Ganglioside GD3, which has been used as "a marker for oligodendrocyte progenitor cells" in vitro, Is present in a few cells under the ependymal lining of the ventricle in adult rat brains. However, recent studies using a monoclonal antibody against GD3 (R24) suggested that GD3-positive cells in adult rat brains are microglia, thus making the cellular localization of GD3 controversial. In this immunohistochemical study, we compared images of immunostained sections of adult rat brains with a monoclonal antibody against GD3 (DSG-1) with those immunostained with microglia-specific (antibody against thymosin beta4), astroglia-specific (antibodies against GFAP or S-100 protein) or with oligodendroglia-specific (antibodies against GalC or MBP) antibodies for the purpose of re-examining the identity of GD3-positive cells in adult rat brains. We observed co-localization of GD3 immunoreactivity with that of GFAP or S-100 protein, but not with that of thymosin beta4. MBP or GalC. These results indicate that the GD3-positive cells in adult rat brains are astroglia, not microglia or oligodendroglia.

NeuroD/BETA2/BHF1 is a basic helix-loop-helix transcriptional factor, which can convert the Xenopus ectoderm to neurons, and activate the transcription of the insulin gene. In addition, the knock-out mice developed diabetes (Lee et al., 1995; Naya et al., 1995; 1997). Human NeuroD (NEUROD1) was mapped in chromosome 2q32 (Tamimi et al., 1996) which was near IDDM7 (Copeman et al., 1995), a human candidate susceptibility locus of insulin dependent diabetes mellitus (IDDM), and a variant of the NeuroD gene (Ala45Thr) is associated with IDDM (Iwata et al., 1999). Further mapping of this gene in other species is expected to accelerate the finding of the IDDM susceptibility loci in the rat and mouse IDDM models. We mapped NeuroD (Neurod1) to rat chromosome 3q24→ q32, and mouse chromosome 2E2–E3, which is consistent with the central region of mouse chromosome 2 determined by interspecific backcross analysis (Tamimi et al., 1996). Materials and methods

Differential hybridization screening of the cDNA libraries derived from the rat facial nucleus was performed, and a number of cDNA clones were isolated and found to be upregulated after facial nerve axotomy. One of the isolated cDNA clones encoded the A‐chain of C1q (C1q‐A) of the rat, whose cloning has not been reported in the literature. Sequence analysis showed that C1q‐A is well conserved in the rat, mouse and human on the DNA and protein levels. In situ hybridization demonstrated that there were strong signals for C1q‐A mRNA within the rat facial nucleus damaged by axotomy, and the signals were localized in activated microglia. Immunohistochemistry showed strong immunoreactivity for C1q protein in the activated microglia of the rat facial nucleus damaged by axotomy. C1q‐immunoreactivity was also found in the extracellular space of the damaged facial nucleus. No significant C1q‐A mRNA signals and C1q‐immunoreactivity were observed in the normal brain tissue. These results suggest that microglia markedly up‐regulate the C1q‐A gene to produce and secrete C1q in response to the neuronal damage caused by axotomy.

Retrograde degeneration of neurons following axotomy is an excellent experimental model of neurodegeneration. Using this model, molecular biological techniques have demonstrated a number of specific genes to be involved in the pathological processes of degeneration, regeneration, and repair which occur after axotomy. Further insight into gene involvement in these processes can be gained using the recently developed methods for screening genes whose expression is up‐ or down‐regulated after injury. These methods have successfully identified many genes to be markedly regulated and thus significantly involved in retrograde degeneration of the rat facial nucleus after axotomy of the facial nerve. Some are novel genes not previously identified, and others are genes already documented but their involvement in neuropathology not previously evaluated. Elucidation of the function of these genes would aid studies on the pathology of retrograde degeneration and provide important information on degenerative disorders of the human nervous system.

Proliferation and differentiation of ependymal cells in the injured carp spinal cord were studied by immunohistochemistry using proliferating cell nuclear antigen (PCNA) and H-3-thymidine (H-3-TdR) autoradiography. A surge of proliferation of ependymal cells occurred in the lesion with a peak around 6th day after surgical operation (complete transection) of the spinal cord. The proportion of PCNA-positive ependymal cells at 6th post-operative day (6 POD) was over 14 times that in the normal state. Electron microscopic autoradiography revealed that most of the ependymal cells incorporating H-3-TdR at 5 POD, especially those located in the caudal side of the transection site, contained numerous free ribosomes in their apical portion, but the other organellae, such as rough endoplasmic reticulum and Golgi apparatus, were poorly developed in this portion. In three weeks thereafter, the ependymal cell layer was reconstructed through the lesion, and the apical part of the H-3-TdR-labeled ependymal cells became elongated and morphologically differentiated: that is, it had free ribosomes decreased and glial filaments increased in number. Many bundles of regenerating axons were observed to course within the reconstructed ependymal cell layer. These results may suggest that proliferation, differentiation and reconstruction of the ependymal cell layer following injury of the carp spinal cord are requisite to make the permissive milieu for elongation of the regenerating axons.

