Search

Abnormal α-synuclein aggregation is a key pathological feature of a group of neurodegenerative diseases known as synucleinopathies, which include Parkinson’s disease (PD), dementia with Lewy bodies and multiple system atrophy (MSA). The pathogenic β-sheet seed conformation of α-synuclein is found in various tissues, suggesting potential as a biomarker, but few studies have been able to reliably detect these seeds in serum samples. In this study, we developed a modified assay system, called immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC), which enables the detection of pathogenic α-synuclein seeds in the serum of individuals with synucleinopathies. In our internal first and second cohorts, IP/RT-QuIC showed high diagnostic performance for differentiating PD versus controls (area under the curve (AUC): 0.96 (95% confidence interval (CI) 0.95–0.99)/AUC: 0.93 (95% CI 0.84–1.00)) and MSA versus controls (AUC: 0.64 (95% CI 0.49–0.79)/AUC: 0.73 (95% CI 0.49–0.98)). IP/RT-QuIC also showed high diagnostic performance in differentiating individuals with PD (AUC: 0.86 (95% CI 0.74–0.99)) and MSA (AUC: 0.80 (95% CI 0.65–0.97)) from controls in a blinded external cohort. Notably, amplified seeds maintained disease-specific properties, allowing the differentiation of samples from individuals with PD versus MSA. In summary, here we present a novel platform that may allow the detection of individuals with synucleinopathies using serum samples.

Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as ‘in-resin CLEM’, have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca

Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins.

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum–like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly

Caffeic acid (CA), a coffee-related natural compound, has various beneficial biological effects, including antiviral effects. Our former studies demonstrated that the CA dose-dependently inhibited the in vitro infection with Dabie bandavirus, which was previously named as severe fever with thrombocytopenia syndrome virus (SFTSV), mainly at the step of virus attachment. Therefore, we studied the structural basis of CA for conferring anti-SFTSV activity to clarify the mechanism of action of CA against SFTSV. In this study, the anti-SFTSV activity of nine CA analogs were examined. The treatment of SFTSV with the 3,4-dihydroxyhydrocinnamic acid (DHCA) as well as CA inhibited the SFTSV infection in a dose-dependent manner, whereas other CA analogs did not. Both CA and DHCA only possessed the o-dihydroxybenzene backbone. When SFTSV was treated with catechol (o-dihydroxybenzene), SFTSV infection was also dose-dependently inhibited. Additionally, four compounds having the o-dihydroxybenzene backbone; CA phenethyl ester, methyl CA, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid, dose-dependently inhibited the viral infection, although these compounds were more toxic or less effective than CA. In conclusion, the o-dihydroxybenzene backbone in CA and its analogs was a critical structure for the anti-SFTSV activity. Based on these findings, modifications of the o-dihydroxybenzene backbone with various other residues might improve the antiviral effect and cytotoxicity for SFTSV.

We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To validate the usability of PK-LC3 mice, we quantified PK-LC3 puncta in Purkinje cells of mice kept in normal feeding conditions and of mice starved for 24 hours. Our results showed a significant increase in autophagosome number and in individual puncta areal size following starvation. To confirm these results, we used morphometry at the electron microscopic level to analyze the volume densities of autophagosomes and lysosomes/autolysosomes in Purkinje cells of PK-LC3 mice. The results revealed that the volume densities of autophagic structures increase significantly after starvation. Together, our data show that PK-LC3 mice are suitable for monitoring autophagy flux in Purkinje cells of the cerebellum, and potentially other areas in the central nervous system.

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

Recently, the genetic variability in lysosomal storage disorders has been implicated in the pathogenesis of Parkinson’s disease. Here, we found that variants in prosaposin (PSAP), a rare causative gene of various types of lysosomal storage disorders, are linked to Parkinson’s disease. Genetic mutation screening revealed three pathogenic mutations in the saposin D domain of PSAP from three families with autosomal dominant Parkinson’s disease. Whole-exome sequencing revealed no other variants in previously identified Parkinson’s disease-causing or lysosomal storage disorder-causing genes. A case-control association study found two variants in the intronic regions of the PSAP saposin D domain (rs4747203 and rs885828) in sporadic Parkinson’s disease had significantly higher allele frequencies in a combined cohort of Japan and Taiwan. We found the abnormal accumulation of autophagic vacuoles, impaired autophagic flux, altered intracellular localization of prosaposin, and an aggregation of α-synuclein in patient-derived skin fibroblasts or induced pluripotent stem cell-derived dopaminergic neurons. In mice, a Psap saposin D mutation caused progressive motor decline and dopaminergic neurodegeneration. Our data provide novel genetic evidence for the involvement of the PSAP saposin D domain in Parkinson’s disease.

