Cheil Moon

Fabrication and Characterization of Magnetic Microrobots for Three‐Dimensional Cell Culture and Targeted Transportation

Magnetically manipulated microrobots are demonstrated for targeted cell transportation. Full three-dimensional (3D) porous structures are fabricated with an SU-8 photoresist using a 3D laser lithography system. Nickel and titanium are deposited as a magnetic material and biocompatible material, respectively. The fabricated microrobots are controlled in the fluid by external magnetic fields. Human embryonic kidney 239 (HEK 239) cells are cultivated in the microrobot to show the possibility for targeted cell transportation.

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.

Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation

The discovery of stem cells in the adult central nervous system raises questions concerning the neurotrophic factors that regulate postnatal neuronal development. Olfactory receptor neurons (ORNs) are a useful model, because they are capable of robust neurogenesis throughout adulthood. We have investigated the role of leukemia inhibitory factor (LIF) in postnatal neuronal development by using ORNs as a model. LIF is a multifunctional cytokine implicated in various aspects of neuronal development, including phenotype determination, survival, and in response to nerve injury. LIF-deficient mice display significant increases, both in the absolute amount and in the number of cells expressing olfactory marker protein, a marker of mature ORNs. The maturation of ORNs was significantly inhibited by LIF in vitro. LIF activated the STAT3 pathway in ORNs, and transfection of ORNs with a dominant negative form of STAT3 abolished the effect of LIF. These findings demonstrate that LIF negatively regulates ORN maturation via the STAT3 pathway. Thus, LIF plays a critical role in controlling the transition of ORNs to maturity. Consequently, a population of ORNs is maintained in an immature state to facilitate the rapid repopulation of the olfactory epithelium with mature neurons during normal cell turnover or after injury.

Plunc, a Member of the Secretory Gland Protein Family, Is Up-regulated in Nasal Respiratory Epithelium after Olfactory Bulbectomy

Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy. We isolated the rat homologue ofplunc, a murine gene highly expressed in lung and nasopharyngeal regions, by this method. Rat plunc encodes a 270-amino acid protein containing a putative signal peptide.plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization.plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis. Antibodies against Plunc detected a 31-kDa protein in lung, heart, and spleen. Rat nasal epithelium displayed robust immunoreactivity that was highly localized to the microvilli layer of respiratory epithelium. The expression of plunc was up-regulated after bulbectomy in respiratory epithelium. We also detected secreted plunc in rat and human mucus. Sequence and homology analyses suggest that Plunc is a member of the secretory gland protein family with putative bactericidal/bacteriostatic function. This is the first protein found in respiratory epithelium whose expression is regulated by olfactory neuronal injury and may provide protection against infection subsequent to injury.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat

Trigeminal primary afferents expressing P2X3 receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X3‐immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X3 immunoreactivity was mainly in small and medium‐sized somata, but also frequently in large somata. Although most P2X3+ somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X3+ fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X3 immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X3+ terminals contained numerous clear, round vesicles and sparse large, dense‐core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X3+ terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X3+ afferents to all TBSN and that it may be processed differently in different TBSN. J. Comp. Neurol. 506:627–639, 2008.

ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.

Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin, respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO‐1 and HO‐2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post‐natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post‐natally. Mice null for either HO‐1 or HO‐2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO‐2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO‐2 null mice. These roles for HO‐1 and HO‐2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO‐cGMP signaling system and bilirubin in neurodevelopment and in response to injury.

Neurotrophin-3 signaling maintains maturational homeostasis between neuronal populations in the olfactory epithelium

Neurons within the olfactory system undergo functional turnover throughout life. This process of cell death and compensatory neurogenesis requires feedback between neuronal populations of different developmental ages. We examined the role of NT-3 in this process. NT-3 was localized within both the olfactory bulb and olfactory epithelium. Mice null for NT-3 showed increased numbers of immature neurons, without change in the number of mature neurons. This was due to compensatory alterations in apoptosis of mature and immature neuronal populations. Using a primary olfactory neuronal culture, NT-3 was found to directly activate the PI3K/Akt pathway and indirectly activate the MAPK and PLC pathways. Activated PI3K/Akt promoted mature neuronal survival and induced the release of secondary factors, which activated the MAPK and PLC pathways to reduce neuronal precursor proliferation and inhibit neuronal maturation. These effects of NT-3 serve to maintain homeostasis between neuronal populations within the olfactory epithelium.

Mild Hypothermia Attenuates Intercellular Adhesion Molecule-1 Induction via Activation of Extracellular Signal-Regulated Kinase-1/2 in a Focal Cerebral Ischemia Model.

Intercellular adhesion molecule-1 (ICAM-1) in cerebral vascular endothelium induced by ischemic insult triggers leukocyte infiltration and inflammatory reaction. We investigated the mechanism of hypothermic suppression of ICAM-1 in a model of focal cerebral ischemia. Rats underwent 2 hours of middle cerebral artery occlusion and were kept at 37°C or 33°C during occlusion and rewarmed to normal temperature immediately after reperfusion. Under hypothermic condition, robust activation of extracellular signal-regulated kinase-1/2 (ERK1/2) was observed in vascular endothelium of ischemic brain. Hypothermic suppression of ICAM-1 was reversed by ERK1/2 inhibition. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) in ischemic vessel was attenuated by hypothermia. STAT3 inhibitor suppressed ICAM-1 production induced by stroke. ERK1/2 inhibition enhanced phosphorylation and DNA binding activity of STAT3 in hypothermic condition. In this study, we demonstrated that hypothermic suppression of ICAM-1 induction is mediated by enhanced ERK1/2 activation and subsequent attenuation of STAT3 action.

Leukemia inhibitory factor promotes olfactory sensory neuronal survival via phosphoinositide 3-kinase pathway activation and Bcl-2

Leukemia inhibitory factor (LIF), a neuropoietic cytokine, has been implicated in the control of neuronal development. We previously reported that LIF plays a critical role in regulating the terminal differentiation of olfactory sensory neurons (OSNs). Here, we demonstrate that LIF plays a complementary role in supporting the survival of immature OSNs. Mature OSNs express LIF, which may be elaborated in a paracrine manner to influence adjacent neurons. LIF null mice display more apoptotic immature neurons than do their wild‐type littermates. LIF treatment of dissociated OSNs in vitro significantly reduces the apoptosis of immature OSNs. Double immunocytochemical analysis indicates that the survival of immature OSNs is dependent on the presence of LIF. LIF activates the phosphoinositide 3‐kinase (PI3K) pathways and induces the expression of the antiapoptotic molecule Bcl‐2 in OSNs, whereas inhibition of the PI3K pathway blocks LIF‐dependent OSN survival and Bcl‐2 induction. Thus, LIF plays a central role in maintaining the size and integrity of the population of immature neurons within the olfactory epithelium; this population is critical to the rapid recovery of olfactory function after injury. LIF may play a similar role elsewhere in the CNS and thus be important for manipulation of stem cell populations for therapeutic interventions.