evaluation-lc3-ii-release-via-extracellular-vesicles-relation-to

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf ...

Okayama University of Science

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.

Kinase activity-tagged western blotting assay.

Progranulin (PGRN) is a glycoprotein that is widely expressed among organs, including the central nervous system. PGRN insufficiency is involved in various neurodegenerative disorders such as frontotemporal dementia, Alzheimer's disease, and neuronal ceroid lipofuscinosis. One of the major causes of neuronal damage is hyperactivation of the cerebrum triggered by upregulation of excitatory systems. In the present study, we examined the possible involvement of PGRN in modulating excitability of the cerebrum using wild type and PGRN-deficient mice. First, we treated wild type and PGRN-deficient mice with seizure-inducible drugs, bicuculline or N-methyl-D-aspartate (NMDA), which provoke hyperexcitement of neurons. PGRN-deficient mice showed higher intensity of seizure and longer duration of convulsive behavior when treated with either bicuculline or NMDA. Next, we quantified the expression of NMDA receptor subunits in the hippocampus and cerebral cortex. The expression level of NR2A subunit protein was significantly higher in the hippocampus of PGRN-deficient mice, while no difference was observed in the cerebral cortex. On the other hand, mRNA levels of NMDA receptor subunits in the hippocampus were comparable or even lower in PGRN-deficient mice. These results suggest that PGRN modulates the excitability of the cerebrum by regulating at least partially the protein level of NMDA receptors in the hippocampus.

Convulsive responses to seizure-inducible drugs are exacerbated in progranulin-deficient mice.

CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals. Here we characterized CPI-17 N- and C-terminal unstructured tails using recombinant proteins that lack the potions. Recombinant CPI-17 proteins at a physiologic level (10 µM) were doped into beta-escin-permeabilized smooth muscle strips for Ca sensitization force measurement. The ectopic full-length CPI-17 augmented the PDBu-induced Ca sensitization force at pCa6.3, indicating myosin phosphatase inhibition. Deletion of N- and C-terminal tails of CPI-17 attenuated the extent of PDBu-induced Ca-sensitization force. The N-terminal deletion dampened phosphorylation at Thr38 by protein kinase C (PKC), and the C-terminal truncation lowered the affinity to the myosin phosphatase. Under the physiologic conditions, PKC and myosin phosphatase may recognize CPI-17 N-/C-terminal unstructured tails inducing Ca sensitization force in smooth muscle cells.

Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca sensitization force of smooth muscle.

Progranulin (PGRN) haploinsufficiency from autosomal dominant mutations in the PGRN gene causes frontotemporal lobar degeneration, which is characterized by cytoplasmic inclusions predominantly containing TDP-43 (FTLD-TDP). PGRN supplementation for patients with a PGRN gene mutation has recently been proposed as a therapeutic strategy to suppress FTLD-TDP. However, it currently remains unclear whether excessive amounts of PGRN are beneficial or harmful. We herein report the effects of PGRN overexpression on autophagic flux in a cultured cell model. PGRN overexpression increased the level of an autophagosome marker without promoting autophagosome formation and decreased the signal intensity of an autolysosome marker, indicating the suppression of autophagic flux due to reductions in the formation of autolysosomes. Assessments of lysosome numbers and biogenesis using LysoTracker and cells stably expressing TFEB-GFP, respectively, indicated that PGRN overexpression increased the lysosome numbers without lysosomal biogenesis. These results suggest that PGRN overexpression suppressed autophagic flux by inhibiting autophagosome-lysosome fusion. Moreover, PGRN overexpression enhanced polyglutamine aggregation and aggregate-prone TDP-43 accumulation, indicating that the suppression of autophagic flux by excessive amounts of PGRN worsens the pathology of neurodegenerative diseases.

Overexpression of progranulin increases pathological protein accumulation by suppressing autophagic flux.

Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades. However, limited information is currently available on the formation of autolysosomes, which may partially be attributed to fewer methods to study the formation of autolysosomes than that of autophagosomes. Abemaciclib (Abe) and vacuolin-1 (Vac) are drugs that suppress the progression of breast cancer and induce characteristic vacuole formation in cells. Since Abe-induced vacuoles have the appearance of autolysosomes, they may be used to examine the formation of autolysosomes. However, it remains unknown whether Abe-/Vac-induced vacuoles are regulated by autophagosome-lysosome fusion. Markers for endosomes, lysosomes, and autophagosomes (Rab7, LAMP1, and mRFP-GFP-LC3, respectively) indicated that Abe-/Vac-induced vacuoles were autolysosomes. Abe and Vac failed to induce vacuolation in ATG16L1-deficient autophagy-null cells. Furthermore, Abe-/Vac-induced vacuolation was suppressed by bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, whereas it was facilitated by rapamycin and the overexpression of Beclin-1, inducers of autophagosome-lysosome fusion. Moreover, vacuole formation was inhibited by the knockdown of progranulin (PGRN), a regulator of autophagosome-lysosome fusion, and promoted by its overexpression. The present results suggest the potential of Abe-/Vac-induced vacuole-like autolysosomes as a tool for evaluating autophagosome-lysosome fusion and examining the effects of PGRN in autophagy.

Abemaciclib and Vacuolin-1 induce vacuole-like autolysosome formation - A new tool to study autophagosome-lysosome fusion.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis.

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux. In the present study, the Abe and Vac treatment dose-dependently reduced the GFP/RFP ratio in SH-SY5Y neuroblastoma cells stably expressing the autophagic flux marker GFP-LC3-RFP-LC3ΔG. Abe and Vac also increased the omegasome marker GFP-ATG13 signal and the autophagosome marker mCherry-LC3 localized to the lysosome marker LAMP1-GFP. The Abe and Vac treatment decreased the intracellular level of the lysosome marker LAMP1-GFP in SH-SY5Y cells stably expressing LAMP1-GFP, but did not increase the levels of LAMP1-GFP, the autophagosome marker LC3-II, or the multivesicular body marker TSG101 in the extracellular vesicle-enriched fraction. Moreover, Abe and Vac treatment autophagy-dependently inhibited GFP-tagged aggregate-prone TDP-43 accumulation. The results of a PI(3)P reporter assay using the fluorescent protein tagged-2 × FYVE and LAMP1-GFP indicated that Abe and Vac increased the intensity of the PI(3)P signal on lysosomes. A treatment with the VPS34 inhibitor wortmannin (WM) suppressed Abe-/Vac-facilitated autophagic flux and the degradation of GFP-tagged aggregate-prone TDP-43. Collectively, these results suggest that Abe and Vac degrade aggregate-prone TDP-43 by accelerating autophagosome formation and autophagosome-lysosome fusion through the formation of PI(3)P.

Article	Year
Kinase activity-tagged western blotting assay. BioTechniques\| PubMed ID: 31939317	2020
Convulsive responses to seizure-inducible drugs are exacerbated in progranulin-deficient mice. Neuroreport\| PubMed ID: 32168097	2020
Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca sensitization force of smooth muscle. Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi\| PubMed ID: 35418530	2022
Overexpression of progranulin increases pathological protein accumulation by suppressing autophagic flux. Biochemical and biophysical research communications\| PubMed ID: 35483222	2022
Abemaciclib and Vacuolin-1 induce vacuole-like autolysosome formation - A new tool to study autophagosome-lysosome fusion. Biochemical and biophysical research communications\| PubMed ID: 35598430	2022
Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43. The Journal of biological chemistry\| PubMed ID: 37739033	2023
PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis. Biomolecules\| PubMed ID: 38136612	2023
Abemaciclib and Vacuolin-1 decrease aggregate-prone TDP-43 accumulation by accelerating autophagic flux. Biochemistry and biophysics reports\| PubMed ID: 38596406	2024

Yoshinori Tanaka

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