Treatment of tubular damage in high-fat-diet-fed obese mice using sodium-glucose co-transporter inhibitors.

A long-term high-fat diet (HFD) causes obesity and changes in renal lipid metabolism and lysosomal dysfunction in mice, causing renal damage. Sodium-glucose co-transporter inhibitors, including phlorizin, exert nephroprotective effects in patients with chronic kidney disease, but the underlying mechanism remains unclear. A HFD or standard diet was fed to adult C57BL/6J male mice, and phlorizin was administered. Lamellar body components of the proximal tubular epithelial cells (PTECs) were investigated. After phlorizin administration in HFD-fed mice, sphingomyelin and ceramide in urine and tissues were assessed and label-free quantitative proteomics was performed using kidney tissue samples. Mitochondrial elongation by fusion was effective in the PTECs of HFD-fed obese mice under phlorizin administration, and many lamellar bodies were found in the apical portion of the S2 segment of the proximal tubule. Phlorizin functioned as a diuretic, releasing lamellar bodies from the apical membrane of PTECs and clearing the obstruction in nephrons. The main component of the lamellar bodies was sphingomyelin. On the first day of phlorizin administration in HFD-fed obese mice, the diuretic effect was increased, and more sphingomyelin was excreted through urine than in vehicle-treated mice. The expressions of three peroxisomal ß-oxidation proteins involved in fatty acid metabolism were downregulated after phlorizin administration in the kidneys of HFD-fed mice. Fatty acid elongation protein levels increased with phlorizin administration, indicating an increase in long-chain fatty acids. Lamellar bodies accumulated in the proximal renal tubule of the S2 segment of the HFD-fed mice, indicating that the urinary excretion of lamellar bodies has nephroprotective effects.

Astrocytic dysfunction induced by ABCA1 deficiency causes optic neuropathy.

Astrocyte abnormalities have received great attention for their association with various diseases in the brain but not so much in the eye. Recent independent genome-wide association studies of glaucoma, optic neuropathy characterized by retinal ganglion cell (RGC) degeneration, and vision loss found that single-nucleotide polymorphisms near the ABCA1 locus were common risk factors. Here, we show that Abca1 loss in retinal astrocytes causes glaucoma-like optic neuropathy in aged mice. ABCA1 was highly expressed in retinal astrocytes in mice. Thus, we generated macroglia-specific Abca1-deficient mice (Glia-KO) and found that aged Glia-KO mice had RGC degeneration and ocular dysfunction without affected intraocular pressure, a conventional risk factor for glaucoma. Single-cell RNA sequencing revealed that Abca1 deficiency in aged Glia-KO mice caused astrocyte-triggered inflammation and increased the susceptibility of certain RGC clusters to excitotoxicity. Together, astrocytes play a pivotal role in eye diseases, and loss of ABCA1 in astrocytes causes glaucoma-like neuropathy.

Synaptic pruning of murine adult-born neurons by microglia depends on phosphatidylserine.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E-based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

Controllable ultraslow optical solitons in a degenerated two-level atomic medium under EIT assisted by a magnetic field.

We proposed a simple model for generation of controllable ultraslow optical solitons of a weak probe laser light in a degenerated two-level atomic medium under electromagnetically induced transparency assisted by a magnetic field. It is shown that bright and dark optical solitons can be formed from a probe light with controllable ultraslow group velocities at a few m/s by tuning the strength of a coupling light and/or the magnetic field. In addition to the ultraslow velocity, the advantage of this model is to use a sole laser for delivering both pump and probe lights. Furthermore, one can switch between bright and dark solitons by reversing the direction of the magnetic field. Such controllable ultraslow solitons are interested in finding applications in optical communications and optical data processing.

Sodium-glucose co-transporter (SGLT) inhibitor restores lost axonal varicosities of the myenteric plexus in a mouse model of high-fat diet-induced obesity.

