Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury ...(TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.
To understand the retinal environmental changes by injured optic nerves, we focused on the major phospholipid species that are enriched in the whole retina. Here, we show the spatial distributions of phospholipid species and the effect by injured optic nerves in mouse retina by using imaging mass spectrometry.
Lamellar corpuscles function as mechanoreceptors in the skin, composed of axon terminals and lamellae constructed by terminal Schwann cells. They are classified into Pacinian, Meissner, and simple ...corpuscles based on histological criteria. Lamellar corpuscles in rat dermal papilla cells have been reported; however, the morphological aspects have yet to be thoroughly investigated. In the present study, we analyzed the enzyme activity, distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin. The lamellar corpuscles exhibiting non-specific cholinesterase were densely distributed in rat footpads, evident as notable skin elevations, especially at the apex, the highest portion of the ridges in each footpad. In contrast, only a few lamellar corpuscles were found in other plantar skin areas. Lamellar corpuscle was considered composed of a flat axon terminal Schwann cell lamellae, which were roughly concentrically arranged in the dermal papilla. These histological characteristics correspond to those of the simple corpuscle. Moreover, the axon tracing method revealed that one trunk axon innervated several simple corpuscles. The territory of the trunk axons overlapped with each other. Finally, the animals’ footprints were analyzed. During the pausing and walking phases, footpads are often in contact with the floor. These results demonstrate that the type of lamellar corpuscles in the dermal papillae of rat plantar skin is a simple corpuscle and implies that their distribution pattern in the plantar skin is convenient for efficient sensing and transmission of mechanical stimuli from the ground.
Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression ...pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M‐phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N‐terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N‐terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.
Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. Bookmarking transcription factors can be retained in mitotic chromosomes and would contribute to a novel third mechanism of cellular memory. Here, we report that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates the proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, is retained in the chromatin during mitosis in the neural tube of mouse embryos. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate the novel function of transcription factors.
Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological ...phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3′ untranslated region (3′UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3′UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.
•Cpeb1 3′UTR represses both Cpeb1 mRNA and protein expression levels.•Multiple elements in the Cpeb1 3′UTR are involved in Cpeb1 mRNA expression.•Serial AREs in the Cpeb1 3′UTR mediate Cpeb1 mRNA level.•Depletion of AUF1 increased the Cpeb1 mRNA levels, but reduced the protein levels.•Cpeb1 expression may be controlled by multimodal mechanisms through its 3′UTR.
In the dorsal root ganglia (DRG), two types of glial cells (Schwann cells and satellite glial cells) have been identified based on cell morphology and expression of specific markers. In the present ...study, we observed unknown glial cells that were positive for p75 neurotrophin receptor (p75NTR), and therefore were immunohistochemically and ultrastructurally characterized for the first time. These cells exhibited stronger immunoreactivity against an anti‐p75NTR antibody than the DRG neurons (hereafter referred to as p75NTR++ cells). Moreover, these cells covered the glial cells surrounding proximal process of the large‐diameter DRG neurons. The proximal process is called “dendro‐axon.” The p75NTR++ cells were predominantly distributed where the first myelinating Schwann cells appear. The p75NTR++ cells were also positive for the pan‐glial cell markers S100, nestin, and Sox10, but negative for fibroblast and macrophage markers. Moreover, they were negative for a satellite glial cell marker, inwardly rectifying potassium channel Kir4.1, as well as a nonmyelinating Schwann cell marker, glial fibrillary acidic protein. In addition, their morphological features were distinct from those of the myelinating Schwann cells. To investigate the three‐dimensional ultrastructure of the p75NTR++ cells, we used array tomography combined with correlative light and electron microscopic observation. Three‐dimensional ultrastructural observation revealed that the p75NTR++ cells loosely covered glial cells around the dendro‐axons with highly ramified processes. Glial cells with these morphological features have not been reported before, indicating that the p75NTR++ glial cells are a new glial cell type in the DRG. Our results will give new insights into cell–cell relationships.
We immunohistochemically and ultrastructurally characterized nonneuronal p75NTR‐positive cells in the dorsal root ganglia for the first time. These cells were positive for pan‐glial cell markers, but their morphological features were distinct from already known glial cells. These morphological results indicate that the p75NTR‐positive glial cells are a new glial cell type.
Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific ...sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.
In the pathogenesis of Alzheimer’s disease (AD), highly neurotoxic amyloid-β (Aβ) oligomers appear early, they are thus considered to be deeply involved in the onset of Alzheimer’s disease. However, ...Aβ oligomer visualization is challenging in human tissues due to their multiple forms (e.g., low- and high-molecular-weight oligomers, including protofibrils) as well as their tendency to rapidly change forms and aggregate. In this review, we present two visualization approaches for Aβ oligomers in tissues: an immunohistochemical (using the monoclonal antibody TxCo1 against toxic Aβ oligomer conformers) and imaging mass spectrometry using the small chemical Shiga-Y51 that specifically binds Aβ oligomers. TxCo1 immunohistochemistry revealed Aβ oligomer distributions in postmortem human brains with AD. Using Shiga-Y51, imaging mass spectrometry revealed Aβ oligomer distributions in the brain of a transgenic mouse model for AD. These two methods would potentially contribute to elucidating the pathological mechanisms underlying AD.
Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional ...regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.
Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed ...that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.
•We established satellite glial cell culture system.•Function of Sox2 in satellite glial cell was examined using siRNA.•Sox2 knockdown downregulated expression level of ErbB2 and ErbB3 mRNA.•Sox2 knockdown increased apoptotic satellite glial cell.•Sox2 promotes satellite glial cell survival through ErbB signaling.
Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the ...mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain‐ and spinal cord‐derived cells of post‐metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord‐derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.
We report the formation of spherical cell aggregates similar to neurospheres in vitro from the brain and spinal cord of post‐metamorphic Pleurodeles waltl. These aggregates expressed several marker proteins of neural stem cells, and cells in the aggregates could differentiate into neurons, astrocytes, and oligodendrocytes in vitro. Based on our findings, we conclude that the observed spherical cell aggregates could be regarded as neurospheres from the central nervous system of P. waltl.
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