Hsiao-Huei Chen, PhD

hchen@uottawa.ca
hchen@ohri.ca

Scientist, Neuroscience, Ottawa Hospital Research Institute

Assistant Professor, Department of Medicine, University of Ottawa

Research Interests:

Using novel transgenic mouse models of congenital neural tube defects and the techniques of chick experimental embryology and electrophysiology, Dr. Chen proposes to establish a paradigm for the characterization of transcriptional control of neural differentiation and synapse specification during development. In addition, understanding the transcriptional mechanisms that regulate neuron apoptosis may promote recovery from brain and spinal ischemia.

Neural tube defects such as anencephaly and spina bifida are a leading cause of death in babies under 1 year of age (incidence of 1/1000 in American Caucasians), but of unknown genetic etiology. LMO4, a LIM-only domain transcription cofactor, is essential for neural tube closure and axonal pathfinding. LMO4 null mutant mice have a marked increase in neural apoptosis and die at birth with anencephaly and aberrant cranial nerve projections.

To understand the development of neural tube and specification of neural circuit, the spinal stretch reflex serves a simple model. Sensory neurons responsive to muscle stretch (Ia afferents) make strong monosynaptic connections with motor neurons supplying their own muscle (homonymous connections) but weak or no connections with motor neurons supplying antagonistic or functionally unrelated muscles (Fig. 1). The specificity of these connections is the hallmark of sensory neuron differentiation and is evident at the time that muscle afferents first make contact with motor neurons (see review by Chen et al, 2003). Dr. Chen has shown that the strength of synaptic connectivity between muscle sensory and motor neurons is regulated by muscle-derived neurotrophin 3 (NT3). By differential screening of single sensory neuron cDNA libraries, Dr. Chen found that LMO4 is a selectively expressed transcription regulatory cofactor in functionally related populations of sensory and motor neurons (Chen et al 2002). Thus, LMO4 may specify neuronal identity and control synapse network formation.

LMO4 is an immediate early gene and its expression is tightly regulated in neurons. LMO4 mRNA and protein has a very short half life (Chen et al 2007). Others have shown that LMO4 is involved CamK/CREB signaling and mediates activity dependent gene activation in neurons. Our recent electrophysiological study on the brain slice using the patch-clamp recording has suggested a role of LMO4 in synaptic plasticity and connections. Understand the molecular mechanism whereby LMO4 is regulating the synaptic plasticity is one of the major projects in the lab.

Another focus of Dr. Chen's laboratory is to understand how transcription factors influence neuron survival from stroke. We showed LMO4 is induced by ischemia in neurons and mice with neuronal ablation are highly susceptible to cerebral damaged in an experimental MCAO model of stroke (Schock et al 2008). Furthermore, we showed that LMO4 is an essential cofactor of PPARgamma. PPARgamma agonist treatment reduces stroke injury in experimental animals. Moreover, patients with type II diabetes show a reduced incidence of recurrent stroke when treated with the PPARgamma agonist pioglitazone. Thus, our studies suggest that the levels of LMO4 may have a profound effect in controlling the efficacy of PPARgamma agonist treatment for stroke. Polymorphisms in the 3'UTR of LMO4 in humans might contribute to the genetic heterogeneity in the response to stroke. We are currently exploring this possibility in collaboration with Dr. Alexandre Stewart at the University of Ottawa Heart Institute Cardiovascular Genetics Centre. Metabolic syndrome is a major risk factor for stroke. Hypothalamus is the neuroendocrine regulation centre of metabolism. We are currently investigating the hypothalamic control of metabolism.

Animal models:

To address how LMO4 participates in sensory neuronal differentiation, axonal pathfinding, and specification of synaptic connections during development, Dr. Chen's laboratory will use LMO4 homozygous null mutant and LMO4 conditional deletion mice, Parvalbumin-CRE/LMO4 floxed transgenic mice that selectively ablate LMO4 expression in proprioceptive neurons, together with the easily accessible chick embryo that allows siRNA-induced LMO4 ablation and LMO4 over-expression during development.

The chick embryo is uniquely suited to experimental manipulations; it comes neatly packaged in a sterile environment, the egg, is easily incubated at 42°C in a humidified file cabinet-sized incubator chamber, can be accessed by opening a window in the egg shell and lies floating on feeder layer of yolk with its own circulatory system. Eggs do not require dedicated animal housing: embryos are used prior to hatching 21 days postfertilization. The brain and spinal cord lie beneath a thin layer of epithelium that can be readily visualized under a stereo-microscope. Microelectrodes can easily target DNA and transgenes to any area of the spinal cord or brain and neurons are transfected by a brief pulse of electrical current by the technique of electroporation. Co-injection of fluorescent markers allows direct visualization of transgene localization in the brain or spinal cord (Fig. 2). Embryos are then allowed to resume their developmental course to any desired embryonic stage by taping the window closed with cellophane tape and returning the egg to the incubator. The transparent cellophane window allows observation of the progress of embryonic development before dissecting the embryo. Knock-down or over-expressing genes in the chick spinal cord or brain allows the assessment of gene function on different developmental processes including neuron differentiation, survival from programmed cell death and axonal pathfinding. The chicken genome has now been fully sequenced and all cDNA probes are now available, making this a highly attractive model to study developmental neurobiology. Moreover, the development of antisense siRNA technology allows direct ablation of target genes at any desired stage during development by electroporation. Evolutionary conservation among vertebrates allows translation of findings between the chick and mouse and ultimately, applicability to human disease.

