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Exp Neurobiol 2018; 27(2): 103-111
Published online April 30, 2018
© The Korean Society for Brain and Neural Sciences
Yeojun Yoon1†, Hasang Park1†, Sangyeon Kim1†, Phuong T. Nguyen2†, Seung Jae Hyeon2†, Sooyoung Chung2, Hyeonjoo Im2, Junghee Lee3,4, Sean Bong Lee5* and Hoon Ryu2,3,4*
1Yonsei University College of Medicine, Seoul 03722, 2Center for Neuromedicine and Neuroscience, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea, 3VA Boston Healthcare System, Boston, MA 02130, 4Boston University Alzheimer’s Disease Center and Department of Neurology, Boston University School of Medicine, Boston, MA 02118, 5Department of Pathology & Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA
Correspondence to: *To whom correspondence should be addressed.
Sean Bong Lee, TEL: 1-504-988-1331, FAX: 1-504-988-7389
Hoon Ryu, TEL: 1-857-364-5910, FAX: 1-857-364-4540
†These authors are contributed equally
A recent study reveals that missense mutations of
Keywords: EWSR1, central nervous system (CNS), neuron, dopamine, DARPP-32, motor function
EWS RNA binding protein 1 (EWSR1) belongs to the FET family of DNA and RNA binding proteins and shares functional homology. FUS, EWSR1, and TAF15, constituting the FET family, have a significant role in transcription and alternative splicing by interacting with transcription pre-initiation complex and various splicing factors [1,2,3,4,5,6,7]. In addition, FET proteins modulate post transcriptional modification through their RBD and RGG domain . The discovery of
While the role of EWSR1 fusion protein in oncogenesis is relatively known, the role of wild-type EWSR1 remains largely unclear. A previous study suggested that EWSR1 is crucial in meiosis.
Recently, several studies have discovered mutations of FET family proteins and cytoplasmic aggregates of FET proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients, suggesting these genes are associated with neurodegenerative diseases [21,22,23,24,25,26,27]. Mutant forms of FET proteins generate prion-like aggregation and alter their subcellular localization . Importantly, a recent study has found missense mutation of
A total of 16 mice (7 homozygous
Neuronal nucleus size was measured from the motor cortex, striatum, and hippocampus. Then their sizes were analyzed by NIH ImageJ program, followed by the principles of unbiased stereology . Neuronal nucleus size was measured as the cross-sectional area of the nucleus. Measurement of neuronal nucleus size was conducted using freehand selection. A large counting box (399×299 µm) was placed over a slide in a systemic and random manner. Nucleus size was measured from at least 100 neurons per slide, which encompasses the average neuronal nucleus size of the area . For comparison, a corresponding region was examined from each animal: M2 region of motor cortex adjacent to the midsagittal line, a CA3 region of the hippocampus and caudate putamen adjoining lateral ventricle. The counting and measurement were conducted by two individual researchers, and each researcher was blinded to the genetic type.
Fore and hind limb movements were assessed as described previously . Using video captures, limb movement was quantified by the number of movements per second (mvmt/sec). Movement of the ventral surface of each mouse was filmed while the mouse was suspended by the tail for 10 seconds, followed by a brief touch down and 10-second suspension, and followed by the second brief touch down and the last suspension for 12 seconds. The total suspension time of each object was not exceeding 32 seconds. Limb movement data was acquired and quantified by blind analysis to the genetic type of mice. Dystonia of both forelimb and hindlimb was recorded at 3 postnatal weeks by counting the number of clasping behavior . In addition, we measured the duration of torso flexion of both WT and
Immunohistochemistry was performed in coronal or sagittal sections of WT and
Western blot was conducted as previous describe . Twenty micrograms of protein was electrophoresed on SDS-PAGE (10%) and blotted with anti-TH antibody (Cat. No.: AB152, Millipore, MA, USA) at 1:1000 dilution or anti-DARPP-32 antibody (Cat. No.: sc-271111, Santa Cruz Biotechnology, CA, USA) at 1:500 dilution. Anti-β-Actin antibody (Cat. No.: a1978, Sigma, MO, USA) at 1: 5000 dilution was used as protein loading control.
Total RNA from WT and
The data are expressed as the mean±standard error of the mean (SEM). Comparison among WT and
At the first series of experiment, we characterized gross anatomical difference of the brain and neuronal nucleus sizes between WT and
In order to determine whether
To address whether the motor dysfunction is associated with impaired dopaminergic signaling in
In the current study, we measured neuronal nucleus size in three different brain regions such as motor cortex, hippocampus, and striatum of
Interestingly, we found that