Summary: Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons. : Genetic variants in the TMEM106B gene, coding for a lysosomal transmembrane protein, are linked to various neurodegenerative diseases. The function of TMEM106B remains enigmatic. Lüningschrör et al. analyze Tmem106b-knockout mice and find drastically enlarged LAMP1-positive vacuoles in proximal axons of selected motoneuron nuclei. Vacuolization is caused by impaired axonal transport. Keywords: TMEM106B, frontotemporal lobar degeneration, lysosome, retrograde, axon, axon initial segment, motoneurons, FTLD

Summary 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., Graphical abstract, Highlights • Successive light microscopy/electron microscopy in optically cleared tissues • Multi-scale neuronal imaging from the entire brain to individual synapses • Simultaneous interrogation of neural circuit structure and synaptic connectivity • Zooming-in to scarce synaptic contacts with the successive imaging, Imaging anatomy; Optical imaging; Neuroscience; Cell biology

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, “ridge-like prominences” (RLPs).

Summary A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner., Graphical Abstract, Highlights • CFLARs Tg mice develop severe ileitis in utero • Intestinal epithelial cells die by apoptosis and necroptosis in CFLARs Tg mice • Blockade of necroptosis rescues lethality of CFLARs Tg mice • Necroptosis activates type 3 innate lymphoid cells, resulting in severe ileitis, Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Nephrocytes are similar in structure to podocytes and play a role in the isolation of toxic substances from hemolymph in insects. Drosophila melanogaster nephrocytes have recently been used to study podocyte function and disease. However, the three-dimensional ultrastructure of nephrocytes is not clearly understood because their surrounding basement membrane makes it difficult to observe using conventional scanning electron microscopy. We reconstructed the three-dimensional ultrastructure of Drosophila pericardial nephrocytes using serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. The basal surfaces were occupied by foot processes and slit-like spaces between them. The slit-like spaces corresponded to the podocyte filtration slits and were formed by longitudinal infolding/invagination of the basal plasma membrane. The basal surface between the slit-like spaces became the foot processes, which ran almost linearly, and had a “washboard-like” appearance. Both ends of the foot processes were usually anastomosed to neighboring foot processes and thus free ends were rarely observed. We demonstrated that FIB/SEM is a powerful tool to better understand the three-dimensional architecture of nephrocytes.

Background Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear. Methods To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. Results Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridge-like prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. Conclusions The three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.

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 microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved 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 the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the 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.

Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

Different conformational strains of tau have been implicated in the clinicopathological heterogeneity of tauopathies. In this study, we hypothesized that distinct strains are degraded in a different manner. Lithium, a drug for bipolar disorder, had previously been reported to reduce aggregation-prone protein content by promoting autophagy. Here, we assessed the effects of lithium on tau aggregates using different tauopathy brain seeds. SH-SY5Y cells were transfected with C-terminal tau fragment Tau-CTF24 (residues 243-441), and Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) brain seeds were introduced. After 48-h lithium treatment, sarkosyl-insoluble fractions were prepared. Lithium treatment was found to reduce the amount of insoluble tau and p62, and increase LC3-II levels along with the number of autophagic vacuoles in AD-seeded cells. The effects were lower in case of CBD seeds, and comparable between PSP and AD seeds. An inhibitor of myo-inositol monophosphatase (IMPase) also demonstrated similar effects. Overall, the study suggested that aggregated tau protein is degraded by lithium-induced autophagy, influencing IMPase in a strain-specific manner.

The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathological conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome human α-synuclein (A53T-BAC-SNCA) transgenic mouse model of Parkinson’s disease, at 1 month, 3 months, 6 months, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice; suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA transgenic mice. Taken together, our data suggest that Parkinson’s disease is accompanied by an abnormality in the mechanisms that control synapse growth and maturity. Significance statement Currently, the clinical diagnosis of Parkinson’s disease is based on the presence of motor symptoms. However, these symptoms only manifest after a significant proportion of dopaminergic cells have degenerated. The latent, prodromal phase is therefore of particular interest for the development of disease-modifying therapies to slow down or reverse the course of neurodegeneration. Although clinical markers to diagnose patients in the prodromal phase are gradually emerging, a structural marker that defines this phase has not yet been identified. Structural changes likely occur in the synapse before the onset of neurodegeneration. Thus, this study performed an ultrastructural analysis of synapses in a mouse model of prodromal Parkinson’s disease and revealed distinct, age-dependent structural changes.

