Autophagy

Macroautophagy (hereafter autophagy) is a cellular recycling pathway that requires different ATG (autophagy-related) proteins to generate double-membraned autophagosomes. ATG9A, a multi-spanning membrane protein, plays a crucial role in this process as the only transmembrane component of the core autophagy machinery. ATG9A functions as a lipid scramblase, redistributing lipids between membrane leaflets for the expanding autophagosome membrane. Structural studies have revealed that ATG9A forms a homotrimer with an interlocked domain-swapped architecture and a network of internal hydrophilic cavities. This configuration underlies its role in lipid transfer and membrane remodeling together with the lipid transporter ATG2A. ATG9A dysfunction has also been linked to human disease, as specific ATG9A mutations cause neurodevelopmental or neurodegenerative phenotypes. Additionally, ATG9A is altered in cancer, promoting pro-tumorigenic traits. However, most missense variants in ATG9A remain uncharacterized, posing a significant challenge for interpreting genomic data. In this study, we employed in silico saturation mutagenesis approach using the MAVISp (Multi-layered Assessment of VarIants by Structure) framework to predict the impact of every missense mutation in ATG9A. By analyzing multiple structural assemblies of ATG9A (monomer, trimer, and the ATG9A-ATG2A complex), we evaluated diverse mechanistic indicators of variant impact, including protein stability, long-range conformational changes, effects on multimerization interfaces, and alterations in post-translational modifications. We integrated the structure-based predictions with Variant Effect Predictors from recent deep-learning or evolutionary-based models and cross-referenced known variants catalogued in ClinVar, COSMIC, and cBioPortal. Finally, we predicted mechanistic indicators for all possible variants with structural coverage not yet reported in the disease-related databases supported by MAVISp. Our analyses identified a group of potentially damaging variants in ATG9A and the possible molecular mechanisms underlying their effects. Together, this work provides a roadmap for interpreting missense variants in autophagy regulators and highlights specific ATG9A mutations that deserve further investigation in the context of human disease.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

The selective autophagy receptors SQSTM1/p62 and NBR1 are evolutionary related and involved in autophagy of different substrates including proteins, protein aggregates and organelles. The two proteins interact via their PB1 domains, and essential for their roles in autophagy is the formation of a specific type of condensates named p62 bodies. The scaffold of these structures is formed by the interaction of polymeric p62 with polyubiquitin, but NBR1 is recruited and essential for their formation. Previous studies have shown that p62 contains nuclear export signal (NES) and nuclear localization signal (NLS) motifs and shuttles between the cytoplasm and the nucleus. Its nuclear roles are not fully understood, but there is evidence that p62 is involved in protein quality control in the nucleus. No previous studies have tested if NBR1 is transported into the nucleus. We show here that NBR1 contains two NES motifs and one NLS motif, and like p62, the protein shuttles between the cytoplasm and the nucleus. NBR1 also accumulates in nuclear p62 bodies and the formation of nuclear p62 bodies depends on NBR1.

Autophagy contributes to normal cells physiology and is essential for progression of malignant tumors. While autophagy is mostly considered as a self-degradative and self-renewal process, it has non-degradative functions whose contribution to tumor progression is poorly explored. Here we use the autophagy dependent Drosophila RasV12, Scrib-/- carcinoma model to examine whether perturbation of distinct steps of autophagy differentially influences tumor progression. We found that inhibition of autophagosome formation, by mutating Atg13 or Atg6 either in the tumor or in the whole animal significantly decreased tumor growth. In contrast, blocking the later autophagosome-lysosome fusion (by loss of Vps39 or Syx17) and thereby autolysosomal degradation, does not reduce tumor size. We observed that an early (Atg13), but not a late (Vps39 or Syx17) block in autophagy showed reduced activity of JAK/STAT signaling, known to be critical for the progression of this tumor type. Importantly, we demonstrated that both Atg13 and Vps39 deficient tumors accumulated Stat92E inhibitor Su(var)2-10/dPIAS, a recently identified autophagic cargo, however in Vps39 mutants Su(var)2-10 is sequestered into autophagosomes. Finally, we found that reduction of Su(var)2-10 partially restores JAK/STAT signaling and rescues the growth of Atg13-deficient tumors, indicating its sequestration is a crucial mechanism to promote tumor progression.

