Research

Neurobiology and Behavior

Outline

Brain functions are achieved by neurons working together in complex nerve circuits. However, unlike the electronic circuits of computers, the bonds between the neurons, or synapses, are not fixed. Rather, they display structural and functional ‘plasticity’. Synaptic plasticity forms the basis of the high order functions of the brain including its learning function, and it is important for understanding the pathology of mental disorders involving retardation of mental development and cognitive impairment. This laboratory focuses on the role of actin cytoskeletal protein in the dendritic spine for the development and plasticity of synapses, in order to elucidate synapse function.

Leading graduate school project assigned to this leading graduate school.

Radiation therapy is widely used as a cancer treatment for malignant brain tumors and so on, but it sometimes causes developmental disorder such as mental retardation. We discovered through animal experiments that X-ray radiation causes acute learning disability and chronic neuron cell death. In addition, through in vitro experiments, we learned that irradiated synapses experience acute structural abnormality. Therefore we believe that abnormal synapses caused by irradiation are one of the reasons for post-radiation learning disabilities, and we are pursuing research to clarify the mechanism. In our research to date, we used primary neuron culture and clarified the fact that (1) the resistance to apoptosis due to radiation of neurons in the development process is significantly strengthened before and after synapse formation, but that in both cases, (2) there are abnormalities in the surviving neurons. Furthermore, in an in vivo experiment using mature mice, we discovered that (3) learning disability occurs immediately after X-ray irradiation.

This laboratory focuses on the role of actin cytoskeletal protein in the dendritic spine for the development and plasticity of synapses, undertaking multifaceted research using approaches from molecular biology, biochemistry, cell biology, morphology, electrophysiology and behavioral pharmacology. In this leading graduate school project, we aim to develop a new and hitherto unknown academic domain of radiation neurobiology, by combining irradiation experiments and researches which are the strengths of this laboratory.

Members

Title Name Research
Professor Tomoaki Shirao, M.D., Ph.D. Synapses and higher brain functions
Assistant Professor Hiroyuki Yamazaki, Ph.D. The molecular mechanisms of synapse formation
Yuta Ishizuka, Ph.D. Postsynaptic signal cascade
Noriko Koganezawa, Ph.D. Neurobiological study of Radiation on the brain
Reiko T Roppongi, Ph.D. The molecular mechanisms of synaptic plasticity

Research

1. Changes in the actin cytoskeleton which occurs due to synaptic plasticity
The primary hippocampal neuron culture is an excellent experimental system in which the dendritic spine, a minute structure of the neuron, can be observed directly. Using this line, we analyzed structural plasticity of the spine caused by synaptic plasticity, focusing on changes in the actin cytoskeleton. We discovered the importance of the regulatory mechanism for the balance of the stable actin (F-actin and drebrin) including drebrin in its structure, and dynamic actin (F-actin and cofilin) with no drebrin as the basis for synaptic plasticity. We are working to clarify the molecular mechanism involved using time lapse imaging and FRAP (fluorescence recovery after photobleaching). Furthermore, we are trying to clarify the molecular dynamics of postsynaptic proteins, such as drebrin in dendritic spine by using super-resolution fluorescence microscope (resolution: 20 nm). 

 

2. The role of drebrin in synaptic plasticity
We prepared a mouse with drebrin knocked out completely (DXKO) and a mouse A with a defective isoform conversion mechanism (DAKO), and elucidated the role of drebrin in synaptic plasticity. The research considered the effects on learning behavior and synaptic plasticity. The DAKO mouse had no obvious morphological abnormality of the brain, but it showed malfunction of fear conditioning memory and synaptic plasticity of the hippocampus. We are currently researching the molecular mechanism involved in these abnormalities. Furthermore, there are suggestions of abnormal neuronal migration in the DXKO mouse.

3. The effects of irradiation on synapses, neurons, and higher brain function
It has been shown using behavioral study and immunohistochemistry that irradiation causes damages on higher brain function because of synaptic dysfunction. In order to reveal the molecular mechanism of this irradiation effects, we have started X-irradiation experiments using the primary hippocampal neuron culture (in vitro) and also animals (in vivo). Furthermore, to compare the effects of X-irradiation and heavy ion-irradiation, we are conducting experiments using heavy ion (carbon ion) beam.

