Genetic Ablation of EWS RNA Binding Protein 1 (EWSR1) Leads to Neuroanatomical Changes and Motor Dysfunction in Mice

Abstract

A recent study reveals that missense mutations of EWSR1 are associated with neurodegenerative disorders such as amyotrophic lateral sclerosis, but the function of wild-type (WT) EWSR1 in the central nervous system (CNS) is not known yet. Herein, we investigated the neuroanatomical and motor function changes in Ewsr1 knock out (KO) mice. First, we quantified neuronal nucleus size in the motor cortex, dorsal striatum and hippocampus of three different groups: WT, heterozygous Ewsr1 KO (+/-), and homozygous Ewsr1 KO (-/-) mice. The neuronal nucleus size was significantly smaller in the motor cortex and striatum of homozygous Ewsr1 KO (-/-) mice than that of WT. In addition, in the hippocampus, the neuronal nucleus size was significantly smaller in both heterozygous Ewsr1 KO (+/-) and homozygous Ewsr1 KO (-/-) mice. We then assessed motor function of Ewsr1 KO (-/-) and WT mice by a tail suspension test. Both forelimb and hindlimb movements were significantly increased in Ewsr1 KO (-/-) mice. Lastly, we performed immunohistochemistry to examine the expression of TH, DARPP-32, and phosphorylated (p)-DARPP-32 (Thr75) in the striatum and substantia nigra, which are associated with dopaminergic signaling. The immunoreactivity of TH and DARPP-32 was decreased in Ewsr1 KO (-/-) mice. Together, our results suggest that EWSR1 plays a significant role in neuronal morphology, dopaminergic signaling pathways, and motor function in the CNS of mice.

Keywords: DARPP-32; EWSR1; central nervous system (CNS); dopamine; motor function; neuron.

Fig. 1. Whole brain sections of WT and Ewsr1 KO (−/−) mice at 3 weeks of age. (A) Sagittal (upper) and coronal (lower) brain sections of WT mice. (B) Sagittal (upper) and coronal (lower) brain sections of Ewsr1 KO (−/−) mice. Sagittal sections were stained with hematoxylin and eosin. Coronal sections were stained with cresyl violet.

Fig. 2. Neuronal nucleus sizes are altered in the cortex, striatum and hippocampus of Ewsr1 KO (−/−) mice. (A) The neuronal nucleus sizes in the motor cortex were reduced in Ewsr1 KO (−/−) mice. (B) The neuronal nucleus sizes in the striatum were reduced in Ewsr1 KO (−/−) mice. (C) The neuronal nucleus sizes in the hippocampus were reduced in Ewsr1 KO (−/−) mice. The tissues were stained with hematoxylin and eosin staining. Scale bar (black): 20 µm. Data are presented as the mean±SEM. Significantly different at ^*p<0.05, ^**p<0.001, ^***p<0.0001.

Fig. 3. Tail suspension test shows an increase of limb clasping behaviors in Ewsr1 KO (−/−) mice. (A) Still images of tail suspended WT and Ewsr1 KO (−/−) mice. (B) The number of forelimb and hindlimb clasping was significantly increased in Ewsr1 KO (−/−) mice at 3 weeks of age. The number of torso flexion was slightly increased Ewsr1 KO (−/−) mice. WT (n=3); KO (−/−) (n=3). (C) The number of forelimb and hindlimb clasping was significantly increased in Ewsr1 KO (−/−) mice at 10 weeks of age. WT (n=6); KO (−/−) (n=6). Data are presented as the mean±SEM. Significantly different at ^*p<0.05, ^**p<0.001, ^***p<0.0001 and n.s, non-significant at p>0.05.

Fig. 4. Dopaminergic signaling pathways are impaired in Ewsr1 KO (−/−) mice at 3 weeks of age. (A) qPCR analysis showed that Th mRNA was significantly lower in the striatum of Ewsr1 KO (−/−) mice (n=4) compared to WT mice (n=4) while Ddc (dopamine decarboxylase) and DARPP-32 mRNAs were not changed noticeably. (B) Western blot analysis showed that protein levels of TH and DARPP-32 were significantly decreased in Ewsr1 KO (−/−) mice (n=4) compared to WT mice (n=4). (C) The immunoreactivity of TH and DARPP-32 was markedly decreased in the striatum and the substantia nigra of Ewsr1 KO (−/−) mice, whereas the immunoreactivity of p-DARPP-32 (Thr75) was highly increased. (d) Densitometry analysis confirmed that the immunoreactivity of TH and DARPP-32 was significantly reduced in the striatum and substantia nigra of Ewsr1 KO (−/−) mice while the immunoreactivity of p-DARPP-32 (Thr75) was increased. Scale bar: 100 µm (black); 20 µm (red). WT (n=3); KO (−/−) (n=3). Data are presented as the mean±SEM. ^*p<0.05, ^**p<0.001, ^***p<0.0001, unpaired Student's t test.

Fig. 5. A schematic diagram showing that EWSR1 deficiency reduces neuronal nucleus size (atrophy) and, in parallel, causes the reduction of tyrosine hydroxylase in the substantia nigra. Non-phosphorylated DARPP-32 and phosphorylated (p)-DARPP32 (Thr75) are differentially regulated in the striatum of Ewsr1 mice, indicating that the dopaminergic signaling pathway is abnormaly regulated by EWSR1 deficiency. Consequently, EWSR1 deficiency leads to motor behavioral dysfunction in mice.