Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.

Protein glycosylation plays a vital role in various cellular functions, many of which occur within the Golgi apparatus. The Golgi pH regulator (GPHR) is essential for the proper functioning of the Golgi apparatus. The lysosomal membrane contains highly glycosylated membrane proteins in abundance. This study investigated the role of the Golgi luminal pH in N-glycosylation of lysosomal membrane proteins and the effect of this protein modification on membrane stability using Gphr-deficient MEFs. We showed that Gphr deficiency causes an imbalance in the Golgi luminal pH, resulting in abnormal protein N-glycosylation, indicated by a reduction in sialylated glycans and markedly reduced molecular weight of glycoproteins. Further experiments using FRAP and PLA revealed that Gphr deficiency prevented the trafficking dynamics and proximity condition of glycosyltransferases in the Golgi apparatus. In addition, incomplete N-glycosylation of lysosomal membrane proteins affected lysosomal membrane stability, as demonstrated by the increased susceptibility to lysosomal damage. Thus, this study highlights the critical role of Golgi pH regulation in controlling protein glycosylation and the impact of Golgi dysfunction on lysosomal membrane stability.

Correlation of the total superoxide dismutase activity between joint fluid and synovium in end-stage knee osteoarthritis.

Recently, we found significantly reduced total superoxide dismutase (SOD) activity in the cartilage of patients with end-stage knee osteoarthritis (OA). In this study, we aimed to evaluate the SOD activity in serum, joint fluid, cartilage, and synovial membrane samples collected from 52 patients with end-stage knee OA who underwent total knee arthroplasty. The relationship between the total SOD activity in each tissue was evaluated using Spearman's rank correlation coefficient. The joint fluid total SOD activity was used as the objective variable, and its association with the serum, cartilage, and synovial total SOD activities was evaluated using multiple linear regression analysis. Univariate analysis revealed that joint fluid total SOD activity was positively correlated with synovial total SOD activity. Multiple linear regression analysis using joint fluid total SOD activity as the objective variable showed a positive association with synovial total SOD activity (ß = 0.493, adjusted R2 = 0.172, P < 0.01). In patients with end-stage knee OA, the state of the synovial total SOD activity is better reflected by the total SOD activity in the joint fluid than that in the cartilage. Joint fluid total SOD activity may serve as a biomarker for the treatment and prevention of synovitis.

SOD2 orchestrates redox homeostasis in intervertebral discs: A novel insight into oxidative stress-mediated degeneration and therapeutic potential.

Low back pain (LBP) is a pervasive global health concern, primarily associated with intervertebral disc (IVD) degeneration. Although oxidative stress has been shown to contribute to IVD degeneration, the underlying mechanisms remain undetermined. This study aimed to unravel the role of superoxide dismutase 2 (SOD2) in IVD pathogenesis and target oxidative stress to limit IVD degeneration. SOD2 demonstrated a dynamic regulation in surgically excised human IVD tissues, with initial upregulation in moderate degeneration and downregulation in severely degenerated IVDs. Through a comprehensive set of in vitro and in vivo experiments, we found a suggestive association between excessive mitochondrial superoxide, cellular senescence, and matrix degradation in human and mouse IVD cells. We confirmed that aging and mechanical stress, established triggers for IVD degeneration, escalated mitochondrial superoxide levels in mouse models. Critically, chondrocyte-specific Sod2 deficiency accelerated age-related and mechanical stress-induced disc degeneration in mice, and could be attenuated by ß-nicotinamide mononucleotide treatment. These revelations underscore the central role of SOD2 in IVD redox balance and unveil potential therapeutic avenues, making SOD2 and mitochondrial superoxide promising targets for effective LBP interventions.

AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models.

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins, and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis, and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate CNS-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno-associated virus (AAV). It expressed efficacy within 6 days, and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for approximately 1 year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter.

α-Synuclein aggregates amplified from patient-derived Lewy bodies recapitulate Lewy body diseases in mice.