A novel gene, designated neurorep 1, was isolated by differential hybridization screening from a complementary DNA library constructed from the rat facial nucleus whose nerve had been transected seven days before sampling. In situ hybridization revealed that this gene was up-regulated in the repair stage after axotomy. The deduced protein, Neurorep 1, consists of 293 amino acid residues, and its molecular mass is approximately 34,000. Protein sequence motif search indicates that this protein has an ecto-5'-nucleotidase consensus sequence at the carboxyl terminal region. In vitro studies showed that Neurorep 1 significantly increased the activity of ecto-5'-nucleotidase, which is considered to be involved in regeneration and repair of the central nervous system. Neurorep 1 might play a significant role in the repair process of nerve tissues by its regulation of ecto-5'-nucleotidase activity.

In order to understand the molecular mechanisms of the repair or regeneration process in the CNS, we tried to detect the genes whose expressions change after axotomy using rat facial nerve-nucleus system. We screened approximately 5,000 clones in the cDNA library of altered facial nuclei obtained at 1 to 7 days after axotomy. With a differential hybridization screening method, 139 genes were identified, and the expression of 27 genes was confirmed to have changed after axotomy by in situ hybridization. Twenty-four were up-and three were down-regulated. Among them three were novel genes (two up-regulated and one down-regulated), The 27 genes encoded the following proteins: beta and gamma actin, alpha and beta tubulin, neurofilament L subunit, alpha internexin, peripherin, calpactin IH chain, thymosin beta 4, cytoplasmic MAP1, cathepsin S, cystatin beta and C, GAP 43, SCG 10, MHC class I, complement Clq, neuroendocrine specific protein, ad-1 antigen, guanylate kinase, lactate dehydrogenase B, enkephalinase, dehydroptyridine reductase, osteonectin, neurodap I (novel), neurorep 1 (novel) and B256 protein (novel).

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (ICa,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca2+ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5–E11.5) than late developmental stage (LDS, E16.5–E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, ICa,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca2+ influx via LTCCs on AC activity, leading to the inability to inhibit AC.

To identify how the gp130-signaling cytokine oncostatin M (OSM), acting alone or in concert with IL-1β or TNFα, affects synovial fibroblast expression of genes relevant to inflammation and bone erosion in inflammatory arthritis.Synovial fibroblasts (SFs) were isolated from non-arthritic wild type (WT) or OSM receptor deficient (OSMR−/−) mice and stimulated with OSM, IL-1β or TNFα and their combinations. Cytokine gene expression was assessed by quantitative RT–PCR. ELISA, flow cytometry and immunohistochemistry identified protein expression. Gene expression patterns were confirmed in SFs isolated from patients with osteoarthritis (OASFs) and rheumatoid arthritis (RASFs).Expression of OSM and its receptors, gp130, OSMR and LIFR, was increased in synovial tissue from the mouse antigen-induced arthritis model. In isolated WT mouse synovial fibroblasts OSM alone, or in synergy with IL-1β, or together with TNFα, potently induced expression of the pro-inflammatory cytokine IL-6. OSM also induced a sustained increase in mRNA levels of the pro-osteoclastic cytokine RANKL. Combining OSM with IL-1β, but not with TNFα, further increased RANKL expression. Importantly these effects of OSM were all dependent on the expression of OSMR. Furthermore, OSM also increased expression of its own receptors, gp130 and OSMR and the IL-1 receptor, IL1-R1; the latter effects were also observed in both human OASFs and RASFs.Together our data suggests that OSM signaling via OSMR in SFs has the potential to contribute significantly to joint destruction in inflammatory arthritis. It not only induces expression of pro-inflammatory and pro-osteoclastic cytokines but can also augment its own actions and that of IL-1 by inducing expression of OSMR and IL-1R1.

Cystatin C, a cysteine proteinase inhibitor, is expressed in the central nervous system (CNS) as well as many other organs of mammals. However, little is known concerning whether its expression is regulated under pathological conditions of the CNS and what types of cells are responsible for this regulation. We performed differential hybridization screening of cDNA libraries derived from the rat facial nucleus and found a cDNA of rat cystatin C to be up-regulated following facial nerve axotomy. In situ hybridization using an RNA probe for rat cystatin C revealed that cystatin C mRNA in the facial nucleus was markedly increased in amount by day 7 after axotomy and was then decreased to the normal level by day 50. The intense signal for cystatin C mRNA in the damaged facial nucleus was localized in the glial cells which had the morphological characteristics of microglia. Light and electron microscopic immunohistochemistry using a rabbit antibody specific for cystatin C confirmed that microglia in the damaged facial nucleus were strongly positive for cystatin C. The immunoreactivity was also found in the extracellular space, consistent with the fact that cells producing cystatin C generally secreted this protein. These results demonstrate that cystatin C is markedly up-regulated by microglia in response to axotomy and is probably secreted by these cells into the extracellular space, suggesting that this proteinase inhibitor has (a) significant function(s) in the processes of neuronal degeneration, regeneration, and/or repair subsequent to axotomy.

