): 20945

Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model.

Catherine Viel, Jennifer Clarke, Can Kayatekin, Amy M Richards, Ming Sum R Chiang, Hyejung Park, Bing Wang, Lamya S Shihabuddin, S Pablo Sardi.

Mutations in GBA, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), represent the greatest genetic risk factor for developing synucleinopathies including Parkinson's disease (PD). Additionally, PD patients harboring a mutant GBA allele present with an earlier disease onset and an accelerated disease progression of both motor and non-motor symptoms. Preclinical studies in mouse models of synucleinopathy suggest that modulation of the sphingolipid metabolism pathway via inhibition of glucosylceramide synthase (GCS) using a CNS-penetrant small molecule may be a potential treatment for synucleinopathies. Here, we aim to alleviate the lipid storage burden by inhibiting the de novo synthesis of the primary glycosphingolipid substrate of GCase, glucosylceramide (GlcCer). We have previously shown that systemic GCS inhibition reduced GlcCer and glucosylsphingosine (GlcSph) accumulation, slowed α-synuclein buildup in the hippocampus, and improved cognitive deficits. Here, we studied the efficacy of a brain-penetrant clinical candidate GCS inhibitor, venglustat, in mouse models of GBA-related synucleinopathy, including a heterozygous Gba mouse model which more closely replicates the typical GBA-PD patient genotype. Collectively, these data support the rationale for modulation of GCase-related sphingolipid metabolism as a therapeutic strategy for treating GBA-related synucleinopathies.

DOI: https://doi.org/10.1038/s41598-021-00404-5
Cells. 2021 Oct 07. pii: 2681. [Epub ahead of print]10(10):

Stem Cell Research Tools in Human Metabolic Disorders: An Overview.

Serena Ricci, Pietro Cacialli.

  Metabolic disorders are very common in the population worldwide and are among the diseases with the highest health utilization and costs per person. Despite the ongoing efforts to develop new treatments, currently, for many of these disorders, there are no approved therapies, resulting in a huge economic hit and tension for society. In this review, we recapitulate the recent advancements in stem cell (gene) therapy as potential tools for the long-term treatment of both inherited (lysosomal storage diseases) and acquired (diabetes mellitus, obesity) metabolic disorders, focusing on the main promising results observed in human patients and discussing the critical hurdles preventing the definitive jump of this approach from the bench to the clinic.

Keywords:  diabetes; lysosomal storage diseases; obesity; stem cell therapy

DOI:  https://doi.org/10.3390/cells10102681
Cells. 2021 Oct 09. pii: 2711. [Epub ahead of print]10(10):

The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

Cecilia Cattelani, Dominik Lesiak, Gudrun Liebscher, Isabel I Singer, Taras Stasyk, Moritz H Wallnöfer, Alexander M Heberle, Corrado Corti, Michael W Hess, Kristian Pfaller, Marcel Kwiatkowski, Peter P Pramstaller, Andrew A Hicks, Kathrin Thedieck, Thomas Müller, Lukas A Huber, Mariana Eca Guimaraes de Araujo.

  Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

Keywords:  KICSTOR; SZT2; autophagy; ciliogenesis; epilepsy; mTORC1; neurodegeneration; neurogenesis

DOI:  https://doi.org/10.3390/cells10102711
J Cell Sci. 2021 Oct 18. pii: jcs.259192. [Epub ahead of print]

His Domain Protein Tyrosine Phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation.

Gabrielle Parkinson, Peristera Roboti, Ling Zhang, Sandra Taylor, Philip Woodman.

  His Domain Protein Tyrosine Phosphatase (HD-PTP) collaborates with Endosomal Sorting Complexes Required for Transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body. Completion of multivesicular body sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase, Rab5. Here we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents multivesicular body sorting, whilst also blocking cargo from exiting Rab5-rich endosomes. HD-PTP depleted cells contain hyperphosphorylated Rabaptin-5, a cofactor for the Rab5 guanine nucleotide exchange factor, Rabex-5, though HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP depleted cells exhibit Rabaptin-5 dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5 and Rab5 binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP and is competed by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternately scaffold ESCRTs and modulate Rabex-5/Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.

Keywords:  ESCRT; Multivesicular body; Rab5; Rabex-5

DOI:  https://doi.org/10.1242/jcs.259192
Glycoconj J. 2021 Oct 23.

Sialidase neu4 deficiency is associated with neuroinflammation in mice.