Extraction of α-Synuclein (αSyn) aggregates from Lewy body disease (LBD) brains has been widely described yet templated fibrillization of LB-αSyn often fails to propagate its structural and functional properties. We recently demonstrated that aggregates amplified from LB-αSyn (ampLB) show distinct biological activities in vitro compared to human αSyn preformed fibrils (hPFF) formed de novo. Here we compare the in vivo biological activities of hPFF and ampLB regarding seeding activity, latency in inducing pathology, distribution of pathology, inclusion morphology, and cell-type preference. Injection of ampLB into mice expressing only human αSyn (male Thy1:SNCA/Snca-/- mice) induced pathologies similar to those of LBD subjects that were distinct from those induced by hPFF-injection or developing spontaneously with aging. Importantly, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca-/- mice maintained the unique biological and conformational features of original LB-αSyn. These results indicate that ampLB-injection, rather than conventional PFF-injection or αSyn overexpression, faithfully models key aspects of LBD.

Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines.

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.

Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter.

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation.

The Effect of Vitamin D3 and Valproic Acid on the Maturation of Human-Induced Pluripotent Stem Cell-Derived Enterocyte-Like Cells.

Cytochrome P450 3A4 (CYP3A4) is involved in first-pass metabolism in the small intestine and is heavily implicated in oral drug bioavailability and pharmacokinetics. We previously reported that vitamin D3 (VD3), a known CYP enzyme inducer, induces functional maturation of iPSC-derived enterocyte-like cells (iPSC-ent). Here, we identified a Notch activator and CYP modulator valproic acid (VPA), as a promotor for the maturation of iPSC-ent. We performed bulk RNA sequencing to investigate the changes in gene expression during the differentiation and maturation periods of these cells. VPA potentiated gene expression of key enterocyte markers ALPI, FABP2, and transporters such as SULT1B1. RNA-sequencing analysis further elucidated several function-related pathways involved in fatty acid metabolism, significantly upregulated by VPA when combined with VD3. Particularly, VPA treatment in tandem with VD3 significantly upregulated key regulators of enterohepatic circulation, such as FGF19, apical bile acid transporter SLCO1A2 and basolateral bile acid transporters SLC51A and SLC51B. To sum up, we could ascertain the genetic profile of our iPSC-ent cells to be specialized toward fatty acid absorption and metabolism instead of transporting other nutrients, such as amino acids, with the addition of VD3 and VPA in tandem. Together, these results suggest the possible application of VPA-treated iPSC-ent for modelling enterohepatic circulation.

Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex.

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

Distinct biological activity of Lewy body α-Synuclein strain in mice.

Extraction of α-Synuclein (αSyn) aggregates from Lewy body disease (LBD) brains has been widely described yet templated fibrillization of LB-αSyn often fails to propagate its structural and functional properties. We recently demonstrated that aggregates amplified from LB-αSyn (ampLB) show distinct biological activities in vitro compared to human αSyn preformed fibrils (hPFF) formed de novo. Here we compare the in vivo biological activities of hPFF and ampLB regarding seeding activity, latency in inducing pathology, distribution of pathology, inclusion morphology, and cell-type preference. Injection of ampLB into mice expressing only human αSyn (Thy1:SNCA/Snca-/- mice) induced pathologies similar to those of LBD subjects that were distinct from those induced by hPFF-injection or developing spontaneously with aging. Importantly, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca-/- mice maintained the unique biological and conformational features of original LB-αSyn. These results indicate that ampLB-injection, rather than conventional PFF-injection or αSyn overexpression, faithfully models key aspects of LBD.

Specific AAV2/PHP.eB-mediated gene transduction of CA2 pyramidal cells via injection into the lateral ventricle.

Given its limited accessibility, the CA2 area has been less investigated compared to other subregions of the hippocampus. While the development of transgenic mice expressing Cre recombinase in the CA2 has revealed unique features of this area, the use of mouse lines has several limitations, such as lack of specificity. Therefore, a specific gene delivery system is required. Here, we confirmed that the AAV-PHP.eB capsid preferably infected CA2 pyramidal cells following retro-orbital injection and demonstrated that the specificity was substantially higher after injection into the lateral ventricle. In addition, a tropism for the CA2 area was observed in organotypic slice cultures. Combined injection into the lateral ventricle and stereotaxic injection into the CA2 area specifically introduced the transgene into CA2 pyramidal cells, enabling us to perform targeted patch-clamp recordings and optogenetic manipulation. These results suggest that AAV-PHP.eB is a versatile tool for specific gene transduction in CA2 pyramidal cells.

Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum.

The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum-core region and dorsal and ventral shell regions-the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.

Characteristics and exchange interactions observed in L-emission spectra of Fe, Mn and their oxides by using a high-energy-resolution soft X-ray emission spectroscopy instrument.

