Intra-Articular Injections of the Adipose-Derived Mesenchymal Stem Cells Suppress Progression of a Mouse Traumatic Knee Osteoarthritis Model.

OBJECTIVE: This study aimed to determine whether the intra-articular injection of human adipose-derived mesenchymal stem cells (ADSCs) protects against the progression of murine post-traumatic osteoarthritis. DESIGN: ADSCs were isolated from human abdomen or buttock adipose tissues. In in vitro study, ADSCs conditioned medium was added to human chondrocytes pre-treated with interleukin-1ß (IL-1ß), and resultant gene expression of target inflammatory genes was measured by real-time quantitative polymerase chain reaction. A mouse model of knee osteoarthritis was generated by unilaterally transecting the medial meniscus in the right hind limb of 20 female C57BL/6 mice. Mice were randomly assigned to 2 treatment groups that received 6 µl intra-articular injections of either phosphate-buffered saline (control) or 2 × 104 cells/µl of ADSCs 14, 28, and 42 days post-surgery. Mice were euthanized 84 days post-surgery and histological and micro-computed tomography evaluation of knee joints were analyzed. Hind limb weight-bearing distribution was measured pre-surgery and 28 and 84 days post-surgery. RESULTS: Conditioned medium from cultured human adipose-derived mesenchymal stem cells suppressed the expression of target inflammatory genes in chondrocytes pre-treated with IL-1ß, suggesting anti-inflammatory properties (P < 0.01). Histological analyses indicated that the progression of destabilization of medial meniscus-induced knee osteoarthritis was suppressed by the administration of ADSCs compared with control group at medial femorotibial joint in vivo. This protective effect was related to a reduction in articular cartilage loss. CONCLUSION: The intra-articular injection of ADSCs suppressed articular cartilage loss in a mouse model of knee osteoarthritis, possible through anti-inflammatory mechanisms.

Intravenous administration of human mesenchymal stem cells derived from adipose tissue and umbilical cord improves neuropathic pain via suppression of neuronal damage and anti-inflammatory actions in rats.

Mesenchymal stem cells (MSCs), which are isolated from adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), or bone marrow, have therapeutic potential including anti-inflammatory and immunomodulatory activities. It was recently reported that MSCs are also effective as a therapeutic treatment for neuropathic pain, although the underlying mechanisms have yet to be resolved. Therefore, in this study, we investigated the effects of human AD- and UC-MSCs on neuropathic pain and its mechanisms using rat models of partial sciatic nerve ligation (PSNL). AD- or UC-MSCs were intravenously administered 4 days after PSNL. Antinociceptive effects were then evaluated using the von Frey and weight-bearing tests. We found that, 3-9 days after the administration of AD- or UC-MSCs to PSNL-exposed rats, both the mechanical threshold and differences in weight-bearing of the right and left hind paws were significantly improved. To reveal the potential underlying antinociceptive mechanisms of MSCs, the levels of activation transcription factor 3- and ionized calcium-binding adapter molecule 1-positive cells were measured by immunohistochemical analysis. AD- and UC-MSCs significantly decreased the levels of these proteins that were induced by PSNL in the dorsal root ganglia. Additionally, UC-MSC significantly improved the PSNL-induced decrease in the myelin basic protein level in the sciatic nerve, indicating that UC-MSC reversed demyelination of the sciatic nerve produced by PSNL. These data suggest that AD- and UC-MSCs may help in the recovery of neuropathic pain via the different regulation; AD-MSCs exhibited their effects via suppressed neuronal damage and anti-inflammatory actions, while UC-MSCs exhibited their effects via suppressed neuronal damage, anti-inflammatory actions and remyelination.

A Subtype of Olfactory Bulb Interneurons Is Required for Odor Detection and Discrimination Behaviors.