Zehra Kevser Timur, Orhan Kerim Inci, Secil Akyildiz Demir, Volkan Seyrantepe.

  Sialidases catalyze the removal of sialic acid residues from glycoproteins, oligosaccharides, and sialylated glycolipids. Sialidase Neu4 is in the lysosome and has broad substrate specificity. Previously generated Neu4-/- mice were viable, fertile and lacked gross morphological abnormalities, but displayed a marked vacuolization and lysosomal storage in lung and spleen cells. In addition, we showed that there is an increased level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in the brain of Neu4-/- mice. In this study, we further explored whether sialidase Neu4 deficiency causes neuroinflammation. We demostrated that elevated level of GD1a and GT1b is associated with an increased level of LAMP1-positive lysosomal vesicles and Tunel-positive neurons correlated with alterations in the expression of cytokines and chemokines in adult Neu4-/- mice. Astrogliosis and microgliosis were also significantly enhanced in the hippocampus, and cerebellum. These changes in brain immunity were accompanied by motor impairment in these mice. Our results indicate that sialidase Neu4 is a novel mediator of an inflammatory response in the mouse brain due to the altered catabolism of gangliosides.

Keywords:  Behavior; Ganglioside; Neu4; Neuroinflammation; Sialidase

DOI:  https://doi.org/10.1007/s10719-021-10017-9
Molecules. 2021 Oct 15. pii: 6235. [Epub ahead of print]26(20):

Patient-Derived Induced Pluripotent Stem Cell Models for Phenotypic Screening in the Neuronal Ceroid Lipofuscinoses.

Ahmed Morsy, Angelica V Carmona, Paul C Trippier.

  Batten disease or neuronal ceroid lipofuscinosis (NCL) is a group of rare, fatal, inherited neurodegenerative lysosomal storage disorders. Numerous genes (CLN1-CLN8, CLN10-CLN14) were identified in which mutations can lead to NCL; however, the underlying pathophysiology remains elusive. Despite this, the NCLs share some of the same features and symptoms but vary in respect to severity and onset of symptoms by age. Some common symptoms include the progressive loss of vision, mental and motor deterioration, epileptic seizures, premature death, and in the rare adult-onset, dementia. Currently, all forms of NCL are fatal, and no curative treatments are available. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type of the human body. Cells reprogrammed from a patient have the advantage of acquiring disease pathogenesis along with recapitulation of disease-associated phenotypes. They serve as practical model systems to shed new light on disease mechanisms and provide a phenotypic screening platform to enable drug discovery. Herein, we provide an overview of available iPSC models for a number of different NCLs. More specifically, we highlight findings in these models that may spur target identification and drug development.

Keywords:  batten disease; drug discovery; induced pluripotent stem cells; model systems; neuronal ceroid lipofuscinosis; screening

DOI:  https://doi.org/10.3390/molecules26206235
Mol Biol Cell. 2021 Oct 20. mbcE21050227

Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole.

Ayelén González Montoro, Prado Vargas Duarte, Kathrin Auffarth, Stefan Walter, Florian Fröhlich, Christian Ungermann.

The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.

DOI: https://doi.org/10.1091/mbc.E21-05-0227
Cells. 2021 Sep 23. pii: 2518. [Epub ahead of print]10(10):

Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases.

Valentina Tedeschi, Daniele La Russa, Cristina Franco, Antonio Vinciguerra, Diana Amantea, Agnese Secondo.

  Located at the level of the endoplasmic reticulum (ER) membrane, stromal interacting molecule 1 (STIM1) undergoes a complex conformational rearrangement after depletion of ER luminal Ca2+. Then, STIM1 translocates into discrete ER-plasma membrane (PM) junctions where it directly interacts with and activates plasma membrane Orai1 channels to refill ER with Ca2+. Furthermore, Ca2+ entry due to Orai1/STIM1 interaction may induce canonical transient receptor potential channel 1 (TRPC1) translocation to the plasma membrane, where it is activated by STIM1. All these events give rise to store-operated calcium entry (SOCE). Besides the main pathway underlying SOCE, which mainly involves Orai1 and TRPC1 activation, STIM1 modulates many other plasma membrane proteins in order to potentiate the influxof Ca2+. Furthermore, it is now clear that STIM1 may inhibit Ca2+ currents mediated by L-type Ca2+ channels. Interestingly, STIM1 also interacts with some intracellular channels and transporters, including nuclear and lysosomal ionic proteins, thus orchestrating organellar Ca2+ homeostasis. STIM1 and its partners/effectors are significantly modulated in diverse acute and chronic neurodegenerative conditions. This highlights the importance of further disclosing their cellular functions as they might represent promising molecular targets for neuroprotection.