Diabetes impairs enteric nervous system functions; however, ultrastructural changes underlying the pathophysiology of the myenteric plexus and the effects of sodium-glucose co-transporter (SGLT) inhibitors are poorly understood. This study aimed to investigate three-dimensional ultrastructural changes in axonal varicosities in the myenteric plexus and the effect thereon of the SGLT inhibitor phlorizin in mice fed a high-fat diet (HFD). Three-dimensional ultrastructural analysis using serial block-face imaging revealed that non-treated HFD-fed mice had fewer axonal varicosities and synaptic vesicles in the myenteric plexus than did normal diet-fed control mice. Furthermore, mitochondrial volume was increased and lysosome number decreased in the axons of non-treated HFD-fed mice when compared to those of control mice. Phlorizin treatment restored the axonal varicosities and organelles in HFD-fed mice. Although HFD did not affect the immunolocalisation of PGP9.5, it reduced synaptophysin immunostaining in the myenteric plexus, which was restored by phlorizin treatment. These results suggest that impairment of the axonal varicosities and their synaptic vesicles underlies the damage to the enteric neurons caused by HFD feeding. SGLT inhibitor treatment could restore axonal varicosities and organelles, which may lead to improved gastrointestinal functions in HFD-induced obesity as well as diabetes.

Mitochondrial Dynamics in Physiology and Pathology of Myelinated Axons.

Mitochondria play essential roles in neurons and abnormal functions of mitochondria have been implicated in neurological disorders including myelin diseases. Since mitochondrial functions are regulated and maintained by their dynamic behavior involving localization, transport, and fusion/fission, modulation of mitochondrial dynamics would be involved in physiology and pathology of myelinated axons. In fact, the integration of multimodal imaging in vivo and in vitro revealed that mitochondrial localization and transport are differentially regulated in nodal and internodal regions in response to the changes of metabolic demand in myelinated axons. In addition, the mitochondrial behavior in axons is modulated as adaptive responses to demyelination irrespective of the cause of myelin loss, and the behavioral modulation is partly through interactions with cytoskeletons and closely associated with the pathophysiology of demyelinating diseases. Furthermore, the behavior and functions of axonal mitochondria are modulated in congenital myelin disorders involving impaired interactions between axons and myelin-forming cells, and, together with the inflammatory environment, implicated in axonal degeneration and disease phenotypes. Further studies on the regulatory mechanisms of the mitochondrial dynamics in myelinated axons would provide deeper insights into axo-glial interactions mediated through myelin ensheathment, and effective manipulations of the dynamics may lead to novel therapeutic strategies protecting axonal and neuronal functions and survival in primary diseases of myelin.

Interactions between mitochondria and endoplasmic reticulum in demyelinated axons.

Demyelination leads to axonal changes that involve the functions and dynamics of axonal mitochondria supporting metabolism and survival of axons. However, the changes in the physical interactions between mitochondria and endoplasmic reticulum, called mitochondria-associated membranes, are poorly understood in demyelinated axons. In this study, we investigated the three-dimensional ultrastructural changes in membrane juxtapositions between mitochondria and endoplasmic reticulum in axons of a chronic progressive demyelination mouse model caused by extra copies of proteolipid protein (PLP4e). In the optic nerve of PLP4e mice, most axons were ensheathed by myelin by age 1 month, but were demyelinated by age 5 months. At age 1 month, mitochondria in PLP4e mice were slightly larger than those in wild-type mice, while the size and frequency of juxtaposition were similar. At age 5 months, the sizes of mitochondria and size of juxtaposition in PLP4e mice were prominently larger than those in wild-type mice. In degenerating axons under demyelination, the enlargement of mitochondria was diminished, while the density and frequency of juxtaposition were similar to those of non-degenerating axons. These results suggest that interactions between mitochondria and ER are enhanced in chronically demyelinated axons and maintained during axonal degeneration in hereditary myelin diseases.

Methodological Improvements With Conductive Materials for Volume Imaging of Neural Circuits by Electron Microscopy.