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Biallelic variants in genes regulating endosomal, lysosomal and autophagy pathways are increasingly implicated in severe neurodevelopmental disorders, yet the contribution of the Rab7 effector WDR91 to human disease remains incompletely defined. We report a child with a severe neurodevelopmental disorder characterized by progressive microcephaly, microlissencephaly, corpus callosum hypoplasia, and early-onset epilepsy, harboring compound heterozygous WDR91 variants: a truncating variant (p.Gln215*) and a missense variant (p.Tyr15Asn). Functional analyses show that p.Gln215* abolishes WDR91 expression, whereas p.Tyr15Asn reduces protein abundance through increased degradation. Reduced WDR91 expression was confirmed in primary patient-derived cells. In silico analyses suggest that p.Tyr15Asn induces a localized change within an N-terminal degron-containing region, potentially affecting protein stability. In cellular models, both variants impair early-to-late endosomal maturation and alter WDR91 localization to Rab7-positive compartments. WDR91 deficiency is further associated with transcriptional and functional evidence of autophagy dysregulation. While the p.Tyr15Asn variant partially restores autophagic flux under overexpression conditions, patient-derived cells display impaired autophagic turnover, consistent with a context-dependent functional and partial loss of function effect of this variant. Together, these findings provide functional evidence supporting the pathogenicity of WDR91 variants and implicate combined defects in endosomal maturation and autophagy in WDR91-related neurodevelopmental disease.

Mitochondria engage in extensive communication with other organelles through membrane contacts. Perturbed mitochondria-organelle interactions are indicated in a variety of neurodegenerative diseases, but the underlying mechanisms remain poorly understood. Here, we report a new class of mitochondria-organelle communication: autophagosome/autophagic vacuole (AV)-mitochondria (Mito) contact, which exhibits hyper-tethering in tauopathy neurons, consequently hampering AV retrograde transport. Such defects are attributed to accelerated turnover of the contact release factor TBC1D15, triggered by mitochondrial bioenergetic deficit-induced hyperactivity of the AMP-activated protein kinase (AMPK). Increasing TBC1D15 levels or repressing AMPK activity normalizes AV-Mito contact release and restores retrograde transport of AVs, thereby increasing autophagic cargo clearance and reducing tau burden in tauopathy axons. Furthermore, overexpression of TBC1D15 enhances autophagic clearance and attenuates tau pathology, alleviating neurodegeneration and cognitive dysfunction in tauopathy mice. Taken together, our study provides new insights into AV-Mito contact dysregulation in tauopathy-related autophagy failure, laying the groundwork for the development of potential therapeutics to combat tauopathy diseases.

Autophagy is a critical neuroprotective mechanism, the impairment of which can lead to severe neurodegenerative diseases. Spinocerebellar ataxia type 1 (SCA1) is a monogenic neurodegenerative disorder, characterised by the presence of protein aggregates and consequent loss of cellular functions. The expression of mutant Ataxin1 (ATXN1) in glial cells has been demonstrated to induce inflammatory responses and loss of supportive functions, thereby exacerbating neuronal degeneration in SCA1. Autophagic dysfunction has been shown to affect both neurons and glial cells, resulting in widespread pathological consequences. In this work, we aimed to evaluate the efficacy of two small-molecule autophagy activators, AUTEN-67 and AUTEN-99, in models of glia-specific SCA1 in Drosophila. Our results demonstrate that AUTEN-99 has a stronger autophagy enhancing effect, with significantly improved response times and survival rates, compared to untreated ATXN1 mutants. Glia-specific assays in mouse primary hippocampal cultures also confirmed that AUTEN-99 is a more effective activator. Ultimately, co-treatment of neuronal and glial cultures did not reveal any synergistic benefits from combining the two AUTEN compounds compared to single-agent treatment. Our findings contribute to a better understanding of the utility of AUTENs and may help to understand the critical role of autophagy in neurodegenerative diseases.