Keywords:  Ca2+ homeostasis; Ca2+-storing organelles; amyotrophic lateral sclerosis (ALS); endoplasmic reticulum (ER); lysosomes

DOI:  https://doi.org/10.3390/cells10102518
J Biol Eng. 2021 Oct 21. 15(1): 24

AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo.

Byung-Hyun Cha, Minjin Jung, Angela S Kim, Victoria C Lepak, Brett A Colson, David A Bull, Youngwook Won.

  Cardiac hypertrophy is one of the most common genetic heart disorders and considered a risk factor for cardiac morbidity and mortality. The mammalian target of rapamycin (mTOR) pathway plays a key regulatory function in cardiovascular physiology and pathology in hypertrophy. AZD2014 is a small-molecule ATP competitive mTOR inhibitor working on both mTORC1 and mTORC2 complexes. Little is known about the therapeutic effects of AZD2014 in cardiac hypertrophy and its underlying mechanism. Here, AZD2014 is examined in in vitro model of phenylephrine (PE)-induced human cardiomyocyte hypertrophy and a myosin-binding protein-C (Mybpc3)-targeted knockout (KO) mouse model of cardiac hypertrophy. Our results demonstrate that cardiomyocytes treated with AZD2014 retain the normal phenotype and AZD2014 attenuates cardiac hypertrophy in the Mybpc3-KO mouse model through inhibition of dual mTORC1 and mTORC2, which in turn results in the down-regulation of the Akt/mTOR signaling pathway.

Keywords:  AZD2014; Cardiac hypertrophy; Cardiomyocyte; mTOR inhibitor

DOI:  https://doi.org/10.1186/s13036-021-00276-3
Biochim Biophys Acta Mol Cell Res. 2021 Oct 13. pii: S0167-4889(21)00215-9. [Epub ahead of print] 119161

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Mariana Juliani do Amaral, Ivone de Andrade Rosa, Sarah Azevedo Andrade, Xi Fang, Leonardo Rodrigues Andrade, Manoel Luis Costa, Claudia Mermelstein.

  Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step toward understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Keywords:  biomolecular condensates; intrinsically disordered protein/region; liquid-liquid phase separation; membraneless organelles; nuclear cloud; perinuclear

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119161
J Clin Med. 2021 Oct 19. pii: 4796. [Epub ahead of print]10(20):

Plasma Neurofilament Light (NfL) in Patients Affected by Niemann-Pick Type C Disease (NPCD).

Andrea Dardis, Eleonora Pavan, Martina Fabris, Rosalia Maria Da Riol, Annalisa Sechi, Agata Fiumara, Lucia Santoro, Maximiliano Ormazabal, Romina Milanic, Stefania Zampieri, Jessica Biasizzo, Maurizio Scarpa.

  (1) Background: Niemann-Pick type C disease (NPCD) is an autosomal recessive lysosomal storage disorder caused by mutations in the NPC1 or NPC2 genes. The clinical presentation is characterized by visceral and neurological involvement. Apart from a small group of patients presenting a severe perinatal form, all patients develop progressive and fatal neurological disease with an extremely variable age of onset. Different biomarkers have been identified; however, they poorly correlate with neurological disease. In this study we assessed the possible role of plasma NfL as a neurological disease-associated biomarker in NPCD. (2) Methods: Plasma NfL levels were measured in 75 healthy controls and 26 patients affected by NPCD (24 NPC1 and 2 NPC2; 39 samples). (3) Results: Plasma NfL levels in healthy controls correlated with age and were significantly lower in pediatric patients as compared to adult subjects (p = 0.0017). In both pediatric and adult NPCD patients, the plasma levels of NfL were significantly higher than in age-matched controls (p < 0.0001). Most importantly, plasma NfL levels in NPCD patients with neurological involvement were significantly higher than the levels found in patients free of neurological signs at the time of sampling, both in the pediatric and the adult group (p = 0.0076; p = 0.0032, respectively). Furthermore, in adults the NfL levels in non-neurological patients were comparable with those found in age-matched controls. No correlations between plasma NfL levels and NPCD patient age at sampling or plasma levels of cholestan 3β-5α-6β-triol were found. (4) Conclusions: These data suggest a promising role of plasma NfL as a possible neurological disease-associated biomarker in NPCD.

