Program for Promoting Academic Excellence of Universities (Phase II)
Annual Report
神經功能表徵之探索：細胞與分子訊號之嶄新詮釋
Novel Neurobiological Phenotypes for
Cellular and Molecular Signals
NSC96-2752-B-110-001-PAE
NSC96-2752-B-110-002-PAE
NSC96-2752-B-075B-001-PAE
NSC96-2752-B-006-001-PAE
NSC96-2752-B-006-002-PAE
Overall Duration: April 1, 2005 ─ March 31, 2009
Annual Duration: April 1, 2007 ─ March 31, 2008
National Sun Yat-sen University
Date: January, 2008
II. (FORM1) BASIC INFORMATION OF THE PROGRAM
Program Title: Novel Neurobiological Phenotypes for Cellular and Molecular Signals
神經功能表徵之探索：細胞與分子訊號之嶄新詮釋 Serial No.: NSC96-2752-B-110-001-PAE Affiliation (in English & Chinese)
Name Chan, Samuel H.H. 陳慶鏗 Name Chang, Alice Y.W. 張雅雯
Tel: 07-5255800 Tel: 07-5253629 Fax: 07-5255801 Fax: 07-5255801 Prin cip al In v estig ato r
E-mail [email protected] Program C
oordi
nat
o
r
E-mail [email protected]
Expenditures1 (in NT$1,000) Manpower2:Full time/Part time(Person-Months)
Projected Actual Projected Actual
FY 94 13000 12991 11/9 11/9
FY 95 15959 15859 12/9 12/9
FY 96 16000 16000 16/9 16/9
FY 97 -
Overall
Serial No. Project Title Principal
Investigator Title Affiliation
Main Project
Novel neurobiological phenotypes for cellular and molecular signals
神經功能表徵之探索：細胞與分子訊號之嶄 新詮釋 Samuel H.H. Chan 陳慶鏗 Professor and Director 教授/主任 Center for Neuroscience, National Sun Yat-sen University 國立中山大學 神經科學 研究中心 Sub-Project 1
Cellular and molecular signals underlying brain stem apoptotic cell death during endotoxemia 參與內毒素敗血症引發腦幹細胞凋亡現象 之細胞及分子訊息研究 Samuel H.H. Chan 陳慶鏗 Alice Y.W. Chang 張雅雯 （共同主持人） Professor and Director 教授/主任 Professor 教授 Center for Neuroscience, National Sun Yat-sen University 國立中山大學 神經科學 研究中心
Sub-Project 2
Oxidative stress and neurogenic hypertension: brain stem cellular and molecular
mechanisms 參與腦幹氧化壓力引發高血壓之細胞及分 子訊息研究 Julie Y.H. Chan 華瑜 Ming-Hong Tai 戴明泓 （共同主持人） Senior Principal Investigator 研究員 Senior Principal Investigator 研究員 Department of Medical Education and Research, Kaohsiung Veterans General Hospital 高雄榮民總醫院 教學研究部 Sub-Project 3
Stress-related modification of amygdala synaptic plasticity and fear memory formation: cellular and molecular mechanisms 參與壓力調控杏仁核神經突觸功能及恐懼 記憶形成之細胞及分子訊息研究 Po-Wu Gean 簡伯武 Professor 教授 Department of Pharmacology, College of Medicine, National Cheng Kung University 國立成功大學 醫學院藥理學科 Sub-Project 4
Cellular and molecular signals mediating normal and stressed hippocampal synaptic function and synaptic plasticity
參與正常及壓力下海馬迴神經突觸功能及 塑性表現之細胞及分子訊息研究 Kuei-Sen Hsu 許桂森 Professor and Director 教授/主任 Department of Pharmacology, College of Medicine, National Cheng Kung University 國立成功大學 醫學院藥理學科
Notes: 1,2 Please explain large differences between projected and actual figures.
III. (FORM 2) LIST OF WORKS, EXPENDITURES, MANPOWER, AND MATCHING SUPPORTS FROM THE PARTICIPATING INSTITUTES（REALITY）.
Serial No.:
NSC96-2752-B-110-001-PAE
Program Title: Novel neurobiological phenotypes for cellular and molecular signals 神經功能表徵之探索：細胞與分子訊號之嶄新詮釋
Expenditures (in NT$1,000) Manpower (person-month) Research Item (Include sub projects) Major tasks and objectives Salary Seminar/ Conference-re lated expenses Project- related expenses Overhead Total Principal Investigators Consultants Research/ Teaching Personnel Supporting Staff Total
Matching Supports from the Participating Institutes (in English & Chinese)
Main Project Novel neurobiological phenotypes for cellular and molecular signals 1668.3 0 1849.5 482.2 4000.0 1 0 1 4 6 None Sub-Project 1: Cellular and molecular signals underlying brain stem apoptotic cell death during endotoxemia 1538.3 180.0 2219.0 562.7 4500.0 2 0 2 2 6 None
Sub-Project 2 Oxidative stress and neurogenic hypertension: brain stem cellular and molecular mechanisms 1054.3 180.0 974.3 291.4 2500.0 2 0 1 3 6 None Sub-Project 3 Stress-related modification of amygdala synaptic plasticity and fear memory formation： cellular and molecular mechanisms. 656.0 0 1694.0 150.0 2500.0 1 0 6 0 7 None Sub-project 4: Cellular and molecular signals mediating normal and stressed hippocampal synaptic function and synaptic plasticity 1322.3 0 868.0 309.7 2500.0 1 0 6 0 7 None SUM 6239.2 360.0 7604.8 1796.0 16000.0 7 0 16 9 32
IV. (FORM 3) STATISTICS ON RESEARCH OUTCOME OF THIS PROGRAM
LISTING TOTAL DOMESTIC INTERNATIONAL SIGNIFICANT1 CITATIONS2 TECHNOLOGYTRANSFER
JOURNALS 13 13 13 3,197 CONFERENCES 28 8 20 28 PUBLISHED ARTICLES TECHNOLOGY REPORTS PENDING - PATENTS GRANTED -
COPYRIGHTED INVENTIONS ITEM
ITEM
WORKSHOPS/CONFERENCES3
PARTICIPANTS
HOURS
TRAINING COURSES
（WORKSHOPS/CONFERENCES） PARTICIPANTS
HONORS/ AWARDS4 9 9
KEYNOTES GIVEN BY PIS 2 2
PERSONAL ACHIEVEMENTS
EDITOR FOR JOURNALS 3 1 2
ITEM LICENSING FEE TECHNOLOGY TRANSFERS ROYALTY
INDUSTRY STANDARDS5 ITEM
ITEM - - -
TECHNOLOGICAL SERVICES6
SERVICE FEE - - -
1 Indicate the number of items that are significant. The criterion for “significant” is defined by the PIs of the program. For example, it may refer to Top journals (i.e., those with impact factors in the upper 15%) in the area of research, or
conferences that are very selective in accepting submitted papers (i.e., at an acceptance rate no greater than 30%). Please specify the criteria in Appendix IV.
2 Indicate the number of citations (1997-2007). The criterion for “citations” refers to citations by other research teams, i.e., exclude self-citations. 3 Refers to the workshop and conferences hosted by the program.
4
Includes Laureate of Nobel Prize, Member of Academia Sinica or equivalent, fellow of major international academic societies, etc.
5 Refers to industry standards approved by national or international standardization parties that are proposed by PIs of the program. 6
V. (FORM4) EXECUTIVE SUMMARY ON RESEARCH OUTCOMES OF THIS PROGRAM
1. General Description of the Program
Note 1: It should be pointed out right from the outset that the funding provided by
the National Science Council for the Program for Promoting Academic Excellence of Universities (Phase II) is substantially less than (approximately 30%) that provided by the Ministry of Education for the Phase I program. To cope with this situation while maintaining the level of scientific sophistication that we envisage for a project of this status and in accordance with our definiton of academic excellence, we have resolved to scale down the scope of our research program.