Techniques: The laboratory is equipped with molecular and electrophysiology (patch-clamp) equipment and a cellular biology imaging system. The lab is funded by the CIHR and HSFC.

Selected Honours and Awards:

2009-2010 Henry J. M. Barnett Research Scholarship award, The Heart and Stroke Foundation of Canada
2009-2014 Early Researcher Award, Ontario Ministry of Research and Innovation
2009-2014 New Investigator Award, Canadian Institutes of Health Research (declined to accept HSFC award).
2009-2014 New Investigator Award, The Heart and Stroke Foundation of Canada

Current Funding:
2006-2007 LMO4 and cytokine signaling in stroke recovery (Heart and Stroke Foundation of Ontario Centre for Stroke Recovery, support for trainee).
2007-2011 Establishment of an experimental embryology, microscopy and electrophysiology laboratory to study the molecular basis of congenital neuronal defects and stroke recovery (Canada Foundation for Innovation & Ontario Research Foundation, support for infra structure)
2006 Control of sensory neuron differentiation and survival. (CIHR Priority Announcement NSA 174808, operating grant).
2008-2011 Mechanisms to reduce injury and improve recovery from stroke. (HSFC Gant-in-Aid NA6301, operating grant)
2008-2013 Control of sensory neuron development. (CIHR NSB 179197, operating grant)

Selected publications (2001-present):

1. Chen, H.-H., Tourtellotte, W.G. and Frank, E. (2002). Muscle spindle-derived neurotrophin 3 (NT3) regulates synaptic connectivity between muscle sensory and motor neurons. J. Neurosci, 23, 3512-3519.

2. Chen, H.-H., Yip, J.W., Stewart, A.F.R. and Frank, E. (2002). Differential expression of a transcription regulatory factor, the LIM-domain only 4 protein LMO4, in muscle sensory neurons. Development, 129, 4879-4889.

3. Chen, H.-H., Mullett, S. J., Stewart, A. F. R. (2004) Vgl-4, a novel member of the Vestigial-like family of transcription cofactors, regulates 1-adrenergic activation of gene expression in cardiac myocytes. Journal of Biological Chemistry, 279, 30800-30806.

4. Chen, H.-H., Maeda, T., Mullett, S. J., Stewart, A. F. R. (2004) The transcription cofactor Vgl-2 is required for skeletal muscle differentiation. Genesis, 39, 273-279.

5. Chen, H.-H., Baty, C., Maeda, T., Saba, S., Ueyama, T., Brooks, S., Baker, L. C., Gursoy, E., Salama, G., London, B., and Stewart, A. F. R. (2004) Transcription Enhancer Factor-1-Related Factor-Transgenic Mice Develop Cardiac Conduction Defects Associated With Altered Connexin Phosphorylation. Circulation, 110, 2980-2987.

6. Chen, H.-H., Xu J, Safarpour F, Stewart AF.LMO4 (2007) mRNA stability is regulated by extracellular ATP in F11 cells. Biochem Biophys Res Commun, 357, 56-61.

7. Chen, H.-H., Schock SC, Xu J, Safarpour F, Thompson CS, Stewart AF. (2007) Extracellular ATP-dependent upregulation of the transcription cofactor LMO4 promotes neuron survival from hypoxia. Exp Cell Res., 313, 3106-16.

8. Schock SC, Xu J, Duquette PM, Qin Z, Rai PS, Lewandowski AJ, Thompson CS, Seifert EL, Harper M, Chen, H.-H. (2008) Rescue of neurons from ischemic injury by PPAR requires a novel essential cofactor LMO4. J. Neurosci. 2008 28: 12433-12444.

Review
Chen, H.-H. Arber, S., and Frank, E. (2003). Development of monosynaptic reflex circuits. Cur. Opinion in Neurobiol, 13, 96-102.

Book Chapter
Chen, H.-H. and Stewart, A. F. R. (2007) Characterization of cardiac gene promoter activity: reporter constructs and heterologous promoter studies. In: Zhang J, Rokosh G, eds. Cardiac Gene Expression: Methods and Protocols. Totowa, New Jersey: Humana Press:217-25.