Keywords:  Niemann–Pick C; biomarkers; neurofilament light; neurological disease

DOI:  https://doi.org/10.3390/jcm10204796
Biomedicines. 2021 Oct 19. pii: 1498. [Epub ahead of print]9(10):

RICTOR Affects Melanoma Tumorigenesis and Its Resistance to Targeted Therapy.

Ahlem Jebali, Maxime Battistella, Céleste Lebbé, Nicolas Dumaz.

  The network defined by phosphatidylinositol-3-kinase (PI3K), AKT, and mammalian target of rapamycin (mTOR) plays a major role in melanoma oncogenesis and has been implicated in BRAF inhibitor resistance. The central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently begun to be unraveled. In the present study, we assessed the role of mTORC2/RICTOR in BRAF-mutated melanomas and their resistance to BRAF inhibition. We showed that RICTOR was significantly overexpressed in melanoma and associated with bad prognoses. RICTOR overexpression stimulated melanoma-initiating cells (MICs) with 'stemness' properties. We also showed that RICTOR contributed to melanoma resistance to BRAF inhibitors and rendered the cells very sensitive to mTORC2 inhibition. We highlighted a connection between mTORC2/RICTOR and STAT3 in resistant cells and revealed an interaction between RAS and RICTOR in resistant melanoma, which, when disrupted, impeded the proliferation of resistant cells. Therefore, as a key signaling node, RICTOR contributes to BRAF-dependent melanoma development and resistance to therapy and, as such, is a valuable therapeutic target in melanoma.

Keywords:  BRAF; BRAF inhibitors; mTOR; mTOR inhibitors; mTORC2; melanoma; resistance

DOI:  https://doi.org/10.3390/biomedicines9101498
Cells. 2021 Oct 08. pii: 2689. [Epub ahead of print]10(10):

SEA and GATOR 10 Years Later.

Yahir A Loissell-Baltazar, Svetlana Dokudovskaya.

  The SEA complex was described for the first time in yeast Saccharomyces cerevisiae ten years ago, and its human homologue GATOR complex two years later. During the past decade, many advances on the SEA/GATOR biology in different organisms have been made that allowed its role as an essential upstream regulator of the mTORC1 pathway to be defined. In this review, we describe these advances in relation to the identification of multiple functions of the SEA/GATOR complex in nutrient response and beyond and highlight the consequence of GATOR mutations in cancer and neurodegenerative diseases.

Keywords:  GATOR complex; SEA complex; amino acid signaling; autophagy; cancer; epilepsy; mTORC1 pathway; neurological disorders

DOI:  https://doi.org/10.3390/cells10102689
Biomolecules. 2021 Oct 06. pii: 1465. [Epub ahead of print]11(10):

Fission Yeast TORC2 Signaling Pathway Ensures Cell Proliferation under Glucose-Limited, Nitrogen-Replete Conditions.

Yusuke Toyoda, Shigeaki Saitoh.

  Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

Keywords:  Gad8/AKT kinase; TORC2; arrestin; endocytosis; glucose limitation; hexose transporter; nitrogen starvation; ubiquitylation

DOI:  https://doi.org/10.3390/biom11101465
Cell Rep. 2021 Oct 19. pii: S2211-1247(21)01334-6. [Epub ahead of print]37(3): 109867

The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.

Damien Toullec, Alberto Elías-Villalobos, Céline Faux, Ambre Noly, Gwendaline Lledo, Martial Séveno, Dominique Helmlinger.

  Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.

Keywords:  HSP90; PIKKs; TTT; assembly; biogenesis; chaperone; folding; multimeric complex; translation

DOI:  https://doi.org/10.1016/j.celrep.2021.109867
Nat Metab. 2021 Oct;3(10): 1284-1285

Nuclear PHGDH protects cancer cells from nutrient stress.

Daniela Annibali, Sarah-Maria Fendt.

DOI: https://doi.org/10.1038/s42255-021-00448-x
NPJ Regen Med. 2021 Oct 22. 6(1): 68

Analysis of the early response to spinal cord injury identified a key role for mTORC1 signaling in the activation of neural stem progenitor cells.

Johany Peñailillo, Miriam Palacios, Constanza Mounieres, Rosana Muñoz, Paula G Slater, Elena De Domenico, Ilya Patrushev, Mike Gilchrist, Juan Larraín.