Note 2: Whereas Project Year 3 should begin on April 1, 2007, funding from the
National Science Council was only made available in August, 2007. Thus, this report
in effect covers only our research efforts for the past 5 months.
This program capitalizes on the momentum set forth by the success of the Phase I Program for Promoting Academic Excellence of University carried out during
2000-2004 entitled “Southern Taiwan Center of Excellence in Neuroscience Research”. 6 neuroscientists, who are responsible for more than 75% of the publications in top journals arising from the Phase I program and are well versed in multidisciplinary and collaborative research of this caliber using state-of-the-art methodologies, again participated in this program.
The conceptual backbone for this program is that any given cellular and molecular signal may subserve a multitude of physiological or pathological phenotypes. It reflects an important message in the post-genomic era that all cells in an organism share the same genome. It is only on up- or downregulation of selected components of this genome that phenotypes unique to individual cells, tissues, organs, systems or organisms are manifested. It follows that quiescent cellular and molecular signaling cascades are potentially operational when suitably triggered; or activated ones are prompt to suppression. Thus, the guiding hypothesis for this program is that a given cellular and molecular signal may subserve brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity. This innovative hypothesis is assessed by consorted and integrative efforts of the participating PIs based on three operational principles.
First, integration through working on the same signaling cascades. We have
taken full advantage of well-established experimental paradigms from our individual laboratories. Thus, each sub-project investigates the engagement of potential target cellular and molecular signals in the neurobiological phenotype that the PI is most familiar with, using stressors that include endotoxin, organophosphate poison,
oxidative stress, fear or neonatal manipulations. This approach allows us to conclude in a relatively short time whether a given cellular and molecular signal may subserve brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity. However, to allow for an integrative link between the 4
sub-projects, all of us will initially target on the same transcription factors (NF-κB and CREB), signaling cascades (PI3K/Akt, PTEN and MAPKs), receptors (angiotensin receptors and glutamate receptors) or protein degradation systems
(ubiquitin-proteasome system and heat shock proteins). These cellular and molecular signals are chosen because the literature and previous or preliminary data strongly suggest that they are reasonable candidates. We anticipate that such a focused effort shall allow us to conclude within 36 months after the commencement of this project on whether they may serve as common cellular or molecular signals for brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity.
Second, integration through scientific interactions. Individual PIs collaborate
with each other by providing technical support or incorporating a mutual scientific question into the sub-projects. On top of this conventional way of integration, we also establish three additional programs of scientific interactions in the main project. (1) A protein-profiling program is responsible for identifying additional cellular and
molecular signals for confirmation by the PIs to be common to our neurobiological phenotypes. (2) A gene delivery program generates, validates and characterizes adenoviral vectors aiming at overexpression or downregulation of selected signaling molecules, to be used by the PIs in their sub-projects. (3) In addition to frequent e-mail or telephone communications, guidance of progress at PI meetings and midterm
progress report provide ample opportunities for face-to-face discussion and interactions between the PIs.
Third, integration through streamlined administrative support. Full-time
administrative personnel at the Center for Neuroscience in National Sun Yat-sen University provide the administrative support and coordination for the scientific and auxiliary activities of the participating PIs, their institutions and the funding agency.
In addition to significant societal impact on Taiwan and the rest of the world, the phenotypes chosen for the proposed study, brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity, represent currently the most recognized neuroscience research areas in Taiwan by the international community. Our program thus has significant scientific impact, leading to high international
competitiveness. On top of validating an innovative concept in contemporary
biomedicine research, it is envisaged that results from our program shall reveal novel neurobiological phenotypes for many established or new cellular and molecular signals. Successful completion of this project is anticipated because of the documented experience and achievements of the participating PIs. These accomplishments are already epitomized in publications in top journals, and invitation to contribute reviews or present lectures or chair sessions at international or national conferences. We thus are confident that such a visibility will marshal our group to the international scene.
2. Breakthroughs and Major Achievements
A. Main Project
In addition to the administrative services, the main project is primarily responsible for encouraging collaboration among the participating PIs and overseeing the
protein-profiling and gene delivery programs.
(1) Collaborations among PIs
6 out of the 13articles published or in press during Project Year 3 are joint efforts between the main and sub-projects, or between individual sub-projects. It must be stressed that this figure is an under-estimate of the collaborative spirit among the 6 participating PIs. This is because we believe that technical support and sharing of experience or information, though vitally important, should not be criteria for authorship.
(2) Common signaling cascades
The fundamental backbone of this program is that a given cellular and
molecular signal may subserve brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity. There are already indications that this innovative concept is justified, based on the candidate cellular and molecular signals chosen for our initial studies.
(1) PI3K/Akt signaling cascade
• “Pro-death” mechanism in brain stem death (rostral ventrolateral medulla, RVLM)
• Genesis of neurogenic hypertension (RVLM) • Fear conditioning (amygdala)
• Stress-induced impairment of long-term synaptic plasticity (hippocampus)
• VEGF-induced synaptic plasticity (hippocampus)
(2) PTEN
• Not involved in brain stem death (RVLM)
• Impairs genesis of neurogenic hypertension (RVLM) • Impairs fear memory (amygdala)
(3) ERK1/2 signaling
• “Pro-life” phase of brain stem death (RVLM)
• Long-term pressor response to angiotensin II (RVLM) • Extinction of conditioend fear by endocannabinoids (mPFC)
• Stress-induced impairment of long-term synaptic plasticity (hippocampus)
(4) p38 MAPK signaling
• Not involved in brain stem death (RVLM)
• Short-term pressor response to angiotensin II (RVLM)
• (S)-3,5-dihydroxyphenylglycine-induced long-term depression
(hippocampus)
(5) Ubiquitin-proteasome system
• Upregulated in brain stem death (RVLM)
• D-cycloserine-induced extinction of fear startle (amygdala)
(6) cAMP response element-binding protein
• Long-term pressor response to angiotensin II (RVLM) • Consolidation of fear memory (amygdala)
2D map of RVLM (
2D map of RVLM (ppII33--10, 10, MMrr<94 <94 kDakDa))
IEF SDS 94 67 43 30 20.1 14.4 M r (kD a ) 3 4 5 6 7 8 9 10 pI
188 Heat shock protein 105 kDa 137 51 47 43 32 34 2 37118 119 120 121 140 62 76 110 111 56 109 7 139 138 137 50 20 80 8 124 45 44 108 105106 107 60 104 59 58 112 9 132 131 113 86 114 117 133 135 134 130 126 127 128 129 125 38 142 141 95 78 26 116 115 65 11 10 31 36 34 53 48 122 851 46 18 17 16 3029 33 55 54 14 13 12 52 49 35 42 21 28 24 22 23 27 39 25 19 40 41 6 145 144 143 206 69 100 204 205 157 101200 15 215 154 150 164 203 202 201 146 147195 199 148 196 197 123 194 217 198 79 190 97 149 216183 156 155 163 187 172 173 17098 214 212 171 169 191 84 186 160 158 161 174 185 162 213 159 193 177 167 168 151 179 178 189 175 184 176 182 181 208 89 152 153 165 166 2D map of RVLM (
2D map of RVLM (ppII33--10, 10, MMrr<94 <94 kDakDa))
IEF SDS 94 67 43 30 20.1 14.4 M r (kD a ) 3 4 5 6 7 8 9 10 pI
188 Heat shock protein 105 kDa 137 51 47 43 32 34 2 37118 119 120 121 140 62 76 110 111 56 109 7 139 138 137 50 20 80 8 124 45 44 108 105106 107 60 104 59 58 112 9 132 131 113 86 114 117 133 135 134 130 126 127 128 129 125 38 142 141 95 78 26 116 115 65 11 10 31 36 34 53 48 122 851 46 18 17 16 3029 33 55 54 14 13 12 52 49 35 42 21 28 24 22 23 27 39 25 19 40 41 6 145 144 143 206 69 100 204 205 157 101200 15 215 154 150 164 203 202 201 146 147195 199 148 196 197 123 194 217 198 79 190 97 149 216183 156 155 163 187 172 173 17098 214 212 171 169 191 84 186 160 158 161 174 185 162 213 159 193 177 167 168 151 179 178 189 175 184 176 182 181 208 89 152 153 165 166 137 51 47 43 32 34 2 37118 119 120 121 140 62 76 110 111 56 109 7 139 138 137 50 20 80 8 124 45 44 108 105106 107 60 104 59 58 112 9 132 131 113 86 114 117 133 135 134 130 126 127 128 129 125 38 142 141 95 78 26 116 115 65 11 10 31 36 34 53 48 122 851 46 18 17 16 3029 33 55 54 14 13 12 52 49 35 42 21 28 24 22 23 27 39 25 19 40 41 6 145 144 143 206 69 100 204 205 157 101200 15 215 154 150 164 203 202 201 146 147195 199 148 196 197 123 194 217 198 79 190 97 149 216183 156 155 163 187 172 173 17098 214 212 171 169 191 84 186 160 158 161 174 185 162 213 159 193 177 167 168 151 179 178 189 175 184 176 182 181 208 89 152 153 165 166 (3) Protein-profiling program
As a baseline for the protein-profiling program, Drs. Samuel Chan and Alice Chang have generated a proteomic map for the RVLM in normotensive
Sprague-Dawley rats (Fig. 1) and identified over 300 protein spots. This experience is currently extended to the hippocampus (Fig. 2). Unfortuanately, this program was substantially handicapped because of the serious delay in availability of funding.