Protein mislocalization, dysfunctional autophagy, and protein aggregation are key features of Huntington disease (HD). Accordingly, a central focus of our work has been identifying and correcting the protein mislocalization that drives the underlying autophagic defects which exacerbates protein aggregation. We previously demonstrated that palmitoylation, or S-acylation, of the autophagy receptor sequestosome 1 (SQSTM1; p62) is significantly reduced in the brains of HD patients and the YAC128 HD mouse model, thereby providing a possible mechanism for the cargo-loading failure observed in HD. Here, we identify the FDA-approved small molecule Vorinostat (suberoylanilide hydroxamic acid, SAHA) as a modulator of this pathway and show that it rescues SQSTM1 palmitoylation and restores autophagic function in HD models. Importantly, we demonstrate that Vorinostat crosses the blood-brain-barrier and significantly increases SQSTM1 palmitoylation in the cortices of YAC128 mice using acyl-biotin exchange and click chemistry assays. We further show that Vorinostat enhances autophagic flux, as evidenced by significant changes in autophagy marker levels and a marked increase in the colocalization of huntingtin with SQSTM1 and lysosomes. Finally, we investigate the mechanism of Vorinostat and propose a dual mode of action involving inhibition of depalmitoylating enzymes and transcriptional regulation of key pathway components. Collectively, these findings underscore SQSTM1 palmitoylation as a promising therapeutic target and support Vorinostat as a strong therapeutic candidate in HD.

Autophagy involves the rapid growth of phagophores through membrane addition. This growth is triggered by vesicles containing the Atg9A protein. However, Atg9A is not incorporated into mature autophagosomes. We now demonstrate that Dynamin-2 (Dnm2) colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1/SH3GLB1) and other autophagy proteins when autophagy is induced. Our data suggest that Atg9A is retrieved from phagophores via fission, with Dnm2 acting as the membrane scission protein. Blocking Atg9A recycling, either by mutating Dnm2, using RNA interference, or applying chemical inhibitors, results in Atg9A remaining in autophagosomes and being degraded during autophagy. Overall, these findings provide new insights into the roles of membrane-scission proteins in autophagy.

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features as well as surrounding and attached vesicles suggest that the enlarged rims are a result of cytosolic vesicles fusing with the growing phagophore. High-resolution imaging in this study shows that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

Mutations in Fused in Sarcoma (FUS), a RNA binding protein, cause Amyotrophic Lateral Sclerosis (ALS). ALS is an aggressive neurodegenerative disease resulting in motor neuron degeneration. Defects in synaptic integrity precede neuronal loss in ALS, but the mechanisms responsible for these early synaptic defects are unclear. To investigate early synaptic defects associated with ALS, we expressed an ALS-linked variant of human FUS in adult motor neurons and assessed synaptic pathology at the neuromuscular junction (NMJ). Here we highlight the accumulation of FUS-positive aggregates at synaptic terminals and subsequent reduction in microtubule stability. We show that inducing autophagy via expression of Rab1 or Fragile-X Mental Retardation Protein 1 (FMR1), or treatment with Rapamycin reduces aggregate formation and restores synaptic structure and function. These findings reveal the utility of inducing autophagy to address early synaptic dysfunction in an ALS model and demonstrate a potential therapeutic target to preventing later stages of disease progression.