Xenopus laevis are able to regenerate the spinal cord during larvae stages through the activation of neural stem progenitor cells (NSPCs). Here we use high-resolution expression profiling to characterize the early transcriptome changes induced after spinal cord injury, aiming to identify the signals that trigger NSPC proliferation. The analysis delineates a pathway that starts with a rapid and transitory activation of immediate early genes, followed by migration processes and immune response genes, the pervasive increase of NSPC-specific ribosome biogenesis factors, and genes involved in stem cell proliferation. Western blot and immunofluorescence analysis showed that mTORC1 is rapidly and transiently activated after SCI, and its pharmacological inhibition impairs spinal cord regeneration and proliferation of NSPC through the downregulation of genes involved in the G1/S transition of cell cycle, with a strong effect on PCNA. We propose that the mTOR signaling pathway is a key player in the activation of NPSCs during the early steps of spinal cord regeneration.

DOI: https://doi.org/10.1038/s41536-021-00179-3
Nat Metab. 2021 Oct;3(10): 1357-1371

The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress.

Chunmin Ma, Ke Zheng, Kun Jiang, Qin Zhao, Nannan Sha, Wang Wang, Man Yan, Tao Chen, Yuzheng Zhao, Yuhui Jiang.

The multifunctional roles of metabolic enzymes allow for the integration of multiple signals to precisely transduce external stimuli into cell fate decisions. Elevation of 3-phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme for de novo serine biosynthesis, is broadly associated with human cancer development; although how PHGDH activity is regulated and its implication in tumorigenesis remains unclear. Here we show that glucose restriction induces the phosphorylation of PHGDH by p38 at Ser371, which promotes the translocation of PHGDH from the cytosol into the nucleus. Concurrently, AMPK phosphorylates PHGDH-Ser55, selectively increasing PHGDH oxidation of malate into oxaloacetate, thus generating NADH. In the nucleus, the altered PHGDH activity restricts NAD+ level and compartmentally repressed NAD+-dependent PARP1 activity for poly(ADP-ribosyl)ation of c-Jun, thereby leading to impaired c-Jun transcriptional activity linked to cell growth inhibition. Physiologically, nuclear PHGDH sustains tumour growth under nutrient stress, and the levels of PHGDH-Ser371 and PHGDH-Ser55 phosphorylation correlate with p38 and AMPK activity, respectively, in clinical human pancreatic cancer specimens. These findings illustrate a previously unidentified nutrient-sensing mechanism with the critical involvement of a non-canonical metabolic effect of PHGDH and underscore the functional importance of alternative PHGDH activity in tumorigenesis.

DOI: https://doi.org/10.1038/s42255-021-00456-x
Trends Immunol. 2021 Oct 15. pii: S1471-4906(21)00205-2. [Epub ahead of print]

Ragulator-Rag and ROS TORment gasdermin D pore formation.

Venkat Giri Magupalli, Pietro Fontana, Hao Wu.

Upon cleavage, the Gasdermin D (GSDMD) N-terminal fragment assembles into pores on the plasma membrane to orchestrate the lytic cell death known as pyroptosis. In a recent article, Evavold et al. showed that the Ragulator-Rag-mTORC1-ROS pathway controls the transition from cleavage and membrane localization to oligomerization and pore formation.

DOI: https://doi.org/10.1016/j.it.2021.09.014
Int J Mol Sci. 2021 Oct 14. pii: 11088. [Epub ahead of print]22(20):

Role of PI3K-AKT-mTOR Pathway as a Pro-Survival Signaling and Resistance-Mediating Mechanism to Therapy of Prostate Cancer.

Thanakorn Pungsrinont, Julia Kallenbach, Aria Baniahmad.

  Androgen deprivation therapy (ADT) and androgen receptor (AR)-targeted therapy are the gold standard options for treating prostate cancer (PCa). These are initially effective, as localized and the early stage of metastatic disease are androgen- and castration-sensitive. The tumor strongly relies on systemic/circulating androgens for activating AR signaling to stimulate growth and progression. However, after a certain point, the tumor will eventually develop a resistant stage, where ADT and AR antagonists are no longer effective. Mechanistically, it seems that the tumor becomes more aggressive through adaptive responses, relies more on alternative activated pathways, and is less dependent on AR signaling. This includes hyperactivation of PI3K-AKT-mTOR pathway, which is a central signal that regulates cell pro-survival/anti-apoptotic pathways, thus, compensating the blockade of AR signaling. The PI3K-AKT-mTOR pathway is well-documented for its crosstalk between genomic and non-genomic AR signaling, as well as other signaling cascades. Such a reciprocal feedback loop makes it more complicated to target individual factor/signaling for treating PCa. Here, we highlight the role of PI3K-AKT-mTOR signaling as a resistance mechanism for PCa therapy and illustrate the transition of prostate tumor from AR signaling-dependent to PI3K-AKT-mTOR pathway-dependent. Moreover, therapeutic strategies with inhibitors targeting the PI3K-AKT-mTOR signal used in clinic and ongoing clinical trials are discussed.