Fig. 1. Proteomic map for RVLM of Sprague-Dawley rats generated by 2-D electrophoresis. More than 300 spots (yellow circle) have been identified.
3 4 5 6 7 8 9 10 94 67 43 30 20.1 14.4 M r (k Da) IEF SDS 2D map of Hippocampus (
2D map of Hippocampus (ppII3-3-10, 10, MMrr<94 <94 kDakDa))
3 4 5 6 7 8 9 10 94 67 43 30 20.1 14.4 M r (k Da) IEF SDS 2D map of Hippocampus (
2D map of Hippocampus (ppII3-3-10, 10, MMrr<94 <94 kDakDa))
Fig. 2. Proteomic map for hippocampus of Sprague-Dawley rats generated by 2-D electrophoresis.
(4) Gene delivery program
Dr. M.H. Tai has generated and provided various adenoviral vectors to the individual PIs. These included AdSOD1, AdSOD2 or Adcatalase to Dr. Julie Chan; AdPTEN to Dr. Alice Chang; and GFP vector to Dr. K.S. Hsu. He is planning to generate dominant negative form of Akt or PTEN for all the PIs. Again, progress in this program was also handicapped because of the serious delay in availability of funding.
(5) Governance
Two PI meetings have been held (see Appendix I for meeting minutes). Efforts were made to ensure timely completion of the annual report.
B. Sub-project 1: Cellular and molecular signals underlying brain stem apoptotic cell death during endotoxemia (PI: Samuel H.H. Chan; Co-PI: Alice Y.W. Chang)
Note: Per instruction by the NSC, the proposed sub-project on brain stem signlaing
pathways in organophosphate poisoning (PI: Alice Y.W. Chang) was merged into Sub-project 1.
(1) Chan, J.Y.H., Cheng, H.L., Chou, J.L.J., Li, F.C.H., Dai, K.Y., Chan, S.H.H. and Chang, A.Y.W. (2007) Heat shock protein 60 or 70 activates NOS I- and inhibits NOS II-associated signaling, and depresses mitochondrial apoptotic cascade during brain stem death. Journal of Biological Chemistry 282:4585-4600.
The cellular and molecular basis of brain stem death remains an enigma. As the origin of a “life-and-death” signal that reflects the progression towards brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this phenomenon. Here, we evaluated the hypothesis that heat shock proteins (HSPs) play a neuroprotective role in the RVLM during brain stem death and delineated the underlying mechanisms, using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, proteomic, Western blot and real-time PCR analyses demonstrated that Mev induced de novo synthesis of HSP60 or HSP70 in the RVLM without affecting HSP90 level. Loss-of-function manipulations of HSP60 or HSP70 in the RVLM using antiserum or antisense oligonucleotide potentiated Mev-elicited cardiovascular depression, alongside reduced NOS I/PKG signaling, enhanced NOS II/peroxynitrite cascade, intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or activated caspase-3, and augmented cytochrome c/caspase-3 cascade of apoptotic signaling in the RVLM. Co-immunoprecipitation experiments further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during Mev intoxication, alongside a dissociation of cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 and HSP70 confer neuroprotection against Mev intoxication by ameliorating cardiovascular depression via an
antiapoptotic action in the RVLM. The possible underlying intracellular processes include enhancing NOS I/PKG signaling and inhibiting NOS II/peroxynitrite cascade. In addition, HSP60 exerts its effects against apoptosis by blunting Mev-induced activation of the Bax/cytochrome c/caspase-3 cascade.
(2) Chan JYH, Chang AYW, Wang LL, Ou CC and Chan SHH. (2007) Protein kinase C-dependent mitochondrial translocation of pro-apoptotic protein Bax on activation of inducible nitric oxide synthase in rostral ventrolateral medulla mediates cardiovascular depression during experimental endotoxemia. Molecular Pharmacology, 71:1129-1139.
Joint publication with the Main Project and Sub-project 2, described under
“Breakthroughs and Major Achievements” in report for Sub-project 2.
(3) Tsai, C.Y., Wu, C.H.Y., Chan, S.H.H. and Chang, A.Y.W. (2007) Muscarinic receptor-independent activation of cAMP-dependent protein kinase in rostral
ventrolateral medulla underlies the sympathoexitatory phase of cardiovascular responses during mevinphos intoxication in the rat. Shock 27: 559-564.
As inhibitors of acetylcholinesterase, clinical presentations of poisoning from organophosphate compounds are generally believed to entail over-stimulation by the accumulated acetylcholine on muscarinic receptors at peripheral and central synapses. That some patients still succumbed to acute organophosphate poisoning despite repeated dosing of atropine suggest that cellular mechanisms that are independent of muscarinic receptor (MR) activation may also be engaged in organophosphate poisoning. The present study was undertaken to test the hypothesis that muscarinic receptor-independent activation of cAMP-dependent protein kinase (PKA) in rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts, is involved in the cardiovascular responses exhibited during organophosphate intoxication. In
Sprague-Dawley rats, microinjection bilaterally of Mev (10 nmol) into the RVLM significantly augmented PKA activity in ventrolateral medulla that was not
antagonized by co-administration of an equimolar concentration (1 nmol) of atropine or selective M1R (pirenzepine), M2R (methoctramine), M3R (4-diphenyl-acetoxy-N- dimethylpiperidinium) or M4R (tropicamide) inhibitor. Co-microinjection of two selective PKA antagonists (100 pmol), H89 or KT5720, significantly blunted the initial sympathoexcitatory cardiovascular response and the accompanying augmentation of nitric oxide synthase (NOS I) expression in ventrolateral medulla exhibited during Mev intoxication; the secondary sympathoinhibitory phase and associated elevation in NOS II expression were unaffected. We conclude that whereas a muscarinic
receptor-independent augmentation of PKA activity in ventrolateral medulla was manifested throughout acute Mev intoxication, this activation was preferentially involved in the sympathoexcitatory phase by an up-regulation of NOS I expression.