4. Development of a synaptic function imaging assay method with drebrin as an index and its application to iPS derived neurons
We are developing a quantitative, high throughput assay method that effectively reproduces the degree of accumulation of drebrin in the spine. In addition, we aim to apply this technology to iPS derived neurons, and prepare guidelines for safety pharmacological testing using human neurons.

5. The role of new drebrin binding protein (spine and karyoplasm protein, spikar) in synapse formation

Many of the molecules involved in synapse and spine formation have been identified and are being researched. However, many of these are cell adhesion molecules present in the cell membrane of the synapse, or low molecular weight G protein regulatory molecules involved in regulating actin polymerization, and little is known about how molecules that bind directly and work with F-actin function or receive signals. Therefore, we are focusing on drebrin which is a postsynaptic region F-actin structural protein, and by analyzing the molecules around it, we are attempting to identify new synapse and spine formation mechanisms. Using the yeast two-hybrid system, we sought a new drebrin binding protein, and focused on a previously unidentified synapse protein for analysis. We discovered that this new drebrin binding protein, spikar participates in spine formation in dendrites, and functions as a transcription regulation factor in the nucleus. Currently, we have prepared a mouse with this protein knocked out, and are undertaking analysis at the individual level.

6. Research into the mechanism of spine vulnerability using hippocampal neurons
We are conducting research into the effects of various stress such as amyloid and oxidation stress at the cellular level on the dendritic spine in particular with a focus on localized changes in drebrin.The figure at bottom (left) shows a model of decrease in the spine concentration of drebrin in Alzheimer’s disease.The figure at bottom (right) shows primary cultured neurons exposed to amyloid beta oligomers.

Publications

Articles

Ishizuka Y, Shimizu H, Takagi E, Kato M, Yamagata H, Mikuni M, Shirao T. “Histone deacetylase
mediates the decrease in drebrin cluster density induced by amyloid beta
oligomers.” Neurochem Int. in press

Tanabe K, Yamazaki H, Inaguma Y, Asada A, Kimura T, Takahashi J, Taoka M, Ohshima T, Furuichi T, Isobe T, Nagata K, Shirao T, Hisanaga S. “Phosphorylation of drebrin by cyclin-dependent kinase 5 and its role in neuronal migration.” PLoS One. 9:e92291 (2014)

Ishikawa M, Shiota J, Ishibashi1 Y, Hakamata T, Shoji S, Fukuchi M, Masaaki Tsuda M, Shirao T, Sekino Y, Baraban JM and Tabuchi A. “Cellular localization and dendritic function of rat isoforms of the SRF coactivator MKL1 in cortical neurons.” NeuroReport. 25:585-592 (2014)

Mizui T, Sekino Y, Yamazaki H, Ishizuka Y, Takahashi H, Kojima N, Kojima M and ShiraoT. “Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines” PLoS ONE 9(1):e85367 (2014)

Yamazaki H, Kojima N, Kato K, Hirose H, Iwasaki T, Mizui T, Takahashi H, Hanamura K, Roppongi RT, Koibuchi N, Sekino Y, Mori N, Shirao T. “Spikar, a novel drebrin-binding protein, regulates the formation and stabilization of dendritic spines.” J Neurochem 128:507-522 (2014)

白尾智明、山崎博幸「樹状突起棘(スパイン)のアクチンフィラメントと微小管」Clinical Neuroscience 31: 1302-1395 (2013)

Mitsuru Ishikawa, Jun Shiota, Yuta Ishibashi, Tomoyuki Hakamata, Shizuku Shoji, Mamoru Fukuchi, Masaaki Tsuda, Tomoaki Shirao, Yuko Sekino, Toshihisa Ohtsuka, Jay M. Baraban and Akiko Tabuchi.
“Identification, expression and characterization of rat isoforms of the SRF coactivator MKL1.” FEBS Open Bio. 3:387-93 (2013)

