Exp Neurobiol 2015; 24(1): 8-16
Published online March 31, 2015
© The Korean Society for Brain and Neural Sciences
Sung-Tae Hong1 and Won Mah1,2*
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 2Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-53-660-6861, FAX: 82-53-426-7731
GIT1, a multifunctional signaling adaptor protein, is implicated in the development of dendritic spines and neuronal synapses. GIT1 forms a signaling complex with PIX, RAC, and PAK proteins that is known to play important roles in brain development. Here we found that
Keywords: GIT1, dGit, Microcephaly, Brain Development
GIT1 is a multifunctional adaptor protein that regulates the PIX-RAC-PAK signaling pathway . GIT1 interacts with various proteins and generates protein-protein interaction networks at pre- and postsynaptic sites, where it modulates spine and synaptic development. GIT1 interacts with liprin-α and stabilizes surface expression of AMPA receptors , and forms signaling complex with βPIX-RAC1-PAK3 to regulate spine morphogenesis and synapse formation [3, 4]. In the presynaptic compartment, GIT1 is involved in the organization of the presynaptic active zone through the interaction with Piccolo, a presynaptic cytomatrix protein . A recent study identified that the interaction between GIT1 and GluN3A impairs synaptic localization of the Git1-PIX signaling complex and thus downregulates RAC1 activation and spine formation . The EphrinB-Eph receptor signaling pathway phosphorylates GIT1 to regulate spine and synapse formation . Synaptic role of GIT1 has been further supported through
GIT1 is also expressed at inhibitory synapses and interneurons and plays an important role in inhibitory transmission. GIT1-βPIX-RAC1-PAK3 signaling pathway is important for the migration of interneurons . Depletion of GIT1 expression results in decreased Rac1 and PAK3 activation and reduced levels of parvalbumin expression . Smith et al. identified the interaction between GIT1 and GABAA receptor, which promotes surface expression of GABAA receptor through the βPIX-RAC1-PAK signaling pathway . Taken together, GIT1 regulates both excitatory and inhibitory synaptic transmission, which is critical for excitatory and inhibitory balance.
The importance of the GIT1-βPIX-RAC1-PAK3 signaling complex in brain development has been supported by a series of studies. A Mutation of
To better understand the functional implication of GIT1 in the development of the brain, we performed immunohistochemical and behavioral analyses to reveal the importance of GIT1 in the brain development. In addition, using adult
For NeuN (Millipore) staining, brains were isolated from adult mice (2~3 months old) after cardiac perfusion (4% paraformaldehyde). After the post-fixation for 12 h, 50 µm brain sections were obtained by vibratome (Leica Biosystems). Brain sections were washed 3 times with PBS for 10 min, permeabilized with 0.5% TritonX-100 for 30 min, blocked with 5% bovine serum albumin (BSA) for 1 h, stained with primary antibodies at 4℃ for 12 h, stained with secondary antibodies for 1 h, and mounted with Vectashield.
For staining with anti-FASII (Hybridoma Bank), adult brains of
Brain slices from the transcardially perfused adult mice (2~3 months old) were labeled by the ballistic delivery of the lipophilic dye DiI as previously described . Dendrites in the proximal stratum radiatum region of the hippocampus were analyzed to quantify spine density, width, and length. For quantification, MetaMorph software (Molecular Devices) was used.
Gait pattern of wild-type and
Motor coordination and motor learning was assessed with a rotarod apparatus. During 4 min session, rotating velocity of the rod was accelerated from 4 rpm to 40 rpm. Experiments were performed during four consecutive days, with two trials per day. Motor performance of the subject mice was measured by the final speed when mice fell off from the rod.
Student's t test was done using Microsoft Excel 2007 (Microsoft). Result of rotarod assay was analyzed by repeated ANOVA using R (http://www.r-project.org/).
In the previous study,
To further investigate the impaired brain development of
Microcephaly-affected human patients showed various behavioral phenotypes including mental retardation and hyperactivity . In previous research,
To further analyze the role of Git1 in brain development, we observed whether the adult brain development is also impaired in
Here, we report a microcephaly-like brain size reduction, decreased neuronal cell body size, and behavioral deficits such as impaired motor coordination and learning in
Microcephaly, a neurodevelopmental disorder, is characterized by the decreased brain to body ratio, which is often associated with mental retardation and epilepsy . Woods suggested two types of microcephaly; primary microcephaly caused by reduced number of neurons and secondary microcephaly mainly associated with reduced dendritic processes . In
In previous works performed by two independent groups using
In conclusion, severe developmental deficits shown in