(4) Chan, J.Y.H., Wu, C.H.Y., Tsai, C.Y., Cheng, H.L., Dai, K.Y., Chan, S.H.H. and Chang, A.Y.W. (2007) Transcriptional upregulation of nitric oxide synthase II by nuclear factor-κB at rostral ventrolateral medulla in mevinphos intoxication model of brain stem death. Journal of Physiology 581:1293-1307
As the origin of a “life-and-death” signal that reflects central cardiovascular
regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this vital phenomenon. Using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, we evaluated the hypothesis that transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene
expression by nuclear factor-κB (NF-κB) on activation of muscarinic receptors in the RVLM underlies brain stem death. In Sprague-Dawley rats maintained under propofol anesthesia, co-microinjection of muscarinic M2R (methoctramine) or M4R
(tropicamide), but not M1R (pirenzepine) or M3R (4-diphenylacetoxy-N-
dimethylpiperidinium) antagonist significantly reduced the enhanced NOS I/protein kinase G signaling (“pro-life” phase) or augmented NOS II/peroxynitrite cascade (“pro-death” phase) in ventrolateral medulla, blunted the biphasic increase and decrease in sympathetic vasomotor tone that reflect the transition from life to death, and diminished the elevated DNA binding activity or nucleus-bound translocation of NF-κB in RVLM neurons induced by microinjection of Mev into the bilateral RVLM. However, NF-κB inhibitors (diethyldithiocarbamate or pyrrolidine dithiocarbamate) or double-stranded κB decoy DNA preferentially antagonized the augmented NOS II/peroxynitrite cascade and the associated cardiovascular depression exhibited during the “pro-death” phase. We conclude that transcriptional upregulation of NOS II gene expression by activation of NF-κB on selective stimulation of muscarinic M2 or M4 subtype receptors in the RVLM underlies the elicited cardiovascular depression during the “pro-death” phase in our Mev intoxication model of brain stem death.
(5) Sheh, Y.L., Hsu, C., Chan, S.H.H. and Chan, J.Y.H. (2007) NADPH oxidase-and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression. Free Radical Biology and Medicine, 42:1610-1623.
Joint publication with the Main Project and Sub-project 2, described under
“Breakthroughs and Major Achievements” in report for Sub-project 2.
(6) Phosphoinositide 3-kinase (PI3K)/Akt cascade in RVLM underlies the “pro-death” phase of brain stem death. Preliminary results indicated that PI3K/Akt
cascade activates NOS II/peroxynitrite at RVLM during mevinphos intoxication. In Sprague-Dawley rats anesthetized with propofol, microinjection bilaterally of Mev into the RVLM induced a progressive increase in PI3K phosphorylation at Tyr85, Akt phosphorylation at Thr308 and Ser473, nuclear translocation of phospho-Akt, and NOS II or nitrotyrosine (an experimental marker for peroxynitrite) level in the
ventrolateral medulla. Co-microinjection bilaterally of PI3K inhibitors (Wortmannin or LY294002) into the RVLM significantly potentiated and prolonged the increased vasomotor activities during Phase I (“pro-life”) Mev intoxication, and blunted the augmented expression of phospho-Akt, NOS II or nitrotyrosine in the ventrolateral medulla. We conclude that PI3K/Akt signaling is upstream to NOS II/peroxynitrite expression in the RVLM, which underlies the elicited cardiovascular depression during the “pro-death” phase in our Mev intoxication model of brain stem death.
(7) Hypoxia-inducible factor-1 (HIF-1)/heme oxygenase-1 (HO-1) cascade in RVLM underlies the “pro-life” phase of brain stem death. Preliminary results also revealed that HIF-1/HO-1 cascade activates NOS I/protein kinase G (PKG) signaling pathway in RVLM during Mev intoxication. In adult male Sprague-Dawley rats anesthetized with propofol, microinjection bilaterally of Mev (10 nmol) into the RVLM induced an initial enhancement of NOS I/PKG signaling, followed by an augmentation of NOS II/peroxynitrite cascade, alongside biphasic alterations in vasomotor tone. Immunofluorescence staining showed significant nucleus-bound translocation of HIF-1 in RVLM neurons during early Phase I and Phase I Mev intoxication, along with Western blot results showing augmented HO-1, heat shock protein 70 (HSP70), NOS I or PKG expression in ventrolateral medulla. Pretreatment by microinjection of anti-HO-1 antiserum or antisense ho-1 oligonucleotide bilaterally into the RVLM significantly blunted the increased vasomotor activities and the
augmented expression of HSP70, NOS I or PKG during Phase I Mev intoxication. However, pretreatment with normal mouse serum or sense ho-1 oligonucleotide, or anti-HO-2 antiserum or antisense ho-2 oligonucleotide, was ineffective. We conclude that HIF-1/HO-1 upregulates NOS I/PKG signaling pathway via HSP70 in the RVLM during the pro-life phase of Mev intoxication model of brain stem death.
C. Sub-project 2: Oxidative stress and neurogenic hypertension: brain stem cellular and molecular mechanisms (PI: Julie Y.H. Chan; Co-PI: M.H. Tai)
(1) Chan, S.H.H., Wang, L.L., Tseng, H.L. and Chan, J.Y.H. (2007) Upregulation of AT1 receptor gene on activation of PKCβ-NADPH Oxidase-ERK1/2-c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla. Journal of Hypertension 25:1845-1861.
Angiotensin II (Ang II) induces phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) via activation of NADPH oxidase on stimulation of angiotensin subtype 1 receptor (AT1R) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone and blood pressure are located. Ang II-activated p38 MAPK in RVLM promotes short-term pressor effect via augmented glutamatergic neurotransmission. Here, we tested the hypothesis that NADPH oxidase-dependent phosphorylation of ERK1/2 following activation of conventional protein kinase C (PKC) mediates AT1R-dependent long-term pressor effects of Ang II via
transcriptional induction of the proto-oncogene c-fos gene in RVLM. In Sprague-Dawley rats, microinjection of Ang II bilaterally into RVLM induced membrane-bound translocation of the conventional PKCα, PKCβ or PKCγ isoform, phosphorylation of the p47phox subunit of NADPH oxidase and ERK1/2, followed by phosphorylation of transcription factor cAMP response element binding protein (CREB), and c-fos induction. PKC inhibitor antagonized Ang II-induced p47phox phosphorylation; and an antisense oligonucleotide (ASON) complementary to PKCβ mRNA suppressed Ang II-induced ERK1/2 activation, phosphorylation or DNA binding activity of CREB, and upregulation of c-fos mRNA expression in ventrolateral medulla. Furthermore, microinjection of ERK1/2, CREB or c-fos ASON into RVLM significantly reduced the long-term pressor effect and augmented AT1R expression in ventrolateral medulla induced by intracerebroventricular infusion of Ang II. We conclude that the PKCβ-NADPH oxidase-ERK1/2-CREB-c-fos cascade represent a novel signaling cascade that mediates long-term pressor effect induced by Ang II in RVLM.
(2) Sheh, Y.L., Hsu, C., Chan, S.H.H. and Chan, J.Y.H. (2007) NADPH oxidase-and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression. Free Radical Biology and Medicine, 42:1610-1623.