Ishizuka Y, Kakiya N, Witters LA, Oshiro N, Shirao T, Nawa H and Takei N. “AMP-activated protein kinase (AMPK) counteracts brain-derived neurotrophic factor (BDNF)-induced mammalian target of rapamycin complex 1 (mTORC1) signaling in neurons.” J Neurochem. 127:66-77 (2013)

Yuta Ishizuka, Naomasa Kakiya, Lee A. Witters, Noriko Oshiro, Tomoaki Shirao, Hiroyuki Nawa and Nobuyuki Takei “AMP-activated protein kinase (AMPK) counteracts brain-derived neurotrophic factor (BDNF)-induced mammalian target of rapamycin complex 1 (mTORC1) signaling in neurons” J Neurochem.in press

Roppongi RT, Kojima N, Hanamura K, Yamazaki H, Shirao T. “Selective reduction of drebrin and actin in dendritic spines of hippocampal neurons by activation of 5-HT2A receptors.” Neurosci. Lett. 547:76-81 (2013)

Shirao T, González-Billault “Actin filaments and microtubules in dendritic spines.” J Neurochem 126: 155-164 (2013)

K Shirai, T Mizui, Y Suzuki, M Okamoto, K Hanamura, Y Yoshida, M Hino, S Noda, W S. AL-Jahdari, A Chakravarti, T Shirao, T Nakano, “X-Irradiation Changes Dendritic Spine Morphology and Density through Reduction of Cytoskeletal Proteins in Mature Neurons” Radiation Research in press (2012)

C.B. Canto, N. Koganezawa, P. Beed, E.I. Moser, M.P. Witter “All layers of medial entorhinal cortex receive presubicular and parasubicular inputs” J.Neuroscience, 32(49): 17620-17631 (2012)

Kenichi Kato, Tomoaki Shiraob, Hiroyuki Yamazaki, Kazuyuki Imamura, and Yuko Sekino. “Regulation of AMPA receptor recruitment by the actin binding protein drebrin in cultured hippocampal neurons.” J. Neurosci. Neuroeng. 1: 153-160(2012)

Okamotoa,T, Shirao, T, Shutoha, F, Suzukia, T, Nagaoa, S“Post-training cerebellar cortical activity plays an important role for consolidation of memory of cerebellum-dependent motor learning.” Neurosci Lett. 504: 53-56. (2011)

Annalisa Mancini, Dario Sirabella, Weijia Zhang, Hiroyuki Yamazaki, Tomoaki Shirao and Robert S Krauss “Regulation of myoblast fusion by the actin-binding factor drebrin” Skeletal Muscle 1-36 (2011)

Kobayashi-Yamazaki C, Shirao T, Sasagawa Y, Maruyama Y, Akita H, Saji M, and Sekino Y. “Lesions of the supramammillary nucleus decrease self-grooming behavior of rats placed in an open field” The Kitakanto Medical Jorunal , 61:287-292 (2011)

Tanaka K, Sato K, Yoshida T, Fukuda T, Hanamura K, Kojima N, Shirao T, Yanagawa T, and Watanabe H. “Evidence for cell density affecting C2C12 myogenesis: possible regulation of myogenesis by cell-cell communication.” Muscle and Nerve, 44:968-977 (2011)

Han W, Takamatsu Y, Yamamoto H, Kasai S, Endo S, Shirao T, Kojima N, Ikeda K. “Inhibitory role of inducible cAMP early repressor (ICER) in methamphetamine-induced locomotor sensitization” PLoS One 6(6):e21637 (2011)

Okamoto T, Endo S, Shirao T, and Nagao S. “Role of Cerebellar Cortical Protein Synthesis in Transfer of Memory Trace of Cerebellum-Dependent Motor Learning” J. Neurosci. 31: 8958-8966 (2011)

Kaminuma T, Suzuki Y, Shirai K, Mizui T, Noda S, Yosida Y, Funayama T, Takahasi T, Kobayashi Y, Shirao T, Nakano T. “Effectiveness of carbon-ion beams for apoptosis induction in rat primary immature hippocampal neurons” J Radiat Res (Tokyo) 51:627-631 (2010)

