Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

김소연,유승준,Gabriele V Ronnett,김은경,문제일 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.6

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3’-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorantinduced Bcl-2 expression, as well as the effects on odorant- induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

AMP-Activated Protein Kinase (AMPK) and Energy-Sensing in the Brain

Santosh Ramamurthy,Gabriele V. Ronnett 한국뇌신경과학회 2012 Experimental Neurobiology Vol.21 No.2

5’-adenosine monophosphate-activated protein kinase (AMPK) is an evolutionarily conserved cellular and organismal energy integrator that responds to numerous stimuli with the overall intention to facilitate energy conservation and enhance energy balance while also aff ecting cellular survival and behaviors. AMPK has been appreciated for many years to function in peripheral organs that contribute to the generation or disposition of cellular energy, while its role in the brain has been only recently elucidated. While acknowledged to respond to organismal energy balance, we now recognize that energy balance within neurons also aff ects the brain’s response to these peripheral signals. In this review, we discuss AMPK’s regulation and its ever-expanding role as a neuronal energy integrator at both the cellular and systems levels.

Protective Effects of Inducible HO-1 on Oxygen Toxicity in Rat Brain Endothelial Microvessel Cells

유승준,Neal K. Nakra,Gabriele V. Ronnett,문제일 대한내분비학회 2014 Endocrinology and metabolism Vol.29 No.3

Background: Reperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1) have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin. Methods: To confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively. Results: HO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant. Conclusion: Our results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.

Direct measurement of extracellular electrical signals from mammalian olfactory sensory neurons in planar triode devices

Kim, Hwajeong,Kim, So Yeun,Nam, Sungho,Ronnett, Gabriele V.,Han, Hyung Soo,Moon, Cheil,Kim, Youngkyoo The Royal Society of Chemistry 2012 The Analyst Vol.137 No.9

<P>An artificial nose was developed to mimic aspects of sensory transduction of the peripheral mammalian olfactory system. We directly cultured and differentiated rat olfactory sensory neurons (OSNs) on indium-tin oxide electrodes of planar triode substrates without a coupling agent. Direct voltage (∼50 μV) and current (∼250 nA) signals were measured simultaneously when OSNs on the planar triode substrates were exposed to odorant mixtures. The response signals were sensitive to the concentration of the odorant mixture, with a typical lifetime, shape, and adaptation profile as seen in responses upon repeated stimulation <I>in vivo</I>. We found that the rising time to the peak current was ∼161 ms, while the signal back to baseline was in 1.8 s, which are in agreement with the natural intracellular electrophysiological responses. These results provide the first evidence that mature OSNs grown in a planar triode device are able to detect direct electrophysiological responses to odorants.</P> <P>Graphic Abstract</P><P>A planar triode-type artificial nose with olfactory sensory neurons (OSNs) generates direct voltage (~50 μV) and current (~250 nA) signals upon odorant stimulation. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c2an16205a'> </P>

Odorant Receptors Containing Conserved Amino Acid Sequences in Transmembrane Domain 7 Display Distinct Expression Patterns in Mammalian Tissues

류상은,Tammy Shim,이주연,김소연,박선화,김성원,Gabriele V. Ronnett,문제일 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.12

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Nu-merous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction.

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Kim, So Yeun,Yoo, Seung-Jun,Ronnett, Gabriele V,Kim, Eun-Kyoung,Moon, Cheil Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.6

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorantinduced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

Effects of unilateral naris occlusion on the olfactory epithelium of adult mice

Suh, Kyung Shik,Kim, So Yeun,Bae, Yong Chul,Ronnett, Gabriele V.,Moon, Cheil Lippincott Williams Wilkins, Inc. 2006 NEUROREPORT - Vol.17 No.11

Sensory stimulation is an essential component of neuronal functions, including information processing, dendritic and axonal refinement, synaptic plasticity, and neuronal survival. We examined the roles of sensory stimulation in the maintenance of olfactory sensory neurons in the olfactory epithelium using the adult mouse olfactory system as the model system. Obstruction of sensory stimulation through unilateral naris occlusion caused downregulation of proliferation and upregulation of apoptosis of neurons in the olfactory epithelium. Sensory stimulation is therefore important in maintaining homeostasis in the adult olfactory epithelium. Our study contributes to further understanding the roles and mechanisms of sensory stimulation in the postnatal neuronal development as well as to the damage and potential treatments of patients who suffer recurrent nasal obstruction.

Odorant Receptors Containing Conserved Amino Acid Sequences in Transmembrane Domain 7 Display Distinct Expression Patterns in Mammalian Tissues

Ryu, Sang Eun,Shim, Tammy,Yi, Ju-Yeon,Kim, So Yeun,Park, Sun Hwa,Kim, Sung Won,Ronnett, Gabriele V.,Moon, Cheil Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.12

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Numerous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction.

Physiological oxygen level is critical for modeling neuronal metabolism in vitro

Zhu, Jing,Aja, Susan,Kim, Eun‐,Kyoung,Park, Min Jung,Ramamurthy, Santosh,Jia, Junling,Hu, Xueying,Geng, Ping,Ronnett, Gabriele V. Wiley Subscription Services, Inc., A Wiley Company 2012 Journal of neuroscience research Vol.90 No.2

<P><B>Abstract</B></P><P>In vitro models are important tools for studying the mechanisms that govern neuronal responses to injury. Most neuronal culture methods employ nonphysiological conditions with regard to metabolic parameters. Standard neuronal cell culture is performed at ambient (21%) oxygen levels, whereas actual tissue oxygen levels in the mammalian brain range from 1% to 5%. In this study, we examined the consequences of oxygen level on the viability and metabolism of primary cultures of cortical neurons. Our results indicate that physiological oxygen level (5% O<SUB>2</SUB>) has a beneficial effect on cortical neuronal survival and mitochondrial function in vitro. Moreover, oxygen level affects metabolic fluxes: glucose uptake and glycolysis was enhanced at physiological oxygen level, whereas glucose oxidation and fatty acid oxidation were reduced. Adenosine monophosphate‐activated protein kinase (AMPK) was more activated in 5% O<SUB>2</SUB> and appears to play a role in these metabolic effects. Inhibiting AMPK activity with compound C decreased glucose uptake, intracellular ATP level, and viability in neurons cultured in 5% O<SUB>2</SUB>. These data indicate that oxygen level is an important parameter to consider when modeling neuronal responses to stress in vitro. © 2011 Wiley Periodicals, Inc.</P>

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

Moon, Cheil,Liu, Bridget Q.,Kim, So Yeun,Kim, Esther J.,Park, Yun Ju,Yoo, Joo-Yeon,Han, Hyung Soo,Bae, Yong Chul,Ronnett, Gabriele V. Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of neuroscience research Vol.87 No.5

<P>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. © 2008 Wiley-Liss, Inc.</P>