We evaluated the contribution of superoxide anion (O2•−) generated by NADPH oxidase or mitochondrion in the rostral ventrolateral medulla (RVLM), where sympathetic promotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase (iNOS)-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2•− production (60-240 min) in the RVLM, alongside phosphorylation of p47phox or p67phox, upregulation of gp91phox or p47phox protein, and increase in Rac 1 or NADPH oxidase activity (60-120 min); and depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of gp91phox or p47phox gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial KATP channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2•− production in RVLM and cardiovascular depression. We conclude that whereas NADPH oxidase-derived O2•− in RVLM participates
predominantly in the early phase, O2•− generated by depression in mitochondrial respiratory enzyme activity or opening of mitoKATP channels mediates the delayed phase of LPS-induced cardiovascular depression.
(3) Chan, J.Y.H., Chang, A.Y.W., Wang, L.L., Ou, C.C. and Chan, S.H.H. (2007) Protein kinase C-dependent mitochondrial translocation of pro-apoptotic protein Bax on activation of inducible nitric oxide synthase in rostral ventrolateral medulla mediates cardiovascular depression during experimental endotoxemia. Molecular
Pharmacology 71:1129-1139.
Sympathetic premotor neurons for the maintenance of vasomotor tone are located in rostral ventrolateral medulla (RVLM). We demonstrated previously that
overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in RVLM, leading to caspase 3-dependent apoptotic cell death, plays a pivotal role in
cardiovascular depression during endotoxemia induced by intravenous administration of Escherichia coli lipopolysaccharide. The interposing intracellular events remain unknown. We evaluated the hypothesis that these events encompass protein kinase C
mitochondrial permeability transition pore by interacting with adenine nucleotide translocase (ANT) or voltage-dependent anion protein (VDAC), followed by cytosolic release of cytochrome c. In Sprague-Dawley rats, co-immunoprecipitation and Western blot analyses revealed sequential manifestations during endotoxemia of
membrane-bound translocation of PKC, dissociation of cytosolic PKC/Bax complex, mitochondrial translocation of activated Bax, augmented Bax/ANT or Bax/VDAC association, elevated cytosolic cytochrome c and caspase 3, and DNA fragmentation in ventrolateral medulla. Microinjection of iNOS inhibitor into bilateral RVLM
significantly retarded PKC and Bax activation. The induced association of translocated Bax with ANT or VDAC and the triggered mitochondrial apoptotic signaling cascade were blunted by blockade in RVLM of PKC, mitochondrial translocation of Bax, Bax channels, ANT or caspase 3, alongside significant amelioration of cardiovascular depression. We conclude that formation of mitochondrial Bax/ANT or Bax/VDAC complex that initiates caspase 3-dependent apoptosis in the RVLM as a result of PKC-dependent mitochondrial translocation of activated Bax activated by iNOS-derived NO play a pivotal role in the manifestation of endotoxin-induced cardiovascular depression.
(4) Tai, M.H., Weng, W.T., Lo, W.C., Chan, J.Y.H., Lin, C.J., Lam, H.C. and Tseng, C.J. (2007) Role of nitric oxide in α-melanocyte-stimulating hormone-
induced hypotension in the nucleus tractus solitarii of the spontaneously hypertensive rats. Journal of Pharmacology and Experimental Therapeutics 321:455-461.
Pro-opiomelanocortin (POMC) is expressed in the nucleus tractus solitarii (NTS) of the brainstem, where nitric oxide (NO) plays an important role in cardiovascular regulation. The POMC derived neuropeptides and their receptors are important regulators of energy homeostasis and cardiovascular functions in the central nervous system. In this study, we investigated the cardiovascular effect of α-melanocyte- stimulating hormone (α-MSH), a POMC-derived neuropeptide, and its relationship with NO pathway in the NTS of spontaneously hypertensive rats (SHR). Unilateral microinjection of α-MSH (0.3–300 pmol) into the NTS resulted in a dose-dependent hypotension and bradycardia in urethane-anesthetized SHR. The α-MSH-induced hypotension was abolished by pretreatment with the antagonist of melanocortin-3/4 receptor (MC-3/4R), Ac-Nle-c[Asp-His-DNal(2’)-Arg-Trp-Lys]-NH2 (SHU9119). Blockade of cAMP/protein kinase A (PKA), the downstream effector of melanocortin receptors, by previous injection of N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) also ablated the cardiovascular effect of α-MSH. To elucidate the role of NO pathway in α-MSH-evoked hypotension, pretreatment with Nω-nitro-L-arginine methyl ester, a universal inhibitor of nitric-oxide synthase (NOS), partially reversed the
depressor and bradycardiac effects of α-MSH. Furthermore, previous application of the inducible NOS (iNOS) inhibitor, aminoguanidine, but not the neuronal NOS inhibitor, 7-nitroindazole, attenuated the cardiovascular effect of α-MSH. Histological analysis revealed the colocalization of MC-4R, but not MC-3R, with iNOS in the NTS of SHR. In summary, intra-NTS injection of α-MSH induces hypotension and bradycardia of SHR via MC-4R signaling, which activates cAMP/PKA and iNOS.
(5) Kung, L.C., Chan, S.H.H., Ou, C.C., Tai, M.H. and Chan, J.Y.H. (2008) Mitochondrial respiratory complexes as cellular targets of NO and superoxide interaction in the rostral ventrolateral medulla of spontaneously hypertensive rats.
American Journal of Physiology Heart and Circulatory Physiology, in revision.
Overproduction of nitric oxide (NO) by gene transfer of endothelial NO synthase (eNOS) in rostral ventrolateral medulla (RVLM), which is responsible for maintenance of vasomotor tone, reduces arterial pressure in spontaneously hypertensive rats (SHR). This NO-induced vasodepression, however, is not sustained and is followed by
rebound hypertension. Since superoxide anion (O2¯) level is increased and synthesis or activity of mitochondrial manganese SOD (SOD2) is reduced in RVLM during
hypertension, we hypothesized that an interplay between NO and O2¯ in RVLM, using mitochondrial respiratory enzyme complexes (MRC) as the cellular target, contributes to those cardiovascular outcomes after eNOS gene transfer in SHR. The present study assessed this hypothesis using adenoviral vectors to overexpress eNOS (AdeNOS) and/or SOD2 (AdSOD2) in RVLM of SHR or normotensive Wistar-Kyoto (WKY) rats. Microinjection of AdeNOS bilaterally into RVLM depressed the enzyme activity of MRC-IV in ventrolateral medulla of SHR and WKY rats. The eNOS transgene also inhibited MRC-I and evoked a significant increase in O2¯ level in RVLM of SHR, but not WKY rats, that was reversed by co-transfected AdSOD2. Co-transfection of AdeNOS and AdSOD2 in RVLM of SHR elicited significantly greater decreases in arterial pressure and heart rate than those promoted by the individual transgene, and prevented the AdeNOS-induced rebound hypertension. We conclude that an interplay between NO and O2¯ on MRC-I in RVLM contributes to the unsustained hypotensive effects of NO after overexpression of eNOS in SHR. The mitochondria-derived O2¯ also mediates the rebound hypertension induced by eNOS transgene in RVLM of SHR.
D. Sub-project 3: Stress-related modification of amygdala synaptic plasticity and fear memory formation: cellular and molecular mechanisms (PI: Po-Wu Gean)
(1) Ou, L.C. and Gean, P.W. (2007) Transcriptional regulation of brain-derived neurotrophic factor in the amygdala during consolidation of fear memory. Molecular
Pharmacology 72:350-358.
