In the brain, glycogen metabolism has been implied in synaptic plasticity and learning, yet the distribution of this molecule has not been fully described. We investigated cerebral glycogen of the ...mouse by immunohistochemistry (IHC) using two monoclonal antibodies that have different affinities depending on the glycogen size. The use of focused microwave irradiation yielded well‐defined glycogen immunoreactive signals compared with the conventional periodic acid‐Schiff method. The IHC signals displayed a punctate distribution localized predominantly in astrocytic processes. Glycogen immunoreactivity (IR) was high in the hippocampus, striatum, cortex, and cerebellar molecular layer, whereas it was low in the white matter and most of the subcortical structures. Additionally, glycogen distribution in the hippocampal CA3‐CA1 and striatum had a ‘patchy’ appearance with glycogen‐rich and glycogen‐poor astrocytes appearing in alternation. The glycogen patches were more evident with large‐molecule glycogen in young adult mice but they were hardly observable in aged mice (1–2 years old). Our results reveal brain region‐dependent glycogen accumulation and possibly metabolic heterogeneity of astrocytes. GLIA 2016;64:1532–1545
Main Points
Glycogen immunohistochemistry reveals the hippocampus, cortex, striatum, and cerebellar molecular layer as glycogen‐rich areas.
Hippocampal glycogen distribution is organized in astrocytic patches, which diminish in aged mice (1‐2 years old).
Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of ...β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.
Neuron-glia interactions contribute to pain initiation and sustainment. Intra-ganglionic (IG) secretion of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) modulates pain ...transmission through neuron-glia signaling, contributing to various orofacial pain conditions. The present study aimed to investigate the role of satellite glial cells (SGC) in TG in causing cytokine-related orofacial nociception in response to IG administration of CGRP. For that purpose, CGRP alone (10 μL of 10
M), Minocycline (5 μL containing 10 μg) followed by CGRP with one hour gap (Min + CGRP) were administered directly inside the TG in independent experiments. Rats were evaluated for thermal hyperalgesia at 6 and 24 h post-injection using an operant orofacial pain assessment device (OPAD) at three temperatures (37, 45 and 10 °C). Quantitative real-time PCR was performed to evaluate the mRNA expression of IL-1β, IL-6, TNF-α, IL-1 receptor antagonist (IL-1RA), sodium channel 1.7 (NaV 1.7, for assessment of neuronal activation) and glial fibrillary acidic protein (GFAP, a marker of glial activation). The cytokines released in culture media from purified glial cells were evaluated using antibody cytokine array. IG CGRP caused heat hyperalgesia between 6⁻24 h (paired-
test,
< 0.05). Between 1 to 6 h the mRNA and protein expressions of GFAP was increased in parallel with an increase in the mRNA expression of pro-inflammatory cytokines IL-1β and anti-inflammatory cytokine IL-1RA and NaV1.7 (one-way ANOVA followed by Dunnett's post hoc test,
< 0.05). To investigate whether glial inhibition is useful to prevent nociception symptoms, Minocycline (glial inhibitor) was administered IG 1 h before CGRP injection. Minocycline reversed CGRP-induced thermal nociception, glial activity, and down-regulated IL-1β and IL-6 cytokines significantly at 6 h (
-test,
< 0.05). Purified glial cells in culture showed an increase in release of 20 cytokines after stimulation with CGRP. Our findings demonstrate that SGCs in the sensory ganglia contribute to the occurrence of pain via cytokine expression and that glial inhibition can effectively control the development of nociception.
Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for ...oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.
Abstract
Background
Analyses of tooth families and tooth-forming units in medaka with regard to tooth replacement cycles and the localization of odontogenic stem cell niches in the pharyngeal ...dentition clearly indicate that continuous tooth replacement is maintained. The
secretory calcium-binding phosphoprotein
(
scpp
) gene cluster is involved in the formation of mineralized tissues, such as dental and bone tissues, and the genes encoding multiple SCPPs are conserved in fish, amphibians, reptiles, and mammals. In the present study, we examined the expression patterns of several
scpp
genes in the pharyngeal teeth of medaka to elucidate their roles during tooth formation and replacement.