Axonal injury and its repair are common and basic neuropathological processes in the CNS, and are composed of a complex of events in a molecular term. In order to get a comprehensive understanding of these processes, we isolated several known and unknown genes which were up-or downregulated in the facial nucleus after transection of the facial nerve by a subtractive/differential screening. Among them, we focus on one downregulated gene, named Neurodap1, because this gene encodes a novel protein carrying the RING-H2 sequence motif categorized in the zinc finger family. Immunoelectron microscopic analysis revealed that the protein encoded by Neurodap1, Neurodap1, was distributed mainly on the cytoplasmic side of the membranes constituting endoplasmic reticulum and Golgi apparatus, supporting the notion of a previously postulated function of RING-H2 motif proteins, that is, involvement in the protein sorting machinery. More interestingly, Neurodap1 was also bound to the postsynaptic density (PSD) region of axosomatic synapses. This fact suggests that Neurodap1 is associated with a specific system sorting proteins to PSD. Therefore, Neurodap1, a newly identified protein as an axotomy-suppressed gene product, might play a significant role in synaptic communication and plasticity through the control of the formation of PSD for maintaining vital functions of nerve cells.

Pathological studies on several neurodegenerative diseases including Alzheimer's disease have revealed common deposition of ubiquitin in many inclusion bodies. This implies a possible association of ubiquitin with neurodegeneration. To address this possibility, we examined histochemically the effect of intraventricular infusion of leupeptin, a thiol proteinase inhibitor, which is known to elevate anti-ubiquitin immunoreactivity in rat Purkinje cells. In the leupeptin-infused rat, an intense anti-ubiquitin immunoreactivity in the cytoplasm of neurons occurred not only in cerebellar Purkinje cells but also elsewhere in a wide area of the rat brain. The increase in the immunoreactivity was followed by a gradual depletion of pyramidal neurons in the hippocampal CA1 and CA3 subfields. The immunoreactive neurons disappeared concurrently. The number of anti-ubiquitin immunoreactive neurons was negatively correlated with that of surviving neurons when the duration of leupeptin infusion was varied. These results suggest that increased anti-ubiquitin immunoreactivity associates with neuronal death in leupeptin-treated rat brain.

In order to get a deeper insight into comprehensive understanding of gene regulation of brain-derived neurotrophic factor (BDNF), we characterized the transcriptional apparatus of this gene on the basis of the genomic structure. The results in this study revealed that there are at least four distinctive promoters in the BDNF gene; two of them are neuron-specific and the rest are active in some non-neuronal tissues as well as neuronal ones. Although the analyses of the promoter usage pattern clarified many characteristic features in controlling these promoter activities, the most notable finding was that administration of kainic acid resulted in great activation of two out of the four promoters in hippocampal neurons in a regionally different manner and thus indicated the presence of two distinct signal transduction pathways for kainate-induced activation of BDNF gene expression in neurons. The analysis of BDNF gene expression in terms of the promoter usage pattern would provide a new and important insight into understanding a molecular control mechanism of this gene expression.

The existence of a neurofilament-deficient mutant of Japanese quail was recently documented (Yamasaki, H., C. Itakura, and M. Mizutani. 1991. Acta Neuropathol. 82:427-434), but the genetic events leading to the neurofilament deficiency have yet to be determined. Our molecular biological analyses revealed that the expression of neurofilament-L (NF-L) gene was specifically repressed in neurons of this mutant. To search for mutation(s) responsible for the shutdown of this gene expression, we cloned and sequenced the NF-L genes in the wild-type and mutant quails. It is eventually found that the NF-L gene in the mutant includes a nonsense mutation at the deduced amino acid residue 114, indicating that the mutant is incapable of producing even a trace amount of polymerization-competent NF-L protein at any situation. The identification of this nonsense mutation provides us with a solid basis on which molecular mechanisms underlying the alteration in the neuronal cytoskeletal architecture in the mutant should be interpreted.

The diameter and conduction velocity of myelinated fibers in the sciatic nerve isolated from neurofilament-deficient quail ('quiver') were compared with those from wild quail. The diameter was significantly reduced in quiver, however, the frequency distribution formed three peaks as did that observed in the wild. The fibers were categorized in three groups possessing different conduction velocity. The conduction velocity appeared to be proportional to the diameter in each strain, and was significantly reduced in quiver. These results indicate that, although the diameter and conduction velocity are reduced in quiver fiber, the correlation between the parameters observed in wild quail is still preserved.