Mitophagy is an essential quality control mechanism that maintains neuronal health by selectively removing damaged mitochondria via autophagosomes. In neurons, mitophagy is mainly driven by Optineurin (OPTN), a selective autophagy receptor that is recruited to damaged mitochondria. Consistent with its role in maintaining mitochondrial integrity, OPTN-mediated mitophagy is upregulated in response to mild oxidative stress. However, many mechanistic studies of mitophagy have relied on non-neuronal systems and acute mitochondrial damage paradigms. Thus, it remains unclear how well these findings translate to physiological stress conditions in neurons. Here, we investigated the temporal dynamics of neuronal mitophagy under mild oxidative stress using live-cell imaging in primary rat hippocampal neurons. Surprisingly, we found that in neurons, autophagosomes failed to readily engulf OPTN-positive (OPTN+) mitochondria, revealing a novel rate-limiting step in neuronal mitophagy. Interestingly, this inefficient engulfment was specific to OPTN+ mitochondria at mitophagy events. Given the slow progression of mitophagy, we extended our time course to define the timescale of OPTN-regulated mitophagy. Using a pulse-chase assay to monitor long-term mitochondrial turnover, we found that OPTN+ mitochondria colocalized with acidified lysosomes over a timescale significantly longer than reported in non-neuronal cells and acute neuronal models. Since inefficient autophagosome engulfment appeared to limit mitophagy, we stimulated autophagosome formation via nutrient deprivation, which increased lysosomal colocalization with damaged mitochondria and enhanced mitophagy flux. Together, these findings indicate that mitophagy proceeds relatively slowly in neurons, a characteristic that may contribute to neuronal vulnerability in neurodegenerative disease by promoting the accumulation of dysfunctional mitochondria.

Autophagy is a recycling pathway that clears cellular constituents, supporting homeostasis. In primary murine neurons, autophagosome biogenesis declines during aging. Importantly, this decline can be restored by the ectopic expression of key autophagy component WIPI2B. The phosphorylation state of WIPI2B serine 395 is critical for this restoration, suggesting that WIPI2B S395 phosphorylation regulates autophagosome biogenesis. Here, we identified protein phosphatase 2A (PP2A) and CDK16 as regulators of WIPI2B S395 phosphorylation and neuronal autophagy. Using Caenorhabditis elegans, we showed that PP2A and CDK16 regulate neuronal autophagy through the same genetic pathway as WIPI2B in vivo. Further, purified mammalian PP2A and CDK16 directly modified WIPI2B S395 phosphorylation in vitro. In primary murine neurons, PP2A and CDK16 colocalized with WIPI2B at autophagosomes, and manipulation of PP2A and CDK16 expression altered WIPI2B puncta formation and rates of autophagosome biogenesis. Altogether, our data support the conclusion that PP2A and CDK16 regulate WIPI2B S395 phosphorylation, modulating autophagosome biogenesis in neurons.

Rhabdomyolysis is the acute breakdown of skeletal muscle resulting from failure of cellular homeostasis in response to metabolic stress. Recurrent forms are frequently linked to inherited defects affecting energy metabolism or calcium handling. Ryanodine receptor type 3 (RyR3) is an intracellular calcium release channel, expressed in skeletal muscle, that contributes to the fine-tuning of calcium signaling. Although variants in other calcium-handling proteins have been implicated in rhabdomyolysis, the role of RyR3 has not been established. In this study, we report rare compound heterozygous missense variants in RYR3 identified in two unrelated individuals with severe, fever-triggered recurrent rhabdomyolysis. Muscle biopsies revealed mild structural changes with triadic disorganization, mitochondrial alterations, lipid accumulation, and autophagic material, while overall muscle architecture was largely preserved. Structural modeling supports the pathogenicity of the variants, and calcium flux analysis demonstrated significantly reduced ryanodine receptor-mediated calcium release in patient-derived myoblasts. Functional analyses showed that RyR3 deficiency impaired starvation-induced autophagy, characterized by defective autophagosome formation and reduced autophagic flux, and increased susceptibility to metabolic stress. Mitochondrial bioenergetic profiling revealed reduced oxidative phosphorylation capacity and decreased membrane potential under stress conditions, consistent with compromised mitochondrial adaptation. In zebrafish, ryr3 knockdown resulted in structural and functional muscle abnormalities, including reduced myotome area and decreased locomotor activity, associated with impaired autophagic flux. This study establishes a novel association between recessive RYR3 variants and recurrent rhabdomyolysis and identifies RyR3 as a critical regulator of skeletal muscle stress adaptation through calcium-dependent control of autophagy and mitochondrial homeostasis. More broadly, our findings further highlight autophagy as a central determinant of muscle resilience in the context of rhabdomyolysis.