We have previously demonstrated that brain-derived neurotrophic factor (BDNF) signaling in the amygdala is required for the consolidation of fear memory. This study is designed to characterize the signal cascades by which fear conditioning modulates transcriptional and translational expression of BDNF. Real-time RT-PCR showed a significant increase in BDNF exon I- and III-containing mRNA in the amygdala of fear-conditioned rats, indicating that fear conditioning was capable of up-regulating BDNF mRNA. Bilateral administration of actinomycin D or anisomycin to the amygdala attenuated conditioning-induced increase in BDNF protein. Inhibitors for NMDA receptor, L-type voltage-dependent calcium channel (L-VDCC), adenylyl cyclase, cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent kinase IV (CaMKIV) significantly reduced the increase. Moreover, DNA affinity precipitation and chromatin immunoprecipitation (ChIP) assays showed that
phosphorylated cAMP response element-binding protein (p-CREB) binding activity in the proximal region of BDNF promoter I and III was significantly increased after fear conditioning. Intra-amygdala administration of CRE decoy DNA prior to training impaired fear learning. Taken together, these results suggest that calcium influx through NMDA receptors and L-VDCCs during fear conditioning activates PKA and CaMKIV resulting in CREB phosphorylation. The phosphorylated CREB binds to
BDNF promoter and up-regulates the expression of BDNF in the amygdala, which
helps the consolidation of fear memory.
(2) Lin, H.C., Mao, S.C., Su, C.L. and Gean, P.W. (2008) A role for prefrontal cortex CB1 receptors in the modulation of fear memory. Cerebral Cortex, in revision.
Understanding the mechanism of how fear memory can be extinguished provides potential therapeutic strategy for the treatment of post-traumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of medial prefrontal cortex (mPFC) retarded extinction of fear-potentiated startle.
Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated extinction of fear memory. Unexpectedly, administration of WIN to the IL without cue alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by the endocannabinoid (eCB) uptake or fatty acid
amidehydrolase (FAAH) inhibitors. Rats were trained with 10 CS+ (yellow light)-shock pairings. CS+ (yellow light) alone but not CS- (blue light) alone trials decreased
fear-potentiated startle suggesting that the rats were able to discriminate between the CS+ and the CS- . Intra-IL infusion of WIN before CS- alone trials decreased startle potentiation. Cannabinoid agonist activated extracellular signal-regulated kinases (ERKs) in the mPFC slices. ERK inhibitor blocked the effect of cannabinoid agonist on fear-potentiated startle at the same dose that inhibited cannabinoid agonist-induced ERK phosphorylation. These results suggest that CB1 receptors in the IL are involved in the extinction of conditioned fear and the adaptation to aversive situations in general.
(3) Mao, S.C., Lin, H.C. and Gean, P.W. (2008) Augmentation of fear extinction by D-cycloserine is blocked by proteasome inhibitors. Neuropsychopharmacology, in revision.
D-cycloserine (DCS) has been shown to facilitate extinction of conditioned fear in rats and to improve fear reduction of social phobia and fear of heights in human studies. Here we investigate the mechanism of DCS effect by measuring internalized GluR1 and GluR2 using cell-surface biotinylation techniques. DCS selectively increased NMDA receptor-mediated synaptic response without affecting AMPA
receptor-mediated synaptic response. Low-frequency stimulation (LFS) when applied in the presence of DCS induced GluR1 and GluR2 internalization in the amygdala slices. Proteasome inhibitors block DCS facilitation of LFS-induced depotentiation and a reduction in surface levels of GluR1 and GluR2. Furthermore, DCS in combination with LFS reduced cellular levels of PSD-95 and synapse-associated protein 97 (SAP97) that were also blocked by proteasome inhibitors. In the in vivo experiments,
DCS-induced reduction of fear-potentiated startle and reversal of conditioning-induced increase in surface expression of GluR1 were blocked by proteasome inhibitors.
DCS-treated rats fail to exhibit reinstatement after US alone presentations. These results suggest that DCS facilitates receptor internalization in the presence of extinction training resulting in augmented reduction of startle potentiation.
(4) Hsiao, Y.H., Chen, P.S., Lin, C.H. and Gean, P.W. (2008) N-acetylcysteine prevents β-amyloid toxicity by retarding proteolytic degradation of a cyclin-dependent kinase 5 activator in cultured cortical neurons. Journal of Neuroscience Research, in revision.
Although previous studies have indicated that the neuroprotective effect of
ERK is not clear. Here we investigated the effect of NAC on Aβ25-35-induced neuronal death. Pretreatment of neurons with NAC 1 hr before application of Aβ prevented Aβ-mediated cell death. NAC increased cyclin-dependent kinase 5 (Cdk5)
phosphorylation and the effect was blocked by Cdk5 inhibitor. Neuroprotective effect of NAC was significantly attenuated by Cdk5 inhibitors or in neurons transfected with Cdk5 or p35 small interfering RNA (siRNA). Conversely, pretreatment of neurons with calpain inhibitors calpeptin or MDL28170 enhanced neuroprotective effect of NAC. Aβ25-35 caused a significant decrease in the level of p35 with a concomitant increase in p25 that was completely prevented by NAC. This effect of NAC was blocked by Cdk5 inhibitors roscovitine and butyrolactone. In addition, NAC increases Cdk5/p35 kinase activity but reduces Cdk5 kinase activity. Aβ25-35 treatment decreases the phosphorylated levels of ERK that could be reversed by NAC. The effect of NAC was completely blocked by Cdk5 inhibitors. NAC reversed Aβ25-35–induced decrease in the expression of Bcl-2 that could be blocked by MAPK kinase (MEK) inhibitor or Cdk5 inhibitors. These results suggest that NAC-mediated neuroprotection against Aβ toxicity is likely mediated by p35/Cdk5-ERKs-Bcl-2 signal pathway.
(5) Lin, C.H. and Gean, P.W. (2008) Glutamate preconditioning prevents neuronal death induced by combined oxygen glucose deprivation in cultured cortical neurons. Submitted to Journal of Neurochemistry.
Study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with NMDA receptor antagonists prevented OGD-induced cell death whereas AMPA receptor and voltage-dependent Ca++ channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca++ was omitted or removed with EGTA. Pre-treatment with staurosporin and KN93 but not LY294002 or U0126 significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of CREB and pretreatment with CRE-decoy
oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that
inducing p-CREB-mediated Bcl-2 expression.
(6) Lin, H.C., Mao, S.C., Chen, P.S. and Gean, P.W. (2008) Resistant to the extinction of fear memory after chronic administration of CB1 receptor agonist to rats. Submitted to Biological Psychiatry.
Endocannabinoid is critically involved in the extinction of fear memory. Tolerance represents an excellent opportunity to explore the neural connection and mechanism involved in the physiological functions of endocannabinoid system. Here we examined the effects of repeated cannabinoid administration on the extinction of fear memory in animal and on the inhibitory synaptic transmission in medial prefrontal (mPFC) slices. Rats were treated with CB1 receptor agonist WIN55212-2 (WIN 10 mg/kg, i.p.) or vehicle for 7 days. Fear conditioning was measured using the potentiated startle paradigm. Intracellular recording technique was applied to record inhibitory synaptic responses in the mPFC slices. Chronically WIN treated rats exhibited much less extinction to cue alone presentations. In addition, reduction of fear-potentiated startle normally seen when CB1 receptor agonists were infused into the mPFC was absent in the chronically WIN treated rats. Afferent stimulation applied to the layer II of rat mPFC slices in the presence of CNQX plus D-APV elicited an IPSP. Application of WIN for 15 min significantly reduced IPSP. In slices from the chronic WIN-treated rats, WIN-induced inhibition of IPSP was significantly less than those of
vehicle-treated control rats. Similarly, WIN failed to induce ERKs phosphorylation after development of cannabinoid tolerance. These results suggest that inhibition of IPSP and activation of ERKs by endocannabinoids within the mPFC plays an important role in the extinction of conditioned fear.