Hanamura K, Mizui T, Kakizaki T, Roppongi TR, Yamazaki H, Yanagawa Y, Shirao T. “Low accumulation of drebrin at glutamatergic postsynaptic sites on GABAergic neurons” Neuroscience 169: 1489-1500 (2010)

Pérez-Martínez M, Gordón-Alonso M, Cabrero JR, Barrero-Villar M, Rey M, Mittelbrunn M, Lamana A, Morlino G, Calabia C, Yamazaki H, Shirao T, Vázquez J, González-Amaro R, Veiga E, Sánchez-Madrid F. “F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse” J. Cell Sci. 123:1160-1170 (2010)

Mercer JC, Mottram LF Qi Q, Lee YC, Bruce D, Iyer A, Yamazaki H, Shirao T, Choe MH, Peterson BR, August A “Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses.” Int. J. Biochem. Cell Biol. 42:337-345 (2010)

Kojima N, Hanamura K, Yamazaki H, Ikeda T, Itohara S, Shirao T. “Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood” Neuroscience 165: 138-150 (2010)

Okamoto M, Suzuki Y, Shirai K, Mizui T, Yoshida Y, Noda S, Al-Jahdari WS, Shirao T, Nakano T. “Effect of Irradiation on the Development of Immature Hippocampal Neurons In Vitro” Radiat Res 172:718-724 (2009)

Aoki C, Kojima N, Sabaliauskas N, Shah L, Oakford J, Ahmed T, Yamazaki H, Hanamura K, Shirao T “Drebrin A Knock-Out Eliminates the Rapid Form of Homeostatic Synaptic Plasticity at Excitatory Synapses of Intact Adult Cerebral Cortex” J Comp Neurol 517:105-121 (2009)

Ito M, Shirao T, Doya K, Sekino Y. “Three-dimensional distribution of Fos-positive neurons in the supramammillary nucleus of the rat exposed to novel environment.” Neurosci. Res. 64: 379-402 (2009)

Mizui T., Kojima N, Yamazaki H, Katayama M, Hanamura K, Shirao T. “Drebrin E is involved in the mechanism regulating axonal growth through actin-myosin interactions.” J Neurochem 109:611-622 (2009).

Takahashi, T, Yamazaki, H, Hanamura K, Sekino Y and Shirao T “AMPA receptor inhibition causes abnormal dendritic spines by destabilizing drebrin” J Cell Sci. 122:1211-1229 (2009)

Ivanov A, Esclapez M, Pellegrino1 C, Shirao T, and Ferhat L. “Drebrin A regulates the dendritic spine plasticity and synaptic function in cultured hippocampal neurons” J. Cell Sci. 122: 524-534 (2009)

Song M, Kojima N, Hanamura K, Sekino Y, Inoue KH, Mikuni M, Shirao T, “Expression of drebrin E in migrating neuroblasts in adult rat brain: coincidence between drebrin E disappearance from cell body and cessation of migration” Neuroscience. 152:670-682 (2008)

Sekino Y, Kojima N, Shirao T. “Role of actin cytoskeleton in dendritic spine morphogenesis” Neurochem. Int. 51: 92-104 (2007)

Presentations

Brain and Health Informatics 2013, 2013, Hideo Shimizu, Yuta Ishizuka, Eiko Takagi, Reiko T Roppongi and Tomoaki Shirao. “Effects of histone Deacetyltransferase inhibitor and allopregnanolone on neuron and synapses. -New analyzing system of neuronal and synaptic function using drebrin as a functional marker-” Maebashi, Japan

Brain and Health Informatics 2013, 2013, Tomoaki Shirao, Noriko Koganezawa, Anggraeini Puspitasari, Katsuyuki Shirai, Yoshiyuki Suzuki, Natsume Tanaka, Sachiko Tanaami, Takashi Nakano, Nobuhiko Kojima “Radiation induced change in the neuronal cytoskeleton and its possible adverse effect on brain function.” Maebashi, Japan Oct. 29-31.

Society for Neuroscience 40th Annual Meeting, 2013, Ohara Y, Yamazaki H, Sato K, Shirao T, Sekino Y. “Morphological development and expression of synaptic proteins of human iPSC-derived neurons”. San Diego, California, November 9-13.