Recent advancements in electron microscope volume imaging, such as serial imaging using scanning electron microscopy (SEM), have facilitated the acquisition of three-dimensional ultrastructural information of biological samples. These advancements help build a comprehensive understanding of the functional structures in entire organelles, cells, organs and organisms, including large-scale wiring maps of neural circuitry in various species. Advanced volume imaging of biological specimens has often been limited by artifacts and insufficient contrast, which are partly caused by problems in staining, serial sectioning and electron beam irradiation. To address these issues, methods of sample preparation have been modified and improved in order to achieve better resolution and higher signal-to-noise ratios (SNRs) in large tissue volumes. These improvements include the development of new embedding media for electron microscope imaging that have desirable physical properties such as less deformation in the electron beam and higher stability for sectioning. The optimization of embedding media involves multiple resins and filler materials including biological tissues, metallic particles and conductive carbon black. These materials alter the physical properties of the embedding media, such as conductivity, which reduces specimen charge, ameliorates damage to sections, reduces image deformation and results in better ultrastructural data. These improvements and further studies to improve electron microscope volume imaging methods provide options for better scale, quality and throughput in the three-dimensional ultrastructural analyses of biological samples. These efforts will enable a deeper understanding of neuronal circuitry and the structural foundation of basic and higher brain functions.

Decreased number and increased volume with mitochondrial enlargement of cerebellar synaptic terminals in a mouse model of chronic demyelination.

Impaired nerve conduction, axonal degeneration, and synaptic alterations contribute to neurological disabilities in inflammatory demyelinating diseases. Cerebellar dysfunction is associated with demyelinating disorders, but the alterations of axon terminals in cerebellar gray matter during chronic demyelination are still unclear. We analyzed the morphological and ultrastructural changes of climbing fiber terminals in a mouse model of hereditary chronic demyelination. Three-dimensional ultrastructural analyses using serial block-face scanning electron microscopy and immunostaining for synaptic markers were performed in a demyelination mouse model caused by extra copies of myelin gene (PLP4e). At 1 month old, many myelinated axons were observed in PLP4e and wild-type mice, but demyelinated axons and axons with abnormally thin myelin were prominent in PLP4e mice at 5 months old. The density of climbing fiber terminals was significantly reduced in PLP4e mice at 5 months old. Reconstruction of climbing fiber terminals revealed that PLP4e climbing fibers had increased varicosity volume and enlarged mitochondria in the varicosities at 5-month-old mice. These results suggest that chronic demyelination is associated with alterations and loss of climbing fiber terminals in the cerebellar cortex, and that synaptic changes may contribute to cerebellar phenotypes observed in hereditary demyelinating disorders.

Polymorphic regulation of mitochondrial fission and fusion modifies phenotypes of microglia in neuroinflammation.

Microglia are the resident macrophages of the central nervous system and play complex roles in the milieu of diseases including the primary diseases of myelin. Although mitochondria are critical for cellular functions and survival in the nervous system, alterations in and the roles of mitochondrial dynamics and associated signaling in microglia are still poorly understood. In the present study, by combining immunohistochemistry and 3D ultrastructural analyses, we show that mitochondrial fission/fusion in reactive microglia is differentially regulated from that in monocyte-derived macrophages and the ramified microglia of normal white matter in myelin disease models. Mouse cerebral microglia in vitro demonstrated that stimulation of TLR4 with lipopolysaccharide, widely used to examine microglial reactions, caused the activation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) and enhanced production of reactive oxygen species (ROS). The increase in the ROS level activated 5' adenosine monophosphate-activated protein kinase (AMPK), and facilitated elongation of mitochondria along the microtubule tracks. These results suggest that the polymorphic regulation of mitochondrial fission and fusion in reactive microglia is mediated by distinct signaling under inflammatory conditions, and modulates microglial phenotypes through the production of ROS.

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging.

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy.

Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.