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ABBREVIATIONS: A:C autophagic membrane:cytosol; ALS amyotrophic lateral sclerosis; ATG4 autophagy related 4; Atg8 autophagy related 8; BafA1 bafilomycin A1; BNIP3L/Nix BCL2 interacting protein 3 like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; EBSS Earle's balanced salt solution; GABARAP GABA type A receptor-associated protein; GST glutathione S transferase; HKO hexa knockout; Kd dissociation constant; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NLS nuclear localization signal/sequence; PE phosphatidylethanolamine; SpHfl1 Schizosaccharomyces pombeorganic solute transmembrane transporter; SQSTM1/p62 SQSTM1/p62; TARDBP/TDP-43 TAR DNA binding protein; TKO triple knockout.

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Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or pathological roles. RNP granules contain RNA and numerous RNA-binding proteins, transiently formed through the liquid-liquid phase separation. The assembly or disassembly of numerous RNP granules is strongly controlled to maintain their homeostasis and perform their cellular functions properly. Normal RNA granules are reversibly assembled, whereas abnormal RNP granules accumulate and associate with various neurodegenerative diseases. This review summarizes current studies on the physiological or pathological roles of post-translational modifications of various cellular RNP granules and discusses the therapeutic methods in curing diseases related to abnormal RNP granules by autophagy.

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Stress granules (SGs) are membraneless cytosolic granules containing dense aggregations of RNA-binding proteins and RNAs. They appear in the cytosol under stress conditions and inhibit the initiation of mRNA translation. SGs are dynamically assembled under stressful conditions and rapidly disassembled after stress removal. They are heterogeneous in their RNA and protein content and are cell type- and stress-specific. In post-mitotic neurons, which do not divide, the dynamics of neuronal SGs are tightly regulated, implying that their dysregulation leads to neurodegeneration. Mutations in RNA-binding proteins are associated with SGs. SG components accumulate in cytosolic inclusions in many neurodegenerative diseases, such as frontotemporal dementia and amyotrophic lateral sclerosis. Although SGs primarily mediate a pro-survival adaptive response to cellular stress, abnormal persistent SGs might develop into aggregates and link to the pathogenesis of diseases. In this review, we present recent advances in the study of neuronal SGs in physiology and pathology, and discuss potential therapeutic approaches to remove abnormal, persistent SGs associated with neurodegeneration.

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The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy- related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CAΔα3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs. [BMB Reports 2021; 54(2): 118-123].

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C-terminal fragments of Tar DNA-binding protein 43 (TDP-43) have been identified as the major pathological protein in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how they affect cellular toxicity and neurodegeneration, including the modulation process remains unknown. This study revealed that the C-terminal fragment of TDP-43 (TDP-25) was localized primarily to mitochondria and caused abnormal mitochondrial morphology, inducing Parkin-mediated mitophagy. Also, we discovered that the knockdown of selective autophagy receptors, such as TAX1BP, Optineurin, or NDP52 caused TDP-25 accumulation, indicating that TDP-25 was degraded by mitophagy. Interestingly, myosin IIB, a nonmuscle type of myosin and actin-based motor protein, is mostly colocalized to TDP-25 associated with abnormal mitochondria. In addition, myosin IIB inhibition by siRNA or blebbistatin induced mitochondrial accumulation of insoluble TDP-25 and Tom20, and reduced neuronal cell viability. Our results suggest a novel role of myosin IIB in mitochondrial degradation of toxic TDP-25. Therefore, we proposed that regulating myosin IIB activity might be a potential therapeutic target for neurodegenerative diseases associated with TDP-43 pathology.

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Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.

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The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways. [BMB Reports 2019; 52(12): 700-705].

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Autophagy allows for lysosomal cellular degradation of cytosolic components. In particular, neuronal autophagy is essential for cellular homeostasis and neuronal survival and is tightly regulated by several autophagy-related (ATG) proteins in post-mitotic neurons. Among these ATG proteins, the LC3/GABARAP proteins are known to regulate autophagosome biogenesis/maturation and cargo recognition. However, little is known about the role of GABARAP family proteins in neuronal autophagy despite their abundant expression in post-mitotic neurons. We have previously developed HyD (Hydrophobic Domain)-LIR (LC3-interacting region)-based autophagosome markers. In this study, to monitor GABARAP family proteins in autophagosomes of post-mitotic neurons, we improved the sensitivity of the probes for specifically detecting endogenous GABARAP family proteins by adding one more LIR motif to the LIR probes. We have tested the efficiency of two different LIRs, from ULK2 and Stbd1, in regard to their cellular localization to autophagosomes. HyD-2xLIR(ULK2)-GFP and HyD-2xLIR(Stbd1)-GFP demonstrated specific localization to GABARAP-positive autophagosomes relative to LC3B-positive autophagosomes in MEF/HeLa cells in an autophagy-dependent manner. Indeed, HyD-2xLIR(Stbd1)-GFP could efficiently detect GABARAP-positive autophagosomes in cultured cortical neurons. Our improved GABARAP-sensitive probes will contribute toward understanding the specific role of GABARAP family proteins in regard to neuronal autophagy.

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Heme oxygenase-1 (HO-1) has anti-apoptotic and anti-inflammatory effects. In this study, the HO-1 gene was delivered into the brain using dexamethasone-conjugated polyamidoamine generation 2 (PAMAM G2-Dexa) for the treatment of ischemic stroke. PAMAM G2-Dexa formed stable complexes with plasmid DNA (pDNA). The pDNA delivery efficiency of PAMAM G2-Dexa was higher than that of polyethylenimine (PEI25k, 25 kDa), dexamethasone-conjugated PEI (PEI-Dexa), and PAMAM G2 in Neuro2A cells. Therapeutic effect of PAMAM G2-Dexa/pHO-1 complexes was evaluated in a stroke animal model. PAMAM G2-Dexa delivered pHO-1 more efficiently into the ischemic brain than PEI25k and PEI-Dexa with higher therapeutic effect. Therefore, PAMAM G2-Dexa/pHO-1 complexes may be useful for ischemic stroke gene therapy.

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Fluorescein-labeled hyaluronic acids (HA) were immobilized on gold nanoparticles for reactive oxygen species (ROS) detection. The efficacy of HA immobilized gold nanoparticles (HHAuNPs) was evaluated in a stroke animal model. The stroke rat model was produced by transient middle cerebral artery occlusion (MCAO), which induced transient ischemia and reperfusion (I/R) in the brain. The increase of ROS in the I/R brain was confirmed by TBARS assay with the brain extracts. For brain imaging, HHAuNPs were injected into the rat brain 1 h before transient MCAO. Five hours after the injection, the rats were sacrificed and the brains were subjected to imaging analysis. The results showed that stronger signals were detected in the I/R brains than in the normal brains. To identify the time window for effective detection of ROS, HHAuNPs were injected into the post-ischemic rat brains at various time points. The results showed that ROS level reached a maximum at 24 h after the transient MCAO. Also, a live imaging study was performed with HHAuNPs in the normal and I/R animals. The results confirmed that ROS level increased in the I/R animal group with time, while the signal was decreased in the normal animal group. Together, our results suggest that HHAuNPs may be useful to monitor ROS level in the ischemic brain and to identify the infarct areas in ischemic brains for the treatment of stroke.

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