Biochemical and Biophysical Research Communications
Crybb2 associates with Tmsb4X and is crucial for dendrite morphogenesis
Introduction
In all vertebrate animals, the brain serves as the center of the nervous system. It is very well organized by neurons and glial cells, in which neurons are mainly responsible for conducting information of whole central nervous system [1]. Neurons are morphologically characterized by the presence of axons, somata, and dendrites. The shape of dendrites is the major indication how neurons integrate and process incoming information, and thus, they play a vital role in the functional properties of neural circuits [2,3]. During the last years, many studies documented considerable evidence that dendrite morphology (dendrite number and branches) is relatively dynamic. Alterations in dendrite morphology have been consistently observed in psychiatric disorders like schizophrenia [4,5]. Numerous studies have been endeavored to explore the mechanism underlying proper dendrite morphology development. The mechanism requires the tight control by neuronal signaling such as calcium as well as proteins involved in cytoskeletal rearrangement [6,7].
As one of the most prominent members of the crystallins, βB2-crystallin (gene symbol: Crybb2/CRYBB2) has been considered for a long time to be just a structural protein of the ocular lens [8]. Several clinical observations in human suggest that mutations in CRYBB2, especially the mutations in exon 6, are highly involved in the pathogenesis of cataract [9,10]. Different mouse mutants also showed that mutations in Crybb2 lead to cataract (for review see Graw, 2009). Besides that, there are a few reports that βB2-crystallin has additional functions. Crybb2 mutants have problems in fertility [11], and Crybb2 participates in axonal regeneration [12]. Most interestingly, βB2-crystallin also has moderate ability to bind calcium, suggesting a role as calcium buffer [13].
In addition, Crybb2 is also found to be expressed in several regions of the mammalian brain, although its function in the brain remains mostly unknown. To uncover its function in the brain, in the previous study we combined behavioral, neuroanatomical, and physiological analyses in a Crybb2 mouse mutant, O377 [14,15]. Altered hippocampal morphology and neuronal functions were observed in these mutants, O377, as well as increased free intracellular Ca2+ levels and expression changes of calcium-channel related proteins (NMDA receptor).
Crybb2 was found to be clearly up-regulated in the regenerating retina compared to the unregenerated retina [12]. Overexpression of Crybb2 in retinal ganglion cells and in hippocampal neurons increased axon formation. However, its function in dendrite morphology has not been analyzed so far. In this study, we focused on the functions of Crybb2 on hippocampal dendrite morphology, mainly on dendrite number and dendrite branching. In addition, by analysis of previous data and further overexpression studies in vitro, we identified TMSB4X as an associated partner of CRYBB2 being involved in the process of dendrite formation.
Section snippets
Plasmid construction
To generate RNAi constructs directed against Crybb2 the following oligonucleotides were annealed and subcloned into pLentilox 3.7 (Biofeng, Shanghai, China). The oligonucleotides used in the RNAi plasmid construction is 5′- TCCCCAAGATCATCATCTTC TTCAAGAGAGAAGATGATGATCTTGGGG TTTTTTC -3′ and 5′-TCGAGAAAAAACCCCAAGATCATCATCTTCTCTCTTGAAGAAGATGATGATCTTGGGGA-3′.
Cell culture and immunofluorescence microscopy
COS-7 cells were maintained and transfected as described [7]. Primary hippocampal cultures were prepared and processed for immunofluorescence
Crybb2 is expressed in dendrites and the over-expression of Crybb2 promotes dendritogenesis
To see whether CRYBB2 might play a role in dendrite formation, we checked its expression in dendrites of mouse hippocampal neurons. GFP-tagged Crybb2 was transfected in hippocampal neurons at 4 days cultured in vitro (DIV4) and grew for 48 h before fixation, a key period for morphogenesis of dendrites [18]. Interestingly, CRYBB2 was found to be co-localized with the microtubule-associated protein 2 (MAP2) (Fig. 1A), a dendrite marker used previously [7].
Given the fact that CRYBB2 is expressed
Discussion
The brain develops in an intricately orchestrated and highly organized manner. The proper development of neurons, especially the dendritic morphology, is the structural basis of the complex architecture of neuronal networks and the connectivity of the neurons, which is also the basis of the function in the brain. Whereas the expression of Crybb2 in the brain is quite clear [15], the function of Crybb2 in the brain, especially in dendrite morphogenesis, is largely unknown. Here we demonstrate
Acknowledgement
This work was supported by the National Natural Science Foundation of China (61675226), Young Scientists Fund of the National Natural Science Foundation of China (31500695), the Jiangsu Province Natural Science Foundation for Youths (BK20150358) and the Chinese Academy of Sciences President's International Fellowship for Visiting Scientists (Nafees Ahmad).
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