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5flox/flox and Cd169cre mouse models to delete ATG5 in CD169+ macrophages, including RPMs. Atg5-deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5{Delta}Cd169 mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

The ULK1 complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) act together to initiate autophagy. Human ULK1C consists of ULK1 itself, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P generated by PI3KC3-C1 is essential to recruit and stabilize ULK1C on membranes for ULK1 to phosphorylate its membrane-associated substrates in autophagy induction, even though ULK1C subunits do not contain any PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, as well as with WIPI2. Bound to WIPI2-3, ATG13:ATG101 aligns with the membrane to insert its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that alignment of WIPIs and the ATG101 WF finger cooperatively stabilizes the complex on membranes, explaining the essential role of the WF residues in autophagy. Biochemical reconstitution and a cell-based assay show that WIPI3:ATG13 engagement is required for ATG16L1 phosphorylation by ULK1, ATG13 puncta formation, and bulk autophagic flux. We further showed that a kinase domain (KD)-proximal PVP motif within the ULK1 IDR docks onto the surface of the ATG13:ATG101 HORMA dimer and used molecular modeling to show how the ULK1 KD is brought close to the membrane surface. Biochemical reconstitution and cell-based assays show that the PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for starvation-induced autophagy and BNIP3/NIX-dependent mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface downstream of PI3KC3-C1.

While LRRK2 mutations modulate systemic glucose homeostasis and metabolic dysfunction precedes Parkinsons disease (PD) motor-symptoms, the impact of pathogenic LRRK2-mutations on astrocytic glucose-uptake and organellar function remains unexplored. Here, we demonstrate that LRRK2-I1371V mutation causes profound metabolic and organellar dysfunction in LRRK2-I1371V PD-iPSC-derived astrocytes and U87 cells overexpressing I1371V variant. LRRK2-I1371V astrocytes exhibit significantly reduced GLUT1 expression and plasma membrane localization, resulting in impaired glucose-uptake and decreased lactate-production. This metabolic insufficiency correlates with cascading mitochondrial dysfunction, characterized by membrane depolarization, elevated reactive-oxygen-species, enhanced ubiquitination and reduced proteasomal-activity. Reduced LAMP1/LAMP2 expression, impaired lysosomal-acidification, and selective cathepsin D deficiency were observed. Accumulation of undegraded cargo was confirmed by transmission-electron-microscopy upon -synuclein exposure. ER stress was evident through upregulated GADD34/CHOP, increased phospho-PERK, and reduced nascent protein synthesis. Our results reveal that LRRK2-I1371V induces glucose-uptake deficits, causing energy depletion and multi-organellar dysfunction, identifying astrocytic metabolic restoration as a promising therapeutic target for I1371V-associated PD.

Rab32 and Rab38 are paralogous small GTPases involved in the biogenesis of lysosome-related organelles (LROs), yet their roles in hepatic lipid metabolism remain poorly defined. Here, Rab32 and Rab38 double-knockout (DKO) male mice exhibited an age-dependent increase in body weight accompanied by hepatic lipid accumulation, suggesting impaired hepatic lipid processing. In AML12 hepatocytes, Rab32 and Rab38 localized to ring-like, LAMP1-positive structures characteristic of LROs, whose size increased with cell confluence. Pharmacological inhibition of lysosomal acid lipase with orlistat led to the accumulation of lipid droplets (LDs) within Rab32/38-positive LROs, indicating that LD degradation occurs in these compartments. Additional treatment with bafilomycin A1 revealed invagination-like internal membrane structures within enlarged LROs. These processes were not affected by artificial inhibition of macroautophagy, highlighting the involvement of microautophagy. Ring-like signals positive for phosphatidylinositol 3-phosphate (PI3P) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) were detected within or adjacent to LRO membranes, and LDs were frequently associated with these structures, suggesting a role for PI3P and PI(3,5)P2 in internal membrane formation. Vps4B was also required for efficient LD incorporation. Consistently, Rab32/38 double-knockdown (DKD) AML12 cells exhibited increased lipid accumulation, indicating impaired LD engulfment. Together, these findings identify Rab32/38-positive LROs as a structural platform for microautophagy-mediated lipid droplet degradation in hepatocytes.