Methods
Himedaka (Japanese medaka,
Oryzias latipes
) of both sexes (body length: 28 to 33 mm) were used in this study. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qPCR) data were evaluated using one-way analysis of variance for multi-group comparisons, and the significance of differences was determined by Tukey’s comparison test. The expression of
scpp
genes was examined using in situ hybridization (ISH) with a digoxigenin-labeled, single-stranded antisense probe.
Results
qPCR results showed that several
scpp
genes were strongly expressed in pharyngeal tissues. ISH analysis revealed specific expression of
scpp1
,
scpp5
, and
sparc
in tooth germ, and
scpp5
was continually expressed in the odontoblasts of teeth attached to pedicles, but not in the osteoblasts of pedicles. In addition, many
scpp
genes were expressed in inner dental epithelium (ide), but not in odontoblasts, and
scpp2
consistently showed epithelial-specific expression in the functional teeth. Taken together, these data indicate that specific expression of
scpp2
and
scpp5
may play a critical role in pharyngeal tooth formation in medaka.
Conclusion
We characterized changes in the expression patterns of
scpp
genes in medaka during the formation and replacement of pharyngeal teeth.
Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural ...characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.
Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that ...differences are due to fiber type-specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1α (PDH-E1α) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1α). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.
•Infraorbital nerve constriction injury induced pain behavior.•Infraorbital nerve constriction injury induced satellite glial cell activation in trigeminal ganglia.•CXCL2 levels increased at day 1 of ...neuropathic pain induction and decreased gradually, with IL-10 levels showing the opposite trend.•Recombinant IL-10 or anti-CXCL2 injection into trigeminal ganglia decreased pain behavior.
Many trigeminal neuropathic pain patients suffer severe chronic pain. The neuropathic pain might be related with cross-excitation of the neighboring neurons and satellite glial cells (SGCs) in the sensory ganglia and increasing the pain signals from the peripheral tissue to the central nervous system. We induced trigeminal neuropathic pain by infraorbital nerve constriction injury (IONC) in Sprague-Dawley rats. We tested cytokine (CXCL2 and IL-10) levels in trigeminal ganglia (TGs) after trigeminal neuropathic pain induction, and the effect of direct injection of the anti-CXCL2 and recombinant IL-10 into TG. We found that IONC induced pain behavior. Additionally, IONC induced satellite glial cell activation in TG and cytokine levels of TGs were changed after IONC. CXCL2 levels increased on day 1 of neuropathic pain induction and decreased gradually, with IL-10 levels showing the opposite trend. Recombinant IL-10 or anti-CXCL2 injection into TG decreased pain behavior. Our results show that IL-10 or anti-CXCL2 are therapy options for neuropathic pain.
Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural ...characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.
Abstract
Autophagy can selectively target protein aggregates, pathogens, and dysfunctional organelles for the lysosomal degradation. Aberrant regulation of autophagy promotes tumorigenesis, while it ...is far less clear whether and how tumor-specific alterations result in autophagic aberrance. To form a link between aberrant autophagy selectivity and human cancer, we establish a computational pipeline and prioritize 222 potential LIR (LC3-interacting region) motif-associated mutations (LAMs) in 148 proteins. We validate LAMs in multiple proteins including ATG4B, STBD1, EHMT2 and BRAF that impair their interactions with LC3 and autophagy activities. Using a combination of transcriptomic, metabolomic and additional experimental assays, we show that STBD1, a poorly-characterized protein, inhibits tumor growth via modulating glycogen autophagy, while a patient-derived W203C mutation on LIR abolishes its cancer inhibitory function. This work suggests that altered autophagy selectivity is a frequently-used mechanism by cancer cells to survive during various stresses, and provides a framework to discover additional autophagy-related pathways that influence carcinogenesis.
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