Upregulation of Neural Cell Adhesion Molecule 1 and Excessive Migration of Purkinje Cells in Cerebellar Cortex.

Purkinje cells (PCs) are large GABAergic projection neurons of the cerebellar cortex, endowed with elaborate dendrites that receive a multitude of excitatory inputs. Being the only efferent neuron of the cerebellar cortex, PCs project to cerebellar nuclei and control behaviors ranging from movement to cognition and social interaction. Neural cell adhesion molecule 1 (NCAM1) is widely expressed in the embryonic and postnatal development of the brain and plays essential roles in neuronal migration, axon pathfinding and synapse assembly. However, despite its high expression levels in cerebellum, little is known to date regarding the role(s) of NCAM1 in PCs development. Among other aspects, elucidating how the expression of NCAM1 in PCs could impact their postnatal migration would be a significant achievement. We analyzed the Acp2 mutant mouse (nax: naked and ataxia), which displays excessive PC migration into the molecular layer, and investigated how the excessive migration of PCs along Bergmann glia could correlate to NCAM1 expression pattern in early postnatal days. Our Western blot and RT-qPCR analysis of the whole cerebellum show that the protein and mRNA of NCAM1 in wild type are not different during PC dispersal from the cluster stage to monolayer formation. However, RT-qPCR analysis from FACS-based isolated PCs shows that Ncam1 is significantly upregulated when PCs fail to align and instead overmigrate into the molecular layer. Our results suggest two alternative interpretations: (1) NCAM1 promotes excessive PC migration along Bergmann glia, or (2) NCAM1 upregulation is an attempt to prevent PCs from invading the molecular layer. If the latter scenario proves true, NCAM1 may play a key role in PC monolayer formation.

Mutations in the Reelin pathway are associated with abnormal expression of microglial IgG FC receptors in the cerebellar cortex.

Microglia are the immune cells of the central nervous system involved in a variety of developmental processes, such as regulation of cell death and survival, spatial patterning, and contribute to the development of Purkinje cells (PCs) during migration. Microglia express immunoglobulin G Fc receptors (FcgRs). In this report, we describe microglial FcgR expression and its relation to abnormal PC migration in the cerebellum during development. To detect microglial FcgR, the direct anti-IgG (secondary antisera) and high concentrations of Triton X-100 were applied as a method for labeling microglial cells without the use of any specific primary antiserum. By using Acp2-/- mice, which show an excessive PC migration into the molecular layer (ml), and 3 different types of mice with a null to alter the Reelin pathway (Reeler-, Dab1 (SCM)-, and Apoer mutant mice), we studied the location of PCs and the expression of FcgRs. Wild type littermates were used as controls in all studies. We show that the expression of microglial FcgRs was absent and PCs were ectopically located in the white matter in the cerebella of all mutant mice, except for the Acp2-/- mice (PCs were located in the ml). These results suggest a role for FcgRs in the Reelin signaling pathway, not in regulating PC migration, but rather in the adaptation to an environment with a relatively large number of ectopically located PCs. However, the exact correlation between the ectopic location of PCs and lack of FcgRs in Reeler, SCM, and Apoer-/- mice and the presence of FcgRs and directed PC location in the ml in Acp2-/- mice are yet to be determined.

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12-18.

C5L2 Silencing in Human Pulp Fibroblasts Enhances Nerve Outgrowth Under Lipoteichoic Acid Stimulation.

INTRODUCTION: We recently reported that caries-associated C5a receptor (C5aR) expression and activation result in up-regulation of brain-derived neurotropic factor secretion by pulp fibroblasts inducing prominent neurite outgrowth toward the carious site. Our data further showed a negative regulation of this brain-derived neurotropic factor secretion by C5L2, another C5aR. C5L2 was considered a nonfunctional receptor and thus has received much less attention than C5aR. The aim of this study was to identify the role of C5L2 in pulp fibroblast-mediated neurite outgrowth. METHODS: In this study, lipoteichoic acid (LTA) was used to mimic dental caries-like inflammation. To evaluate the role of C5L2 in pulp neurite outgrowth, human pulp fibroblasts were C5L2 small interfering RNA silenced and cocultured with human neurons in a nerve growth assay system. RESULTS: C5L2 silencing drastically increased the neurite outgrowth toward the LTA-stimulated pulp fibroblasts. The number of neurites detected was increased in the LTA-treated pulp fibroblasts. CONCLUSIONS: Our results show that C5L2 constitutes a negative regulator of the neurite outgrowth under LTA stimulation. Of the events occurring during dentin-pulp regeneration, nerve regeneration is the key factor for maintaining tooth viability after infection or injury. Our study provides a foundation for creating therapeutic tools that target pulp fibroblasts during pulp/nerve regeneration.