Keywords:  AR antagonist resistance; PI3K; PKB/AKT; androgen receptor; castration-resistance; mTOR; prostate cancer

DOI:  https://doi.org/10.3390/ijms222011088
Nat Commun. 2021 Oct 22. 12(1): 6144

RIPK1 regulates starvation resistance by modulating aspartate catabolism.

Xinyu Mei, Yuan Guo, Zhangdan Xie, Yedan Zhong, Xiaofen Wu, Daichao Xu, Ying Li, Nan Liu, Zheng-Jiang Zhu.

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1-/- cells enhances the TCA flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1-/- deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance.

DOI: https://doi.org/10.1038/s41467-021-26423-4
Exp Eye Res. 2021 Oct 14. pii: S0014-4835(21)00359-6. [Epub ahead of print] 108793

Quantifying inter-organelle membrane contact sites using proximity ligation assay in fixed optic nerve sections.

Jared Ching, Andrew Osborne, Richard Eva, Julien Prudent, Patrick Yu-Wai-Man.

  Membrane contact sites (MCS) play crucial roles in cell physiology with dysfunction in several MCS proteins being linked with neurological and optic nerve diseases. Although there have been significant advances in imaging these interactions over the past two decades with advanced electron microscopy techniques, super-resolution imaging and proximity-dependent fluorescent reporters, a technique to observe and quantify MCS in mammalian optic nerve tissues has not yet been reported. We demonstrate for the first time that proximity ligation assay (PLA), a technique already used in mammalian cell lines, can be used as an efficient method of quantifying inter-organelle contact sites, namely mitochondria-endoplasmic reticulum (ER) and mitochondria-late-endosomes, in mammalian optic nerve tissues treated with adeno-associated virus (AAV) gene therapy with wild-type or phosphomimetic (active) protrudin. PLA utilises complementary single-stranded DNA oligomers bound to secondary antibodies that hybridise and complete a circular piece of DNA when the primary antibodies of interest interact. These interactions can be detected by amplifying the circular DNA and adding fluorescent probes. We show that PLA is a useful method that can be used to quantify MCS in optic nerve tissues. We have found that upregulation of protrudin with gene therapy significantly increases the number of mitochondria-ER and mitochondria-Rab7-late endosomes contact sites in optic nerves.

Keywords:  Membrane contact sites; Optic nerve; Protrudin; Proximity ligation assay; Retinal ganglion cells

DOI:  https://doi.org/10.1016/j.exer.2021.108793
Cells. 2021 Oct 01. pii: 2630. [Epub ahead of print]10(10):

A Novel Likely Pathogenic Variant in the BLOC1S5 Gene Associated with Hermansky-Pudlak Syndrome Type 11 and an Overview of Human BLOC-1 Deficiencies.

Doris Boeckelmann, Mira Wolter, Barbara Käsmann-Kellner, Udo Koehler, Lea Schieber-Nakamura, Barbara Zieger.

  Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.

Keywords:  BLOC1S5; HPS-11; Hermansky-Pudlak syndrome; bleeding tendency; hypopigmentation; oculocutaneous albinism

DOI:  https://doi.org/10.3390/cells10102630
Genes (Basel). 2021 Oct 08. pii: 1585. [Epub ahead of print]12(10):

Renal Cell Carcinoma in Tuberous Sclerosis Complex.

Elizabeth P Henske, Kristine M Cornejo, Chin-Lee Wu.