UNLABELLED: Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. The OB possesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors. SIGNIFICANCE STATEMENT: Neuronal circuits in the brain include glutamatergic principal neurons and GABAergic interneurons. Although the latter is a minority cell type, they are vital for normal brain function because they regulate the activity of principal neurons. If interneuron function is impaired, brain function may be damaged, leading to behavior disorder. The olfactory bulb (OB) possesses various types of interneurons, including granule cells (GCs); however, the contribution that each type of interneuron makes to the control of olfactory behavior remains unknown. Here, we analyzed electrophysiologically and behaviorally the function of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic branching in OB GCs. We found that, among the various types of OB interneuron, the 5T4 GC subtype is required for odor detection and odor discrimination behaviors.

Ion channels, guidance molecules, intracellular signaling and transcription factors regulating nervous and vascular system development.

Our sophisticated thoughts and behaviors are based on the miraculous development of our complex nervous network system, in which many different types of proteins and signaling cascades are regulated in a temporally and spatially ordered manner. Here we review our recent attempts to grasp the principles of nervous system development in terms of general cellular phenomena and molecules, such as volume-regulated anion channels, intracellular Ca(2+) and cyclic nucleotide signaling, the Npas4 transcription factor and the FLRT family of axon guidance molecules. We also present an example illustrating that the same FLRT family may regulate the development of vascular networks as well. The aim of this review is to open up new vistas for understanding the intricacy of nervous and vascular system development.

Molecular Mechanisms Regulating the Dendritic Development of Newborn Olfactory Bulb Interneurons in a Sensory Experience-Dependent Manner.

Inhibitory interneurons in the olfactory bulb are generated continuously throughout life in the subventricular zone and differentiate into periglomerular and granule cells. Neural circuits that undergo reorganization by newborn olfactory bulb interneurons are necessary for odor detection, odor discrimination, olfactory memory, and innate olfactory responses. Although sensory experience has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and hippocampus, little is known about how sensory experience regulates the dendritic development of newborn olfactory bulb interneurons. Recent studies revealed that the 5T4 oncofetal trophoblast glycoprotein and the neuronal Per/Arnt/Sim domain protein 4 (Npas4) transcription factor regulate dendritic branching and dendritic spine formation, respectively, in olfactory bulb interneurons. Here, we summarize the molecular mechanisms that underlie the sensory input-dependent development of newborn interneurons and the formation of functional neural circuitry in the olfactory bulb.

Npas4 regulates Mdm2 and thus Dcx in experience-dependent dendritic spine development of newborn olfactory bulb interneurons.

Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB). Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs), although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4, which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2, which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.

Goofy coordinates the acuity of olfactory signaling.

The basic scheme of odor perception and signaling from olfactory cilia to the brain is well understood. However, factors that affect olfactory acuity of an animal, the threshold sensitivity to odorants, are less well studied. Using signal sequence trap screening of a mouse olfactory epithelium cDNA library, we identified a novel molecule, Goofy, that is essential for olfactory acuity in mice. Goofy encodes an integral membrane protein with specific expression in the olfactory and vomeronasal sensory neurons and predominant localization to the Golgi compartment. Goofy-deficient mice display aberrant olfactory phenotypes, including the impaired trafficking of adenylyl cyclase III, stunted olfactory cilia, and a higher threshold for physiological and behavioral responses to odorants. In addition, the expression of dominant-negative form of cAMP-dependent protein kinase results in shortening of olfactory cilia, implying a possible mechanistic link between cAMP and ciliogenesis in the olfactory sensory neurons. These results demonstrate that Goofy plays an important role in establishing the acuity of olfactory sensory signaling.

5T4 glycoprotein regulates the sensory input-dependent development of a specific subtype of newborn interneurons in the mouse olfactory bulb.