E. Sub-project 4: Cellular and molecular signals mediating normal and stressed hippocampal synaptic function and synaptic plasticity (PI: Kuei-Sen Hsu)
(1) Huang, C.C., Lin, H.J. and Hsu, K.S. (2007) Repeated cocaine administration facilitates long-term potentiation induction in medial prefrontal cortex pyramidal neurons. Cerebral Cortex 17:1877-1888.
Although drug-induced adaptations in the prefrontal cortex (PFC) may contribute to several core aspects of addictive behaviors, it is not clear yet whether drugs of abuse elicit changes in synaptic plasticity at the PFC excitatory synapses. Here we report that, following repeated cocaine administration (15 mg/kg/day intraperitoneal injection for 5
consecutive days) with a 3-day withdrawal, excitatory synapses to layer V pyramidal neurons in rat medial prefrontal cortex (mPFC) become highly sensitive to the induction of long-term potentiation (LTP) by repeated correlated presynaptic and postsynaptic activity. This promoted LTP induction is caused by cocaine-induced reduction of gamma-aminobutyric acid (GABA)A receptor-mediated inhibition of mPFC pyramidal neurons. In contrast, in slices from rats treated with saline or a single dose of cocaine, the same LTP induction protocol did not induce significant LTP unless the blockade of GABAA receptors. Blockade of the D1-like receptors
specifically prevented the cocaine-induced enhancement of LTP. Repeated cocaine exposure reduced the GABAA receptor-mediated synaptic currents in mPFC pyramidal neurons. Biotinylation experiments revealed a significant reduction of surface GABAA receptor alpha1 subunit expression in mPFC slices from repeated cocaine-treated rats. These findings support an important role for cocaine-induced enhancement of synaptic plasticity in the PFC in the development of drug-associated behavioral plasticity.
(2) Huang, C.C., Yang, B.J., Lin, H.J. and Hsu, K.S. (2007) Repeated cocaine administration blocks group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex. Journal of Neuroscience 27:2958-2968.
Drug-induced neuroadaptations within the medial prefrontal cortex (mPFC) are thought to underlie the development of cocaine sensitization. Here, we report that repeated cocaine administration in vivo impaired the long-term depression (LTD) induced by bath application of group II metabotropic glutamate receptor (mGluR) agonists DCG-IV [2S, 2'R, 3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine] or LY379268 [(1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid] at
excitatory synapses onto layer V pyramidal neurons of rat mPFC. In contrast, this impairment was not found in slices from rats treated with saline or a single dose of cocaine. Such effect of cocaine was selectively prevented when cocaine was coadministered with the selective D1-like receptor antagonist SCH23390
[(R)-(+)-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine]. In slices from control rats, a brief application of either protein kinase C (PKC)
activator phorbol-12,13-dibutyrate or adenosine A3 receptor agonist
2-chloro-N6-(3-iodobenzyl)-adenosine-5-N-methyluronamide mimicked the effect of repeated cocaine treatment to impair the induction of LTD. Bilateral intra-mPFC infusion of PKC inhibitor bisindolylmaleimide I or adenosine A3 receptor antagonist MRS1220 (N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-
benzeneacetamide) before cocaine injection prevented cocaine-induced impairment of LTD induction. Furthermore, endogenous adenosine tone is greater in slices from cocaine-treated rats than from the saline-treated controls. When the metabolism of
cAMP to adenosine was blocked, the extent of LTD in slices from saline and cocaine-treated rats was similar. These results suggest that cocaine-induced
impairment of group II mGluR-mediated LTD is caused, at least in part, by an increase in adenosine subsequent to the rise in cAMP after D1-like receptor activation, which leads to an adenosine A3 receptor-mediated upregulation of PKC activity and thereby triggers an inhibition of group II metabotropic glutamate receptor function.
(3) Huang, C.C., Lin, H.J., Liang, Y.C. and Hsu, K.S. (2007) Effects of neonatal corticosteroid treatment on hippocampal synaptic function. Pediatric Research 62:267-270.
There is growing concern about long-term neurodevelopmental outcomes after neonatal corticosteroid treatment for chronic lung disease (CLD). Here, we use a protocol with tapering doses of dexamethasone (DEX) or hydrocortisone (HC)
proportional to those used in preterm infants to examine the long-term consequences of these treatments on hippocampal synaptic plasticity and associative memory in later life. We found that neonatal DEX, but not HC, treatment impairs long-term
potentiation (LTP) but enhances long-term depression (LTD) induction in adolescent rats. The effects of neonatal DEX treatment on LTP and LTD were prevented when the animals were given glucocorticoid receptor antagonist, RU38486, before DEX
administration. We also found that neonatal DEX, but not HC, treatment induces a profound increase in the autophosphorylation of a isoform of
Ca2+/calmodulin-dependent protein kinase II at threonine-286 and a decrease in the protein phosphatase 1 expression. In addition, only neonatal DEX treatment disrupts memory retention in rats subjected to passive avoidance learning tasks. These results demonstrate that only neonatal DEX treatment alters the hippocampal synaptic
plasticity and associative memory formation in later life and thus suggest that HC may be a safer alternative to DEX for the treatment of CLD in the neonatal period.
(4) Lee, C.C., Kuo, Y.M., Huang, C.C. and Hsu, K.S. (2008) Insulin rescues amyloid β-protein-induced impairment of hippocampal long-term potentiation by influencing oligomer formation. Neurobiology of Aging, in press.
Cerebral accumulation of amyloid β-protein (Aβ) is generally believed to play a critical role in the pathogenesis of Alzheimer's disease (AD). Recent evidence suggests that Aβ-induced synaptic dysfunction is one of earliest pathogenic events observed in AD. Here we report that synthetic Aβ 1-42 strongly inhibited the induction of
which Aβ 1-42 sequences contribute to the impairment of LTP, we compared actions of several Aβ fragments and found that the sequence within 25-35 region of Aβ mainly contributes to the expression of LTP impairment. Importantly, we show that insulin and insulin-like growth factor-1 significantly inhibit Aβ oligomer formation,
particularly dimers and trimers, and ameliorate the synthetic Aβ-induced suppression of LTP. Furthermore, dithiothreitol was found to be capable of significantly preventing the inhibitory effect of insulin on Aβ oligomer formation. In contrast, hemoglobin promotes Aβ oligomer formation and enhances Aβ-mediated inhibition of LTP induction. These results suggest that insulin may have utility in treating the earliest stages of Aβ-induced synaptic dysfunction in AD patients.
(5) Yang P.C., Yang, C.H., Huang, C.C. and Hsu, K.S. (2008) Phosphatidylinositol 3 kinase is required for stress protocol-induced modification of hippocampal synaptic plasticity. Journal of Biological Chemistry, in press.