  Tuberous sclerosis complex (TSC) is an autosomal dominant disorder in which renal manifestations are prominent. There are three major renal lesions in TSC: angiomyolipomas, cysts, and renal cell carcinoma (RCC). Major recent advances have revolutionized our understanding of TSC-associated RCC, including two series that together include more than 100 TSC-RCC cases, demonstrating a mean age at onset of about 36 years, tumors in children as young as 7, and a striking 2:1 female predominance. These series also provide the first detailed understanding of the pathologic features of these distinctive tumors, which include chromophobe-like features and eosinophilia, with some of the tumors unclassified. This pathologic heterogeneity is distinctive and reminiscent of the pathologic heterogeneity in Birt-Hogg-Dube-associated RCC, which also includes chromophobe-like tumors. Additional advances include the identification of sporadic counterpart tumors that carry somatic TSC1/TSC2/mTOR mutations. These include unclassified eosinophilic tumors, eosinophilic solid cystic RCC (ESC-RCC), and RCC with leiomyomatous stroma (RCCLMS). A variety of epithelial renal neoplasms have been identified both in patients with tuberous sclerosis complex (TSC) and in the nonsyndromic setting associated with somatic mutations in the TSC1 and TSC2 genes. Interestingly, whether tumors are related to a germline or somatic TSC1/2 mutation, these tumors often display similar morphologic and immunophenotypic features. Finally, recent work has identified molecular links between TSC and BHD-associated tumors, involving the TFEB/TFE3 transcription factors.

Keywords:  RCC with leiomyomatous stroma; TSC1; TSC2; chromophobe RCC; eosinophilic solid cystic RCC; hybrid oncocytic chromophobe tumor; renal cell carcinoma; tuberous sclerosis complex

DOI:  https://doi.org/10.3390/genes12101585
Cold Spring Harb Mol Case Stud. 2021 Oct;pii: a006106. [Epub ahead of print]7(5):

Prenatal hydrops fetalis associated with infantile free sialic acid storage disease due to a novel homozygous deletion in the SLC17A5 gene.

Afia Hasnain, Sherri Burnett, Ronald Agatep, Elizabeth Spriggs, Bernard Chodirker, Aizeddin Aziz A Mhanni.

  Nonimmune hydrops fetalis, the excessive accumulation of serous fluid in the subcutaneous tissues and serous cavities of the fetus, has many possible etiologies, providing a diagnostic challenge for the physician. Lysosomal storage diseases have been reported in up to 5%-16% of nonimmune hydrops fetalis pregnancies. Infantile free sialic acid storage disease (ISSD) (OMIM #269920) is a severe form of autosomal recessive sialic acid storage disease. ISSD is caused by mutations in SLC17A5 (OMIM #604322), which encodes sialin, a lysosomal-membrane sialic acid transporter. We describe a case of fetal hydrops due to a novel homozygous deletion in the SLC17A5 gene. Prenatal single-nucleotide polymorphism (SNP) array analysis was performed on amniocytes after the discovery of fetal hydrops at 24 wk gestation revealing no copy-number variants. The SNP array, however, reported several regions of homozygosity (ROHs) including one on Chromosome 6 encompassing the SLC17A5 gene. High levels of urine sialic acid in the newborn were detected. SLC17A5 gene sequencing was initiated with no sequence variants identified; however, the assay failed to amplify exons 8 and 9, prompting an exon-level copy-number analysis that revealed a novel homozygous deletion of exons 8 and 9, inherited from heterozygous carrier parents. ISSD should be considered in the workup of patients with nonimmune hydrops fetalis, and analysis for SLC17A5 deletions should be carried out when variants are not detected by gene sequencing.

Keywords:  hydrops fetalis; nonimmune hydrops fetalis; severe hydrops fetalis

DOI:  https://doi.org/10.1101/mcs.a006106
Nat Metab. 2021 Oct;3(10): 1327-1341

Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice.

Heidi H Pak, Spencer A Haws, Cara L Green, Mikaela Koller, Mitchell T Lavarias, Nicole E Richardson, Shany E Yang, Sabrina N Dumas, Michelle Sonsalla, Lindsey Bray, Michelle Johnson, Stephen Barnes, Victor Darley-Usmar, Jianhua Zhang, Chi-Liang Eric Yen, John M Denu, Dudley W Lamming.

Calorie restriction (CR) promotes healthy ageing in diverse species. Recently, it has been shown that fasting for a portion of each day has metabolic benefits and promotes lifespan. These findings complicate the interpretation of rodent CR studies, in which animals typically eat only once per day and rapidly consume their food, which collaterally imposes fasting. Here we show that a prolonged fast is necessary for key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissect the effects of calories and fasting, and proceed to demonstrate that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that daily prolonged fasting, and not solely reduced caloric intake, is likely responsible for the metabolic and geroprotective benefits of a CR diet.