The p38α MAPK Deletion in Oligodendroglia does not Attenuate Myelination Defects in a Mouse Model of Periventricular Leukomalacia.

Periventricular leukomalacia (PVL) is a severe type of white matter damage in premature infants and the most common cause of cerebral palsy. It is generally known to be caused by hypoxia and inflammation. Currently there is no effective treatment available, in part due to that the pathogenesis of the disease has not been well understood. The p38α mitogen-activated protein kinase (MAPK) is the serine/threonine kinase and several in vitro studies demonstrated that p38 MAPK is essential for oligodendroglial differentiation and myelination. Indeed, our nerve/glial antigen 2 (NG2)-specific oligodendroglial p38α MAPK conditional knockout (CKO) mice revealed its complex roles in myelination and remyelination. To identify the specific in vivo roles of oligodendroglial p38α MAPK in PVL, we generated a mouse PVL model by combination of LPS-mediated inflammation and hypoxia-ischemia in NG2-p38α MAPK CKO mice. Our results demonstrate that a selective deletion of p38α MAPK in oligodendrocyte did not attenuate myelination defects in the mouse model of PVL. Myelination phenotype revealed by MBP immunostaining was not significantly affected in the p38α MAPK CKO mice compared to the wildtype after PVL induction. The electron microscopic images demonstrated that the microstructure of myelin structures was not significantly different between the wild-type and p38α MAPK CKO mice. In addition, oligodendrocyte degeneration in the corpus callosum white matter area was unaffected in the p38α MAPK CKO during and after the PVL induction. These data indicate that p38α MAPK in oligodendrocyte has minimal effect on myelination and oligodendrocyte survival in the mouse PVL model.

Poly(Adenosine Phosphate Ribose) Polymerase 1 Inhibition Enhances Brain-derived Neurotrophic Factor Secretion in Dental Pulp Stem Cell-derived Odontoblastlike Cells.

INTRODUCTION: The nuclear enzyme poly(adenosine phosphate ribose) polymerase 1 (PARP-1) has been implicated in the maintenance and differentiation of several stem cells. The role of PARP-1 in dental pulp stem cell (DPSC) differentiation, especially in the context of its ability to modulate nerve regeneration factors, has not been investigated. Regeneration of neuronal components in pulp tissue is important for the assessment of tooth vitality. Brain-derived neurotrophic factor (BDNF) is known to play an integral signaling factor during nerve regeneration. In this study, we identified the role of PARP-1 in the modulation of BDNF in DPSC differentiation into odontoblastlike cells. METHODS: Human DPSCs were prepared from healthy molars and cultured in regular and osteogenic media treated with PARP-1 antagonist and PARP-1 exogeneous protein. Polymerase chain reaction and immunohistochemistry analysis for BDNF and various differentiation markers were performed. RESULTS: Our polymerase chain reaction results showed that differentiated cells show odontoblastlike properties because they express odontogenic markers such as dentin sialophosphoprotein and dentin matrix protein 1. Both PARP-1 inhibitor and protein did not affect odontogenic differentiation and proliferation because the number of the differentiated cells was unaffected, and the expression of dentin sialophosphoprotein and dentin matrix protein 1 was not significantly changed. There is the possibility that PARP-1 treatment induces DPSCs into the unique cell lineage. Some differentiated cells show a very unique morphology with large irregular cytoplasm and an oval nucleus. Moreover, PARP-1 inhibition significantly increased BDNF secretion in DPSC-derived odontoblastlike cells. This observation was also confirmed by immunohistochemistry. CONCLUSIONS: Taken together, our results indicate PARP-1 as a negative regulator in BDNF secretion during odontogenic DPSC differentiation, showing its potential application for translational nerve regeneration strategies to improve dental pulp tissue vitality assessments.