Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

An Extensive Study of the Influence of Key Flow Variables on Printed Line Quality Outcomes during Aerosol Jet Printing Using Coupled Three-Dimensional Numerical Models.

A three-dimensional (3D) numerical model was developed to explore the intricate aerodynamic mechanisms associated with aerosol jet printing (AJP). The proposed approach integrates computational fluid dynamics and discrete phase modeling, offering a comprehensive understanding of the deposition mechanisms of the AJP process. Initially, numerical solutions of the governing equations were obtained under the assumptions of compressible and laminar flows, facilitating an analysis of certain key flow variables, in this case, the sheath gas flow rate and carrier gas flow rate across the fluid domain. Subsequently, incorporating a Lagrangian discrete phase model allowed a detailed examination of the droplet behavior after nozzle ejection, considering the influence of the Saffman lift force. Finally, experiments were performed to elucidate the influence of key flow variables on the printed width. Generally, the measured printed line morphology and corresponding line electrical performance exhibited close conformity with the numerical model, demonstrating that the proposed numerical model is important for making well-informed decisions during process optimization.

L-serine restored lysosomal failure in cells derived from patients with BPAN reducing iron accumulation with eliminating lipofuscin.

Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2'-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether l-serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. l-serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and l-serine treatment reduced the increase of lipofuscin. These data suggest that l-serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity.

FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase.

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Mitochondrial matrix RTN4IP1/OPA10 is an oxidoreductase for coenzyme Q synthesis.

Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.

Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca2+ dynamics linked to DEE66.

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.

Super-resolution proximity labeling reveals anti-viral protein network and its structural changes against SARS-CoV-2 viral proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates in human cells by interacting with host factors following infection. To understand the virus and host interactome proximity, we introduce a super-resolution proximity labeling (SR-PL) method with a "plug-and-playable" PL enzyme, TurboID-GBP (GFP-binding nanobody protein), and we apply it for interactome mapping of SARS-CoV-2 ORF3a and membrane protein (M), which generates highly perturbed endoplasmic reticulum (ER) structures. Through SR-PL analysis of the biotinylated interactome, 224 and 272 peptides are robustly identified as ORF3a and M interactomes, respectively. Within the ORF3a interactome, RNF5 co-localizes with ORF3a and generates ubiquitin modifications of ORF3a that can be involved in protein degradation. We also observe that the SARS-CoV-2 infection rate is efficiently reduced by the overexpression of RNF5 in host cells. The interactome data obtained using the SR-PL method are presented at https://sarscov2.spatiomics.org. We hope that our method will contribute to revealing virus-host interactions of other viruses in an efficient manner.

Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics.

The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice.

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease.

Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.

Monoclonal antibody Y01 prevents tauopathy progression induced by lysine 280-acetylated tau in cell and mouse models.

The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer's disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies, including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau-Tg mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.

A multifunctional peroxidase-based reaction for imaging, sensing and networking of spatial biology.

Peroxidase is a heme-containing enzyme that reduces hydrogen peroxide to water by extracting electron(s) from aromatic compounds via a sequential turnover reaction. This reaction can generate various aromatic radicals in the form of short-lived "spray" molecules. These can be either covalently attached to proximal proteins or polymerized via radical-radical coupling. Recent studies have shown that these peroxidase-generated radicals can be utilized as effective tools for spatial research in biological systems, including imaging studies aimed at the spatial localization of proteins using electron microscopy, spatial proteome mapping, and spatial sensing of metabolites (e.g., heme and hydrogen peroxide). This review may facilitate the wider utilization of these peroxidase-based methods for spatial discovery in cellular biology.

Urinary Exosomal Cystatin C and Lipopolysaccharide Binding Protein as Biomarkers for Antibody-Mediated Rejection after Kidney Transplantation.