Society for Neuroscience 40th Annual Meeting, 2013, Puspitasari A, Koganezawa N, Tanaka N, Kojima N, Shirao T. “Acute effects of X-irradiation on learning, adult neurogenesis and the accumulation of drebrin.” San Diego, California, November 9-13.

Society for Neuroscience 40th Annual Meeting, 2013, M.-J. Xie, H. Yagi, T. Iguchi, Y. Oka, K. Kuroda, M. Yuzaki, S. Matsuda, T. Shirao, Y. Ishikawa, M. Sato, “Phldb2 regulates the maturation of dendritic spines and AMPA receptor endocytosis during long-term depression” San Diego, CA, Nov. 9-13.

ISN 2013 Synapse Satellite Meeting “Emerging Topics in Synapse Function: Molecular Mechanisms, Circuit Functions and Disease”. 2013, T Shirao “Role of Spikar in drebrin-mediated spine formation” (invited) Cancun, Mexico, April

24th ISN Biennial Meeting, 2013, Yamazaki, H., Shirao, T. “Spikar induces the formation of dendritic spines and filopodia in a drebrin-dependent manner.” Cancun, Mexico, April 20 -24.

The 24rd Biennial Meeting of ISN-ESN, 2013, Ishizuka Y, Shimizu T, Takagi E, Shirao T. “Histone deacetylase (HDAC) inhibitor attenuates amyloid beta-induced synaptic dysfunction.” Cancun, Mexico. April 20 -24.

The 24rd Biennial Meeting of ISN-ESN, 2013, Ishizuka Y, Shimizu T, Takagi E, Shirao T. “Acute effect of X-irradiation on adult mouse brain.” Cancun, Mexico. April 20 -24.

The 24th ISN Biennial Meeting, 2013, Roppongi RT, Hanamura K, Ishizuka Y, Shirao T. “5-HT2A receptor activation reduces the accumulation of drebrin in dendritic spines.” Cancun, Mexico. April 20 -24.

Third Conference of International Society of Radiation Neurobiology, 2013, N Koganezawa, A Puspitasari, N Kojima, T Shirao “Time-dependent effect of X-irradiation on fear conditioning and its underlying cellular mechanism.” In the Symposium “Effect on the neural cells and plasticity of the central nervous system.”, Naha, Okinawa, Jan 26. (invited)

Third Conference of International Society of Radiation Neurobiology, 2013, A Puspitasari, N Koganezawa, N Kojima, T Shirao “Acute effect of X-irradiation on adult mouse brain” Naha, Okinawa, Jan 26.

Society for Neuroscience 39th Annual Meeting, , 2012, Roppongi R T., Shirao T. “5-HT2A receptor activation reduced the accumulation of drebrin in dendritic spines.” New Orleans, LA, Oct 13-18.

5th Special Conference of the International Society for Neurochemistry: Synapses and Dendritic Spines in Health and Disease. T Shirao. “Myosin-II-mediated transient translocation of F-actin-drebrin A complex during the initial phase of synaptic plasticity.” Buenos Aires, Argentina, 12–15 September 2012 (invited)

53rd Annual Meeting of American Society for Radiation Oncology, 2011 Y. Suzuki, M. Hino, K. Shirai, Y. Yoshida, T. Mizui, K. Hanamura, T. Shirao, T. Nakano. “X ray Irradiation Induces Acute Depolymerization of Axonal and Dendritic Microfilaments in Cultured Neuron”, Miami, Florida, October 2-6.

The Synapse-From physiology to pathology, 2011, Shirao T. “Activity-dependent accumulation of F-actin associated with drebrin A facilitates spine formation.” Stresa, Italy, September 4-7.(invited)

The 23rd Biennial Meeting of ISN-ESN, 2011 Ishizuka Y, Nawa H, Takei N. (2011) AMPK regulates BDNF-induced activation of mTOR signaling and enhancement of translation in cortical neurons. Athens, Greece, August 28 – September 1

23rd ISN Biennial Meeting, 2011, Fujieda, T., Shirao, T., Sekino, Y. “Voltage-sensitive dye imaging of GABAB receptors mediated responses in the lateral nucleus of the mice amygdala.” Athens, Greece, August 28 – September 1