DOI: https://doi.org/10.1038/s42255-021-00466-9
Nat Commun. 2021 Oct 18. 12(1): 6065

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans.

Darshini Jeyasimman, Bilge Ercan, Dennis Dharmawan, Tomoki Naito, Jingbo Sun, Yasunori Saheki.

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

DOI: https://doi.org/10.1038/s41467-021-26177-z
Elife. 2021 Oct 18. pii: e67900. [Epub ahead of print]10

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain.

Shahzad S Khan, Yuriko Sobu, Herschel S Dhekne, Francesca Tonelli, Kerryn Berndsen, Dario R Alessi, Suzanne R Pfeffer.

  Activating LRRK2 mutations cause Parkinson's disease, and pathogenic LRRK2 kinase interferes with ciliogenesis. Previously, we showed that cholinergic interneurons of the dorsal striatum lose their cilia in R1441C LRRK2 mutant mice (Dhekne et al., 2018). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway.

Keywords:  cell biology; mouse; neuroscience

DOI:  https://doi.org/10.7554/eLife.67900
Genes (Basel). 2021 Sep 29. pii: 1545. [Epub ahead of print]12(10):

Comparative Transcriptome Analysis in Monocyte-Derived Macrophages of Asymptomatic GBA Mutation Carriers and Patients with GBA-Associated Parkinson's Disease.

Tatiana Usenko, Anastasia Bezrukova, Katerina Basharova, Alexandra Panteleeva, Mikhail Nikolaev, Alena Kopytova, Irina Miliukhina, Anton Emelyanov, Ekaterina Zakharova, Sofya Pchelina.

  Mutations of the GBA gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are the greatest genetic risk factor for Parkinson's disease (PD) with frequency between 5% and 20% across the world. N370S and L444P are the two most common mutations in the GBA gene. PD carriers of severe mutation L444P in the GBA gene is characterized by the earlier age at onset compared to N370S. Not every carrier of GBA mutations develop PD during one's lifetime. In the current study we aimed to find common gene expression signatures in PD associated with mutation in the GBA gene (GBA-PD) using RNA-seq. We compared transcriptome of monocyte-derived macrophages of 5 patients with GBA-PD (4 L444P/N, 1 N370S/N) and 4 asymptomatic GBA mutation carriers (GBA-carriers) (3 L444P/N, 1 N370S/N) and 4 controls. We also conducted comparative transcriptome analysis for L444P/N only GBA-PD patients and GBA-carriers. Revealed deregulated genes in GBA-PD independently of GBA mutations (L444P or N370S) were involved in immune response, neuronal function. We found upregulated pathway associated with zinc metabolism in L444P/N GBA-PD patients. The potential important role of DUSP1 in the pathogenesis of GBA-PD was suggested.

Keywords:  GBA; Parkinson’s disease; RNA-seq; macrophages; transcriptome

DOI:  https://doi.org/10.3390/genes12101545
Nat Commun. 2021 Oct 18. 12(1): 6064

Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.

Idil Ulengin-Talkish, Matthew A H Parson, Meredith L Jenkins, Jagoree Roy, Alexis Z L Shih, Nicole St-Denis, Gergo Gulyas, Tamas Balla, Anne-Claude Gingras, Péter Várnai, Elizabeth Conibear, John E Burke, Martha S Cyert.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

DOI: https://doi.org/10.1038/s41467-021-26326-4
Blood Cancer Discov. 2021 Mar;2(2): 116-118

MYC and TFEB Control DNA Methylation and Differentiation in AML.

Xiaoying Wu, Robert N Eisenman.

Although the MYC transcription factor has been consistently implicated in acute myeloid leukemia (AML), its gene targets and precise role in leukemogenesis remain unknown. In this issue of Blood Cancer Discovery, Yun and colleagues provide evidence that MYC directly suppresses the expression of TFEB, an mTORC1-regulated transcription factor. They show that, in the context of the myelocytic/granulocytic lineage, TFEB acts as a tumor suppressor by inducing the IDH1/2-TET pathway, which in turn, leads to altered DNA methylation and increased expression of genes involved in myeloid differentiation and apoptosis. Therefore, high levels of MYC suppress an epigenetic pathway that should normally act to attenuate leukemic progression. Identification of the components of this pathway is likely to inform new therapeutic tactics for AML and possibly other cancers. See related article by Yun et al., p. 162.

DOI: https://doi.org/10.1158/2643-3230.BCD-20-0230