For examining the characteristics of L-emission spectra of Fe, Mn and their oxides, a larger energy-dispersion spectrometer for an electron probe microanalyser was constructed. The energy resolution was evaluated to be 0.3 eV at the Fermi edge observed for the B K-emission of LaB6. The Lα,ß-emission profiles and peak positions of those oxides were different from those of pure metals, reflecting the different density of states of valence bands and different charge states of metal elements. The Lℓ-emission profiles of the oxides showed shoulder structures, even though the emission is caused by transitions between two inner shell levels. The presence of the shoulder structures was assigned to the result of the 3s3d exchange interaction in the final state of the Lℓ emission, in which the 3s state has a spin. The Lℓ profiles were decomposed into two peaks by Lorentz fit, and the energy separation was evaluated to be ∼3 eV.

Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

GPHR-mediated acidification of the Golgi lumen is essential for cholesterol biosynthesis in the brain.

The Golgi pH regulator (GPHR) is essential for maintaining the function and morphology of the Golgi apparatus through the regulation of luminal acidic pH. Abnormal morphology of the Golgi apparatus is associated with neurodegenerative diseases. Here, we found that knockout of GPHR in the mouse brain led to morphological changes in the Golgi apparatus and neurodegeneration, which included brain atrophy, neuronal cell death, and gliosis. Furthermore, in the GPHR knockout mouse brain, transcriptional activity of sterol regulatory element-binding protein 2 (SREBP2) decreased, resulting in a reduction in cholesterol levels. GPHR-deficient cells exhibited suppressed neurite outgrowth, which was recovered by exogenous expression of the active form of SREBP2. Our results show that GPHR-mediated luminal acidification of the Golgi apparatus maintains proper cholesterol levels and, thereby, neuronal morphology.

Protocol for multi-scale light microscopy/electron microscopy neuronal imaging in mouse brain tissue.

An imaging technique across multiple spatial scales is required for extracting structural information on neurons with processes of meter scale length and specialized nanoscale structures. Here, we present a protocol combining multi-scale light microscopy (LM) with electron microscopy (EM) in mouse brain tissue. We describe tissue slice preparation and LM/EM dual labeling with EGFP-APEX2 fusion protein. We then detail ScaleSF tissue clearing and successive LM/EM imaging. Our protocol allows for deciphering structural information across multiple spatial scales on neurons. For complete details on the use and execution of this protocol, please refer to Furuta et al. (2022).

Clinical Efficacy of Melon GliSODin® for the Treatment of Aging-Related Dysfunction in Motor Organs-A Double Blind, Randomized Placebo-Controlled Study.

BACKGROUND: Locomotive syndrome is a concept proposed in Japan involving decreased mobility due to osteoarthritis, osteoporosis, and sarcopenia. This double-blind, randomized study aimed to investigate the effects of superoxide dismutase (SOD)-rich melon extract (Melon GliSODin®) on locomotive syndrome. METHODS: For 6 months, we administered oral Melon GliSODin® (500.4 mg/day) or a placebo to 24 and 22 women, respectively (aged 50-80 years), with knee or lower back discomfort or pain. Using baseline and 6-month data, changes in the Verbal Rating Scale and in subjective symptoms (determined using the Japanese Knee Osteoarthritis Measure, Locomo 25, the Roland-Morris Disability questionnaire, and the Chalder Fatigue Scale) were assessed, along with various oxidative markers, antioxidants, inflammatory markers, renal and liver function biochemical markers, bone metabolism markers, body composition, and motor function. RESULTS: Oral Melon GliSODin® administration tended to be associated with a larger improvement in subjective symptom scores, a reduction in oxidative markers (malondialdehyde and diacron reactive oxygen metabolites) and tumor necrosis factor-α, and a significant increase in non-fat mass between baseline and 6 months. However, no statistically significant differences were observed between the groups for outcomes at 6 months. CONCLUSIONS: Melon GliSODin® tended to improve the subjective symptoms of participants who had knee or lower back pain or discomfort. Melon GliSODin® administration may help to prevent the progression of locomotive syndrome. Future studies involving larger sample sizes and more stringent randomization protocols are needed to determine differences between the placebo and Melon GliSODin® groups.

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (-), and ScaleS4 solution, for a total of 10.5-14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

An optimized protocol for immuno-electron microscopy of endogenous LC3.

MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.Abbreviations: BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen's phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature.

Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.