23rd ISN Biennial Meeting, 2011, Shirao, T., Hanamura, K., Yasuda, H., Kajita, Y., Kamata, Y., Sekino, Y., Kojima, N. “Regulatory role of drebrin in hippocampus-dependent learning.” Athens, Greece, August 28 – September 1

Emerging Concepts of Neuronal Cytoskeleton, 2011, Shirao T, Hanamura K. “Regulatory mechanism of drebrin localization at dendritic spines.” Santa Cruz, Chile, April 24-27 (invited)

Society for Neuroscience 37th Annual Meeting, 2010, Kojima N, Yasuda H, Hanamura K, Shirao T. “Neuron-specific drebrin isoform is necessary for hippocampus-dependent learning in adulthood but not in young age of mice” San Diego, USA, November 13-17

The 10th Biennial Meeting of the APSN Meeting, 2010. Shirao T, Hanamura K. “Low accumulation of drebrin at glutamatergic postsynaptic sites on GABAergic neurons.” Phuket, Thailand, October 17-20

22nd ISN Biennial Meeting, 2009, Shirao, T., Hanamura, K., Takahashi, H., Mizui, T., Sekino, Y.”Regulation of dendritic spine morphology by changing drebrin-A dynamics” Busan, Korea, August 23-28 (invited)

36th International Congress of Physiological Sciences (IUPS2009), 2009, Kojima N, Yasuda H, Hanamura K, Yamazaki H, Shirao T “Specific role of neuronal isoform of drebrin in hippocampal synaptic plasticity and fear memory.” Kyoto, July 27-August 1

36th International Congress of physiological Sciences (IUPS2009), 2009, Kato K, Yamazaki H, Shirao T, Sekino Y. “Maturation of AMPA receptors activity is regulated by expression of drebrin, an actin-binding protein, in cultured hippocampal neurons.” Kyoto, July 27-August 1

36th International Congress of physiological Sciences (IUPS2009), 2009, Shirao T “Role of synaptic activity in spine morphogenesis” in the symposium “Intercellular communications in the brain.” Kyoto, July 27-August 1 (Invited)

Society for Neuroscience 37th Annual Meeting, 2008 Hiroyuki Yamazaki, Kenichi Kato, Yuko Sekino, Tomoaki Shirao “Regulation of dendritic spine formation by Spikar-Drebrin interaction.” Washington, DC, Nov. 15-19

Society for Neuroscience 37th Annual Meeting, 2008 Kenji Hanamura, Nobuhiko Kojima, Hiroyuki Yamazaki, Tomoaki Shirao “Isoform conversion of drebrin during neuronal development regulates synapse formation and fear memory.” Washington, DC, Nov. 15-19

8th Biennial Meeting of the Asia-Pacific Society for Neurochemistry (APSN 2008) Tomoaki Shirao “Ionotropic glutamate receptors modify dendritic spine morphology by regulating drebrin dynamics.” Shanghai, PR China, June 24-26th (Invited)

Society for Neuroscience 37th Annual Meeting, 2007. Mizui T, Katayama M, and Shirao T “Roles of drebrin in the neurite outgrowth” San Diego, CA, Nov. 3-7

Society for Neuroscience 37th Annual Meeting, 2007. Takahashi H, Yamazaki H, and Shirao T “AMPA-type glutamate receptors regulate drebrin turnover during development” San Diego, CA, Nov. 3-7

7th IBRO World Congress of Neuroscience, Shirao, T. “Bidirectional regulation of NMDA receptor and actin cytoskeleton in dendritic spine” in the symposium of “Mechanisms of Structural Plasticity at Exciatory Synapses.” Melbourne, Australia July 12-17 2007 (Invited)

21th ISN Biennial Meeting,. Shirao, T., Takahashi, H., Mizui, T., Sekino, Y. “Genetic and activity-dependent control of spinous actin cytoskeleton in spine formation.” in the Symposium on “Cytoskeletal dynamics and signal transduction in neurons.” Cancun, Mexico August 23, 2007 (Invited)