The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. In this study, using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, we revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy—they filled an enormous gap, which can be called “missing link,” in the evolutionary diversity of podocytes and nephrocytes. Thus, we concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.

Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.

Neurons in the central nervous system display a great diversity of synaptic architecture. While much of our knowledge on the excitatory synapse morphology derives from the prototypical asymmetric synapses, little has been studied about the atypical crest-type synapse that exists in the restricted brain regions. Here, we used focused ion beam scanning electron microscopy (FIB/SEM) to image a neuropil volume of interpeduncular nucleus (IPN) and manually reconstructed several dendrites to obtain an insight about the topography and quantitative features of crest synapses. Three-dimensional reconstruction showed numerous U-shaped structures protruding from the IPN dendrites. On either faces of the U-shaped structure, a pair of crest synapses are aligned in parallel such that there exists a positive correlation between the postsynaptic density (PSD) area of synapses that participate in pair formation. Interestingly, mitochondria are excluded from the site of crest synapses. Several presynaptic axons run through the hollow, cylindrical space of the U-shape grooves such that the plasma membrane of the axon and the dendrite are organized in a tight opposition without any intervening glial membrane. Unlike the peculiar dendritic morphology, IPN neurons possess typical somatic morphology with an oval, centrally located nucleus. In conclusion, our data reveals a hitherto unknown unique topographical feature of crest synapses in the IPN.

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases., Ogawa, Takada et al. show that Streptococcus pneumoniae triggers the formation of pneumococcus-containing LC3-associated phagosome-like vacuoles in an early stage of infection. This study suggests a hierarchical autophagy mechanism activated by intracellular bacterial pathogens.

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

Parkin‐mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N‐terminal SKICH domain and C‐terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly‐ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52‐mediated mitophagy, and the NDP52–MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

Renal proximal tubular epithelial cells are significantly damaged during acute kidney injury. Renal proximal tubular cell-specific autophagy-deficient mice show increased sensitivity against renal injury, while showing few pathological defects under normal fed conditions. Considering that autophagy protects the proximal tubular cells from acute renal injury, it is reasonable to assume that autophagy contributes to the maintenance of renal tubular cells under normal fed conditions. To clarify this possibility, we generated a knock out mouse model which lacks Atg7, a key autophagosome forming enzyme, in renal proximal tubular cells (Atg7flox/flox, KAP-Cre+). Analysis of renal tissue from two months old Atg7flox/flox, KAP-Cre+ mouse revealed an accumulation of LC3, binding protein p62/sequestosome 1 (a selective substrate for autophagy), and more interestingly, Kim-1, a biomarker for early kidney injury, in the renal proximal tubular cells under normal fed conditions. TUNEL (TdT-mediated dUTP Nick End Labeling)-positive cells were also detected in the autophagy-deficient renal tubular cells. Analysis of renal tissue from Atg7flox/flox, KAP-Cre+ mice at different age points showed that tubular cells positive for p62 and Kim-1 continually increase in number in an age-dependent manner. Ultrastructural analysis of tubular cells from Atg7flox/flox, KAP-Cre+ revealed the presence of intracellular inclusions and abnormal structures. These results indicated that autophagy-deficiency in the renal proximal epithelial tubular cells leads to an increase in injured cells in the kidney even under normal fed conditions.

ATG9 is a membrane protein that is essential for autophagy and is considered to be directly involved in the early steps of autophagosome formation. Yeast Atg9 is mainly localized to small vesicles (Atg9 vesicles), whereas mammalian ATG9A is reportedly localized to the trans-Golgi network, the endosomal compartment, and other unidentified membrane structures. To dissect the ATG9A-containing membranes, we examined the subcellular localization of ATG9A and performed immunoisolation of those membranes. ATG9A-green fluorescent protein in human culture cells was observed as numerous puncta that move rapidly throughout the cytoplasm. We isolated these cytoplasmic membranes and found that they were small vesicles that resemble the yeast Atg9 vesicle. One of the proteins obtained via proteomic analyses of the mammalian ATG9A vesicle was Rab1, a small GTPase that is essential in endoplasmic reticulum-to-Golgi vesicle trafficking. Knockdown studies of Rab1B showed a suppression of autophagy. In these Rab1B-depleted cells, ATG9A accumulated in intermediate membrane structures at autophagosome formation sites. These results indicate that Rab1B is involved in regulating the proper development of autophagosomes.-Kakuta, S., Yamaguchi, J., Suzuki, C., Sasaki, M., Kazuno, S., Uchiyama, Y. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.