Sensory input has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor-evoked activity dependent. However, little is known about the developmental role of sensory input in the other major OB population of the GABAgenic interneurons, such as granule cells and periglomerular cells. Here, we identified, with DNA microarray and in situ hybridization screenings, a trophoblast glycoprotein gene, 5T4, whose expression in a specific subtype of OB interneurons is dependent on sensory input. 5T4 is a type I membrane protein, whose extracellular domain contains seven leucine-rich repeats (LRR) flanked by characteristic LRR-N-flanking and C-flanking regions, and a cytoplasmic domain. 5T4 overexpression in the newborn OB interneurons facilitated their dendritic arborization even under the sensory input-deprived condition. By contrast, both 5T4 knockdown with RNAi and 5T4 knockout with mice resulted in a significant reduction in the dendritic arborization of 5T4(+) granule cells. Further, we identified the amino acid sequence in the 5T4 cytoplasmic domain that is necessary and sufficient for the sensory input-dependent dendritic shaping of specific neuronal subtypes in the OB. Thus, these results demonstrate that 5T4 glycoprotein contributes in the regulation of activity-dependent dendritic development of interneurons and the formation of functional neural circuitry in the OB.

Neuropilin-2 is required for the proper targeting of ventral glomeruli in the mouse olfactory bulb.

Recent evidence shows that olfactory sensory neurons expressing a given odorant receptor (OR) are not necessarily confined to one of four zones, rather arranged in an overlapping manner in the olfactory epithelium (OE). In this study, in situ hybridization of OE sections with the OR probes indicated that the OR genes, the mRNAs of which were detected in an array of glomeruli on olfactory bulb (OB) along the anterodorsal/posteroventral (AD/PV) axis, are expressed in subareal zones within the most ventral zone, zone 4, along the dorsomedial/ventrolateral (DM/VL) axis. We also found that Neuropilin-2 (Nrp2) is expressed in a DM-low to VL-high gradient within zone 4 of OE. Furthermore, in Nrp2 mutant mice, we observed multiple glomeruli for zone 4 ORs in OB. These results suggest that the graded expression of Nrp2 in OE is required for the proper targeting of ventral glomeruli along the AD/PV axis in OB.

BIG-2 mediates olfactory axon convergence to target glomeruli.

Olfactory sensory neurons expressing a given odorant receptor converge axons onto a few topographically fixed glomeruli in the olfactory bulb, leading to establishment of the odor map. Here, we report that BIG-2/contactin-4, an axonal glycoprotein belonging to the immunoglobulin superfamily, is expressed in a subpopulation of mouse olfactory sensory neurons. A mosaic pattern of glomerular arrangement is observed with strongly BIG-2-positive, weakly positive, and negative axon terminals in the olfactory bulb, which is overlapping but not identical with those of Kirrel2 and ephrin-A5. There is a close correlation between the BIG-2 expression level and the odorant receptor choice in individual sensory neurons. In BIG-2-deficient mice, olfactory sensory neurons expressing a given odorant receptor frequently innervate multiple glomeruli at ectopic locations. These results suggest that BIG-2 is one of the axon guidance molecules crucial for the formation and maintenance of functional odor map in the olfactory bulb.

Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously.

Fez is a zinc-finger gene encoding a transcriptional repressor that is expressed in the olfactory epithelium, hypothalamus, ventrolateral pallium and prethalamus at mid-gestation. To reveal its function, we generated Fez-deficient mice. The Fez-deficient mice showed several abnormalities in the olfactory system: (1) impaired axonal projection of the olfactory sensory neurons; (2) reduced size of the olfactory bulb; (3) abnormal layer formation in the olfactory bulb; and (4) aberrant rostral migration of the interneuron progenitors. Fez was not expressed in the projection neurons, interneurons or interneuron progenitors. Transgene-mediated expression of Fez in olfactory sensory neurons significantly rescued the abnormalities in olfactory axon projection and in the morphogenesis of the olfactory bulb in Fez-knockout mice. Thus, Fez is cell-autonomously required for the axon termination of olfactory sensory neurons, and Fez non-cell-autonomously controls layer formation and interneuron development in the olfactory bulb. These findings suggest that signals from olfactory sensory neurons contribute to the proper formation of the olfactory bulb.

Arx homeobox gene is essential for development of mouse olfactory system.

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.

Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map.

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.