HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.

Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.

The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.

Membrane rupture of lysosomes results in leakage of their contents, which is harmful to cells. Recent studies have reported that several systems contribute to the repair or elimination of damaged lysosomes. Lysophagy is a type of selective autophagy that plays a crucial role in the lysosomal damage response. Because multiple pathways are involved in this response, an assay that specifically evaluates lysophagy is needed. Here, we developed the TMEM192-mKeima probe to evaluate lysophagy. By comparing the use of this probe with the conventional galectin-3 assay, we showed that this probe is more specific to lysophagy. Using TMEM192-mKeima, we showed that TFEB and p62 are important for the lysosomal damage response but not for lysophagy, although they have previously been considered to be involved in lysophagy. We further investigated the initial steps in lysophagy and identified UBE2L3, UBE2N, TRIM10, 16, and 27 as factors involved in it. Our results demonstrate that the TMEM192-mKeima probe is a useful tool for investigating lysophagy.

Neuronal MML-1/MXL-2 regulates systemic aging via glutamate transporter and cell nonautonomous autophagic and peroxidase activity.

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

Opposing roles of RUBCN isoforms in autophagy and memory B cell generation.

RUBCN (also known as Rubicon) was originally identified as a negative regulator of autophagy, a process by which cells degrade and recycle damaged components or organelles and that requires the activity of the class III PI3K VPS34 and the mTORC1 protein complex. Here, we characterized the role of a shorter isoform, RUBCN100, as an autophagy-promoting factor in B cells. RUBCN100 was translated from alternative translation initiation sites and lacked the RUN domain of the longer, previously characterized RUBCN130 isoform. Specific deficiency of RUBCN130 in B cells enhanced autophagy, which promoted memory B cell generation. In contrast to RUBCN130, which is localized in late endosomes and lysosomes and suppresses the enzymatic activity of VPS34, an effect thought to mediated by its RUN domain, RUBCN100 was preferentially located in early endosomes and enhanced VPS34 activity, presumably because of the absence of the RUN domain. Furthermore, RUBCN100, but not RUBCN130, enhanced autophagy and suppressed mTORC1 activation. Our findings reveal that the opposing roles of two RUBCN isoforms are critical for autophagy regulation and memory B cell differentiation.

Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.

Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes. For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

TFEB-mediated lysosomal exocytosis alleviates high-fat diet-induced lipotoxicity in the kidney.

Obesity is a major risk factor for end-stage kidney disease. We previously found that lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in obesity-related kidney disease, in both humans and experimental animal models. However, the regulatory factors involved in countering renal lipotoxicity are largely unknown. Here, we found that palmitic acid strongly promoted dephosphorylation and nuclear translocation of transcription factor EB (TFEB) by inhibiting the mechanistic target of rapamycin kinase complex 1 pathway in a Rag GTPase-dependent manner, though these effects gradually diminished after extended treatment. We then investigated the role of TFEB in the pathogenesis of obesity-related kidney disease. Proximal tubular epithelial cell-specific (PTEC-specific) Tfeb-deficient mice fed a high-fat diet (HFD) exhibited greater phospholipid accumulation in enlarged lysosomes, which manifested as multilamellar bodies (MLBs). Activated TFEB mediated lysosomal exocytosis of phospholipids, which helped reduce MLB accumulation in PTECs. Furthermore, HFD-fed, PTEC-specific Tfeb-deficient mice showed autophagic stagnation and exacerbated injury upon renal ischemia/reperfusion. Finally, higher body mass index was associated with increased vacuolation and decreased nuclear TFEB in the proximal tubules of patients with chronic kidney disease. These results indicate a critical role of TFEB-mediated lysosomal exocytosis in counteracting renal lipotoxicity.

How cells recognize and remove the perforated lysosome.

Macroautophagy (hereafter autophagy) is a highly conserved intracellular degradation system to maintain cellular homeostasis by degrading cellular components such as misfolded proteins, nonfunctional organelles, pathogens, and cytosol. Conversely, selective autophagy targets and degrades specific cargo, such as organelles, bacteria, etc. We previously reported that damaged lysosomes are autophagy targets, via a process called lysophagy. However, how cells target damaged lysosomes through autophagy is not known. We performed proteomics analysis followed by siRNA screening to identify genes involved in targeting damaged lysosomes and identified a new E3 ligase complex, involving CUL4A (cullin 4A), as a regulatory complex in lysophagy. We also found that this complex mediates K48-linked poly-ubiquitination on lysosome protein LAMP2 during lysosomal damage; particularly, the lumenal side of LAMP2 is important to recruit the complex to damaged lysosomes. This protein modification is thus critical to initiate the clearance of damaged lysosomes.

The mechanisms and roles of selective autophagy in mammals.

Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.

V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy.

Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.

Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.

Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence.

Senescent cells accumulation is associated with aging and age-related diseases, and recent findings suggest that autophagy, the activity of the intracellular degradation system, decreases during senescence. In this protocol, we detail steps to induce cellular senescence in response to DNA damage, evaluate the senescent state using SA-ß-gal staining and western blot for p21, LAMP1, and Lamin B1, and detect autophagy via LC3 western blotting. This protocol can be used in most cell lines and for various types of senescent cells. For complete details on the use and execution of this protocol, please refer to Yamamoto-Imoto et al. (2022).

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.

Loss of RUBCN/rubicon in adipocytes mediates the upregulation of autophagy to promote the fasting response.

Upon fasting, adipocytes release their lipids that accumulate in the liver, thus promoting hepatic steatosis and ketone body production. However, the mechanisms underlying this process are not fully understood. In this study, we found that fasting caused a substantial decrease in the adipose levels of RUBCN/rubicon, a negative regulator of macroautophagy/autophagy, along with an increase in autophagy. Adipose-specific rubcn-knockout mice exhibited systemic fat loss that was not accelerated by fasting. Genetic inhibition of autophagy in adipocytes in fasted mice led to a reduction in fat loss, hepatic steatosis, and ketonemia. In terms of mechanism, autophagy decreased the levels of its substrates NCOA1/SRC-1 and NCOA2/TIF2, which are also coactivators of PPARG/PPARγ, leading to a fasting-induced reduction in the mRNA levels of adipogenic genes in adipocytes. Furthermore, RUBCN in adipocytes was degraded through the autophagy pathway, suggesting that autophagic degradation of RUBCN serves as a feedforward system for autophagy induction during fasting. Collectively, we propose that loss of adipose RUBCN promotes a metabolic response to fasting via increasing autophagic activity.

Age-associated decline of MondoA drives cellular senescence through impaired autophagy and mitochondrial homeostasis.

Accumulation of senescent cells affects organismal aging and the prevalence of age-associated disease. Emerging evidence suggests that activation of autophagy protects against age-associated diseases and promotes longevity, but the roles and regulatory mechanisms of autophagy in cellular senescence are not well understood. Here, we identify the transcription factor, MondoA, as a regulator of cellular senescence, autophagy, and mitochondrial homeostasis. MondoA protects against cellular senescence by activating autophagy partly through the suppression of an autophagy-negative regulator, Rubicon. In addition, we identify peroxiredoxin 3 (Prdx3) as another downstream regulator of MondoA essential for mitochondrial homeostasis and autophagy. Rubicon and Prdx3 work independently to regulate senescence. Furthermore, we find that MondoA knockout mice have exacerbated senescence during ischemic acute kidney injury (AKI), and a decrease of MondoA in the nucleus is correlated with human aging and ischemic AKI. Our results suggest that decline of MondoA worsens senescence and age-associated disease.

Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.

Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).