Taro Koike

Correlation of enhanced cell proliferation with decreased density of vitamin D receptor in parathyroid hyperplasia in chronic dialysis patients

Summary: The decreased density of the vitamin D receptor (VDR) plays an important role in the pathogenesis and progression of parathyroid hyperplasia in renal failure. In chronic dialysis patients, VDR density is less in nodular hyperplasia than in diffuse hyperplasia and the difference of cell proliferation has been also suggested by DNA analysis. to prove a more direct correlation between VDR density and cell proliferation, VDR density and proliferating cell nuclear antigen (PCNA) were detected in situ by immunohistochemistry in serial sections of surgically excised parathyroid glands from 10 chronic dialysis patients. Among 28 excised glands, 20 glands were nodular hyperplasia and eight glands were diffuse hyperplasia. Vitamin D receptor positive cells were much fewer in nodular hyperplasia (13.1 ± 4.8%) than in diffuse hyperplasia (383 ± 5.6%). In contrast, mean PCNA positive cell numbers per one 400 x field were much higher in nodular hyperplasia (2.0± 1.2) than in diffuse hyperplasia (0.1±0.2). These two parameters, simultaneously detected in the same area of the serial sections, showed strong negative correlation (r= ‐0.719, P<0.0001). Remarkable differences in VDR and PCNA were evident between nodules and the surrounding diffuse hyperplasia in the same section. These data suggest more direct relationship between the decrease of VDR density and parathyroid cell proliferation in chronic renal failure as a pathophysiological mechanism.

Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.

How many hair follicles are innervated by one afferent axon? A confocal microscopic analysis of palisade endings in the auricular skin of thy1-YFP transgenic mouse

Hairs are known as a sensory apparatus for touch. Their follicles are innervated predominantly by palisade endings composed of longitudinal and circumferential lanceolate endings. However, little is known as to how their original primary neurons make up a part of the ending. In this study, innervation of the palisade endings was investigated in the auricular skin of thy1-YFP transgenic mouse. Major observations were 1) Only a small portion of PGP9.5-immunopositive axons showed YFP-positivity, 2) All of thy1-YFP-positive sensory axons were thick and myelinated, 3) Individual thy1-YFP-positive trunk axons innervated 4–54 hair follicles, 4) Most palisade endings had a gap of lanceolate ending arrangement, 5) PGP9.5-immunopositive 10–32 longitudinal lanceolate endings were closely arranged. Only a part of them were thy1-YFP-positive axons that originated from 1–3 afferents, and 6) Single nerve bundles of the dermal nerve network included both bidirectional afferents. Palisade endings innervated by multiple sensory neurons might be highly sensitive to hair movement.

Sox2 promotes survival of satellite glial cells in vitro

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.

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry.

HSO3‐3‐galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro‐oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4‐positive cells. In this study, we identified a sulfatide species from the pro‐oligodendroblast‐to‐myelination stage by imaging mass spectrometry. The results demonstrated that short‐chain sulfatides with 16 carbon non‐hydroxylated fatty acids (C16) and 18 carbon non‐hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18‐OH) existed in restricted regions of the early embryonic spinal cord, where pro‐oligodendroblasts initially appear, and co‐localized with Olig2‐positive pro‐oligodendroblasts. C18 and C18‐OH sulfatides also existed in isolated pro‐oligodendroblasts. C22‐OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short‐chain fatty acids in pro‐oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination.

Increased Histone H3 Phosphorylation in Neurons in Specific Brain Structures after Induction of Status Epilepticus in Mice

Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3+ (PH3+) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation.

Differential responses of endogenous adult mouse neural precursors to excess neuronal excitation.

Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess as well as mild neuronal excitation, such as chemoconvulsant‐induced brief seizures. Because most studies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear. Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle progression of precursors was analysed by systemic administration of thymidine analogues. We found that brief seizures immediately resulted in cell cycle retardation in the SVZ. However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ was followed by enhanced cell cycle re‐entry after the first round of mitosis, leading to precursor pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient. Cell cycle progression in the PTZ‐treated group returned to normal after one cell cycle. The numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendants of SVZ precursors, were not significantly different from those in control mice more than 2 days after seizures. Because similar effects were observed following electroconvulsive seizures, these responses are likely to be general effects of brief seizures. These results suggest that neurogenesis in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the SGZ.

A Device for Ribbon Collection for Array Tomography with Scanning Electron Microscopy

“Array tomography” is a method used to observe the fine structure of cells and tissues in a three-dimensional view. In this method, serial ultrathin sections in the ribbon state (ribbons) are mounted on a solid substrate and observed by scanning electron microscopy (SEM). The method may also be used in conjunction with post-embedding immunocytochemistry. However, it is difficult to mount many serial ribbons on a substrate manually. We developed an inexpensive laboratory-made device that mounts ribbons by pulling a nylon fishing line and lifting the substrate up from the water in a knife boat. Using this device, we succeeded in mounting several ribbons consisting a mean of 205.6 (SD: 37.7) serial ultrathin sections on 1.25 (SD: 0.06) × 1.25 (SD: 0.06)-cm silicon substrates. Furthermore, it was confirmed that our method is suitable for ribbons derived from water-soluble resin blocks. We were also able to stain the specimens by post-embedding immunocytochemistry. Thus, our method is useful in mounting numerus sections on a substrate for array tomography with SEM.

Hypocretin/orexin loss changes the hypothalamic immune response

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy.

Identification of NeuN immunopositive cells in the adult mouse subventricular zone

In the adult rodent subventricular zone (SVZ), there are neural stem cells (NSCs) and the specialized neurogenic niche is critical to maintain their stemness. To date, many cellular and noncellular factors that compose the neurogenic niche and markers to identify subpopulations of Type A cells have been confirmed. In particular, neurotransmitters regulate adult neurogenesis and mature neurons in the SVZ have been only partially analyzed. Moreover, Type A cells, descendants of NSCs, are highly heterogeneous and more molecular markers are still needed to identify them. In the present study, we systematically classified NeuN, commonly used as a marker of mature and immature post‐mitotic neurons, immunopositive (+) cells within the adult mouse SVZ. These SVZ‐NeuN+ cells (SVZ‐Ns) were mainly classified into two types. One was mature SVZ‐Ns (M‐SVZ‐Ns). Neurochemical properties of M‐SVZ‐Ns were similar to those of striatal neurons, but their birth date and morphology were different. M‐SVZ‐Ns were generated during embryonic and early postnatal stages with bipolar peaks and extended their processes along the wall of the lateral ventricle. The second type was small SVZ‐Ns (S‐SVZ‐Ns) with features of Type A cells. They expressed not only markers of Type A cells, but also proliferated and migrated from the SVZ to the olfactory bulb. Furthermore, S‐SVZ‐Ns could be classified into two types by their spatial locations and glutamic acid decarboxylase 67 expression. Our data indicate that M‐SVZ‐Ns are a new component of the neurogenic niche and S‐SVZ‐Ns are newly identified subpopulations of Type A cells.