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RESEARCH
We are interested in understanding how the cytoskeleton influences neuronal development during normal or pathological conditions (such as nerve injury). We use a variety of techniques, including optogenetics, biochemistry, bioinformatics, biophysics, cell biology, molecular biology, as well as material science to study this crucial process in neurons.
Neuronal Development and Disorders (神經發育與疾病)
Neuronal morphogenesis is the process through which a neuron (nerve cell) generates its elaborated axon and dendrites. It is the fundamental process underlying the establishment and plasticity of neuronal networks. Neuronal morphogenesis relies on fiber-like scaffolds called microtubule and actin cytoskeleton. While nearly all microtubules inside an undifferentiated cell originate from a microtubule-organizing center (MTOC) called centrosome, the centrosome in post-mitotic neurons does not seem to act as the MTOC. We discovered a microtubule-associated protein called TPX2, which is an important component of the non-centrosomal MTOC. Additionally, we found that the activity of TPX2 is regulated by the small GTPase Ran.
Genetic mutations disrupting neuronal morphogenesis can lead to serious neuronal developmental disorders (such as lissencephaly, microcephaly, polymicrogyria, and/or epilepsy). We are collaborating with Dr. Meng-Han Tsai from Epilepsy Division of Kaohsiung Chang Gung Memorial Hospital to tackle these debilitating disorders. The images below show dissociated mouse hippocampal neurons undergoing neuronal morphogenesis with microtubules shown in red, actin filaments in green, and DNA-containing nuclei in blue.
In the video above, we established an in vitro system using primary cortical neurons to mimic seizure onset. The electric behavior of neurons is detected using calcium imaging. In addition, we artificially created the audio output to make the audience aware of the synchronous firing phenomenon. See if you can spot the difference between these three different types of firing.
Neuroregeneration (神經再生)
The other interest of the lab is the axon/dendrite regeneration process, which happens after extended axons/dendrites are severed. The central nervous system (CNS) in mammals is incapable of regeneration while the peripheral nervous system (PNS) shows various degrees of regeneration. We are interested in the cellular basis of this CNS-PNS difference in neuroregeneration. Additionally, we are developing novel methods (e.g. microRNA, surface nanotopography, nanoparticles, natural compounds) to accelerate neuroregeneration. The video below shows axons of dorsal root ganglion neurons undergoing the regeneration process.
Optical Manipulation of Cells (光遺傳學與光學操控)
In order to study the role of specific cytoskeleton or organelles in neuronal morphogenesis and regeneration, we utilize a variety of optical manipulation methods. For example, we have constructed a laser optical tweezer system to position micrometer-sized cellular structures or to change cell shape in living cells or neurons. In addition, we are developing photoactivatable or photoinactivatable proteins to study the function of specific proteins in a spatial- and temporal-specific manner. The image series below shows the spatially controlled protein aggregation inside the cell. Note that when light irradiation ceases, the aggregation disappears (showing that our system is reversible). The video below shows another cell undergoing the same procedure. We can use this system to study how pathogenic protein aggregation (such as those seem in ALS) affects neurons.
High-content Drug or Biomolecule Screening (藥物篩選)
We are using high-content screening technology to look for drugs, RNAi molecules (siRNA, shRNA, or miRNA), or proteins that can enhance neuroregeneration or alleviate epilepsy. The high-content screen is a microscopy-based high-throughput screen, which takes advantage of the automated image acquisition machinery and image analysis software to screen large volumes of drugs or biomolecules within a short amount of time. Think of it as using a graduation picture to figure out if the student is a boy or a girl, if she/he is short-sighted or not, etc. By combining primary neuronal culture and lentivirus-mediated transduction, we can efficiently knock down endogenous genes in a physiologically relevant model system. We can also use this system to screen for drugs that can promote axon/dendrite regeneration.
High-throughput Quantification/Classification of Neurons
In the bioinformatics section of the lab, we are developing high-throughput image analysis methods for quantifying and classifying neurons based on their morphology.
Due to the advancement of image acquisition machinery, a large volume of data can be generated over a short period of time. How to accurately and efficiently extract morphological features from these images has become a challenge for modern medical centers or research labs.
We are developing image analysis algorithms which can automatically detect neuronal morphological features (our freely available software can be download from here) or pathological conditions.
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