Stress dramatically affects the induction of hippocampal synaptic plasticity; however, the molecular details of how it dose so remain unclear. Phosphatidylinositol 3 kinase (PI3K) signaling plays a crucial role in promoting neuronal survival and neuroplasticity, but its role, if any, in stress-induced alterations of long-term
potentiation (LTP) and long-term depression (LTD) is unknown. We found here that inhibitors of PI3K signaling blocked the effects of acute restraint-tailshock stress protocol on LTP and LTD. Therefore the purpose of the present study is to explore the signaling events involving PI3K in terms of its role in mediating stress
protocol-induced alterations of LTP and LTD. We found that stress protocol-induced PI3K activation can be blocked by various inhibitors including RU38486 for
glucocorticoid receptors, LY294002 for PI3K, DL-2-amino-5- phosphonopentanoic acid for NMDA receptors or BDNF antisense oligonucleotides. Also, immunoblotting analyses revealed that stress protocol induced a profound and prolonged
phosphorylation of numbers of PI3K downstream effectors, including
3-phosphoinositide-dependent protein kinase-1, protein kinase B, mammalian target of rapamycin (mTOR), p70S6 kinase and eukaryotic initiation factor 4B in hippocampal CA1 homogenate, which were prevented by the PI3K inhibitor pretreatment. More importantly, we found that stress protocol significantly increased the protein
expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95), which is known to involve in LTP and LTD, in an mTOR-dependent manner. These results identify a key role of PI3K signaling in mediating the stress protocol-induced
modification of hippocampal synaptic plasticity and further suggest that PI3K may do so by invoking the protein expression of PSD-95.
3. Categorized Summary of Research Outcomes
As stipulated in our proposal, the research accomplishment of this program will be epitomized in publications in top journals, and invitation to present keynote or invited lectures at key international or national meetings.
A. Publications in Top Journals
We define top journals as those with impact factors in the upper 20% of the
research area, or with impact factors ≥ 5.0. All 13 journal articles published or in press during Project Year 3 (listed in Appendix II and III) belong to this category.
(1) Based on percentile in the research area
Number of articles with IF in the upper 10% 3 Number of articles with IF in the upper 15% 7 Number of articles with IF in the upper 20% 3
(2) Based on impact factor
Number of articles with IF ≥ 5.0 6 Number of articles with IF ≥ 4.0 4 Number of articles with IF ≥ 3.0 2 Number of articles with IF ≥ 2.5 1
B. Publications in Top Conferences
We define publications at key conferences as those that are associated with keynote or invited lectures at international or national conferences. (See list in Appendix II and III)
(1) Invited lectures at international conferences
Number of keynote lectures 2 Number of invited lectures 18
(2) Invited lectures at national conferences
C. Publications in Other Conferences
Only the 28 invited lectures at international or national meetings are listed.
D. Citations
Other laboratories have cited publications of the 6 PIs 3,197 times during
1997-2007. Thus, the average annual citation of each PI over the past 11 years is 48.4. In addition, other laboratories have cited publications of the 6 PIs 1,010 times in 2006-2007. Thus, the average citation of each PI during 2006-2007 is 84.2.
4. Program Management
On top of the conventional practice for individual PIs to collaborate with each other by providing technical support or incorporating a mutual scientific question into the sub-projects, three additional levels of scientific interaction are incorporated into this program to foster collaborations and integrations among the institutes involved.
A. Protein-Profiling Program (First Additional Scientific Interaction)
To search for new protein entities other than those selected in our initial studies that may subserve our neurobiological phenotypes, Drs. Samuel Chan and Alice Chang are responsible for proteomic analysis on relevant brain samples submitted by the PIs to identify common proteins that may be involved in brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity, using state-of-the-art analytical chemical techniques. Individual PIs can then confirm the functional engagements of these potential signaling proteins using their respective test paradigms.
B. Gene Delivery Program (Second Additional Scientific Interaction)
As a complement to pharmacological interventions, a powerful tool in
contemporary biomedical research to delineate the distinct functions of specified genes entails gene delivery. Dr. M.H. Tai has initiated and overseen this program in which gene delivery vectors aiming for overexpression or downregulation of selected signaling molecules in the PI3K/Akt or MAPK cascades are generated, validated and characterized. These vectors are shared by the PIs in their sub-projects to ascertain the presence of a causative relationship between these signaling molecules and brain death,
neurogenic hypertension and memory formation or eradication.
C. Governance (Third Additional Scientific Interaction)
In addition to frequent e-mail or telephone communications, PI meetings are organized for self-evaluation of progress. Whether our two target signaling cascades under study are associated with brain death, neurogenic hypertension and memory formation or eradication, a crucial integrative link between the sub-projects, are
discussed. In addition, new signaling protein entities identified by the protein-profiling program are reviewed, and strategies designed to confirm their common cellular and molecular roles. This exercise forms another integrative link between the individual sub-projects. The diversified background in neuroscience of the 6 PIs contributes a wide spectrum of expertise to allow for multi-faceted dialogue during the PI meetings. In addition to methodological support, interactions of this nature bring out potential synergism and new perspectives that can be transformed into further scientific advancement and multidisciplinary collaboration.
Because of the drastic cut in funding by the NSC, we have to regrettably eliminate the planned annual retreat when the PIs present their progress to the
Advisory Committee. The annual scientific conference aiming at providing interactions between the PIs and the biomedical community has been changed to a bi-annual
Progress Report, using funds from other sources.
D. Administrative Support
Full-time administrative personnel at the Center for Neuroscience in National Sun Yat-sen University provided the administrative support and coordination for the scientific and auxiliary activities of the participating PIs, their institutions and the funding agency. They are also responsible for setting up the governance program.
5. Summary of Post-Program Plan
With encouraging results from Project Year 3, the individual sub-projects will continue their current efforts towards delineating whether the identified cellular and molecular signals may subserve brain stem death, neurogenic hypertension, formation and eradication of fear memory or synaptic plasticity, with the aid of the gene delivery program. Detailed analysis of regulators or modulators up- or downstream to these
the baseline proteomic profile of the hippocampus, amygdala of normotensive rats, and RVLM of spontaneously hypertensive rats. This will be prelude to identifying
additional cellular and molecular signals for confirmation by the PIs to be common to our neurobiological phenotypes.
6. International Activities
A. The Inaugural Cross-Strait Conference on Neuroscience
The Southern Taiwan Center of Excellence in Neuroscience Research, of which all 6 PIs are members, the Brain Science Institute of Fadan University, and the State Key Laboratory of Neurobiology, Shanghai Medical School, Fadan University, jointly organized the Inaugrual Cross-Strait Conference on Neuroscience. Co-organized by Dr. Samuel Chan and Dr. Xiong-Li Yang, Director of the Brain Science Institute at Fadan University and member of the Chinese Academy of Science, this Conference was held on September 3-4, 2007 at Fadan University in Shanghai, China. All 6 PIs presented an invited lecture, along with 18 lectures from members of our host institutes.
Whereas informal interchanges between neuroscientists in Taiwan and China have been carried out for more than 2 decades, a formal cross-strait conference on neuroscience has not been forthcoming. Thus, this Conference represents the begining of a new stage of development in neuroscience. It is anticipated that through this process, a new wave of communications between neuroscientists from Taiwan and China will be initiated, leading eventually to collaborative research.
The titles of the lectures by the 6 PIs at this Conference are:
(1) Julie Y.H. Chan: Mitogen-activated protein kinase as novel sighals in brain stem that determine short-term and long-term pressor response to antiogensin II.
(2) Samuel H.H. Chan: Multiple roles of the mitochondrion in an experimental endotoxemia model of brain stem death.
(3) Alice Y.W. Chang: Brain stem neuomodulatory signaling mechanisms in an organophosphate poisoning model of brain stem death.
(4) Po-Wu Gean: Neural activity in the prefrontal cortex is essential for the extinction of fear memory.
(5) Kuei-Sen Hsu: Phosphatidylinoditol 3 kinase activation is required for stress-induced modification of hippocampal synaptic plasticity.
(6) Ming-Hong Tai: Enhanced expression of and cardiovascular response to melanocortin system in spontaneously hypertensive rats.
B. Lectures at International Conferences
As indicated above, all 6 PIs were regularly invited to lecture at international conferences.