We aimed to discover and validate urinary exosomal proteins as biomarkers for antibody-mediated rejection (ABMR) after kidney transplantation. Urine and for-cause biopsy samples from kidney transplant recipients were collected and categorized into the discovery cohort (n = 36) and a validation cohort (n = 65). Exosomes were isolated by stepwise ultra-centrifugation for proteomic analysis to discover biomarker candidates for ABMR (n = 12). Of 1820 exosomal proteins in the discovery cohort, four proteins were specifically associated with ABMR: cystatin C (CST3), serum paraoxonase/arylesterase 1, retinol-binding protein 4, and lipopolysaccharide-binding protein (LBP). In the validation cohort, the level of urinary exosomal LBP was significantly higher in the ABMR group (n = 25) compared with the T-cell-mediated rejection (TCMR) group and the no major abnormality (NOMOA) group. Urinary exosomal CST3 level was significantly higher in the ABMR group compared with the control and NOMOA groups. Immunohistochemical staining showed that LBP and CST3 in the glomerulus were more abundant in the ABMR group compared with other groups. The combined prediction probability of urinary exosomal LBP and CST3 was significantly correlated with summed LBP and CST3 intensity scores in the glomerulus and peritubular capillary as well as Banff g + ptc scores. Urinary exosomal CST3 and LBP could be potent biomarkers for ABMR after kidney transplantation.

Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

Clusterin Binding Modulates the Aggregation and Neurotoxicity of Amyloid-ß(1-42).

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aß aggregation is essentially involved. We observed close colocalization of CLU and Aß(1-42) (Aß42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aß42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aß42 aggregates with very high affinity (Kd = 2.647 nM) in vitro to form solid CLU-Aß42 complexes. This CLU binding prevented further aggregation of Aß42 into larger oligomers or fibrils, enriching the population of smaller Aß42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aß42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aß42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aß42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aß42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aß aggregation.

FTIR study of the surface-ligand exchange reaction with glutathione on biocompatible rod-shaped CdSe/CdS semiconductor nanocrystals.

Semiconductor nanocrystals (SNCs) are an essential optical tool in life sciences. Application of SNCs to living systems requires that their surfaces be covered with biocompatible molecules. The surface capping of SNCs by glutathione (GSH) is an effective means to prepare biocompatible SNCs and involves replacement of the initial surface ligands with GSH. However, molecular insight into such ligand-exchange reactions remains elusive. Molecular insight into this process is important, because surface ligands significantly impact physical properties such as the stability and quantum yield of SNCs. In this study, we investigate the ligand-exchange reaction of GSH on rod-shaped CdSe/CdS SNCs by Fourier-transform infrared (FTIR) absorption spectroscopy. The structure and interactions of GSH on SNC surfaces are clarified. Quantitative determination of the GSH molar fraction on SNC surfaces reveals that ∼3% of the initial trioctylphosphine oxide (TOPO) ligand is retained. Concentration-dependent experiments show that the surface molar fraction of GSH impacts the physical properties, solubilization yields, and quantum yields of SNCs in a linear manner.

Estrogen-Related Receptor γ Maintains Pancreatic Acinar Cell Function and Identity by Regulating Cellular Metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.

SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.

ABBREVIATIONS: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Neuromyelitis optica (NMO)-IgG-driven organelle reorganization in human iPSC-derived astrocytes.

Neuromyelitis optica (NMO) is an autoimmune disease that primarily targets astrocytes. Autoantibodies (NMO-IgG) against the water channel protein, aquaporin 4 (AQP4), are a serologic marker in NMO patients, and they are known to be responsible for the pathophysiology of the disease. In the brain, AQP4 is mainly expressed in astrocytes, especially at the end-feet, where they form the blood-brain barrier. Following the interaction between NMO-IgG and AQP4 in astrocytes, rapid AQP4 endocytosis initiates pathogenesis. However, the cellular and molecular mechanisms of astrocyte destruction by autoantibodies remain largely elusive. We established an in vitro human astrocyte model system using induced pluripotent stem cells (iPSCs) technology in combination with NMO patient-derived serum and IgG to elucidate the cellular and functional changes caused by NMO-IgG. Herein, we observed that NMO-IgG induces structural alterations in mitochondria and their association with the endoplasmic reticulum (ER) and lysosomes at the ultrastructural level, which potentially leads to impaired mitochondrial functions and dynamics. Indeed, human astrocytes display impaired mitochondrial bioenergetics and autophagy activity in the presence of NMO-IgG. We further demonstrated NMO-IgG-driven ER membrane deformation into a multilamellar structure in human astrocytes. Together, we show that NMO-IgG rearranges cellular organelles and alter their functions and that our in vitro system using human iPSCs offers previously unavailable experimental opportunities to study the pathophysiological mechanisms of NMO in human astrocytes or conduct large-scale screening for potential therapeutic compounds targeting astrocytic abnormalities in patients with NMO.