Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I

Abstract

Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src (thl/thl) ) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src (thl/thl) mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

Keywords: Src tyrosine kinase; Williams-Beuren syndrome; calcium channel; general transcription factor 2 I; mouse behavior.

Figure 1.

Craniofacial dysmorphology and growth retardation in Src^thl/thl mice. A, Whole genome scan in (S129 x C57BL/6) F2 toothless mice demonstrated linkage to D2Mit411. Additional markers were used to refine the critical interval (highlighted in yellow). S, Homozygous for S129 allele; H, heterozygous for S129 and C57BL6 alleles; NT, not tested. B, Mutant mice have insertion of a C nucleotide in exon 12 of the Src gene. C, No Src protein was detected in immunoblot of whole-brain lysates from Src^thl/thl mice. D, Faxitron images of WT and Src^thl/thl mouse skulls in lateral (L) and superior (S) views with landmarks used for cephalometric analysis indicated and described in Materials and Methods. Abbreviations are defined in Table 1’s footnote. Analysis of the data is summarized in Table 1. E, Weight of WT and Src^thl/thl mice shown as mean ± SEM (n = 10 WT, n = 9 Src^thl/thl).

Figure 2.

Social approach behaviors are increased and social recognition is impaired in Src^thl/thl mice. A, Src^thl/thl mice spent more time sniffing a social cage versus a nonsocial cage (n = 9 WT males, n = 8 Src^thl/thl males). B, During the second phase of the test to measure social recognition, Src^thl/thl mice did not show a preference for a novel mouse versus a familiar mouse from the first phase (n = 9 WT males, n = 8 Src^thl/thl males). *p < 0.05, compared to the chamber with empty cage, between genotypes and within genotypes, respectively. C, In a direct social interaction test, Src^thl/thl mice showed an increased frequency of interactions with a stranger mouse. Data are presented as mean ± SEM (n = 9 WT males, n = 8 Src^thl/thl males). *p < 0.05, between genotypes. D, Src^thl/thl mice showed social dominance over their WT littermates in a tube test (n = 20 WT males, n = 20 Src^thl/thl males). *p < 0.05, between genotypes. E, Src^thl/thl mice were able to habituate to a smell over time and dishabituate toward a novel smell, demonstrating intact olfaction (n = 10 WT (5 males, 5 females), n = 9 Src^thl/thl (5 males, 4 females)). Data are presented as mean ± SEM.

Figure 3.

Enhanced social reunion of female Src^thl/thl mutant mice. A, Time course of total number of USVs in WT and Src^thl/thl male mice when introduced to females for a 5 min trial. A gradual decline in total number of calls was observed across time but the total number of calls was still high, with no significant difference between WT and Src^thl/thl mice. Data are presented as mean ± SEM for each time point (n = 10 WT, n = 11 Src^thl/thl). B, Time course of total number of USVs in WT and Src^thl/thl females when reunited with a litter mate for a 5 min trial. A gradual decline in total number of calls is observed across time with a significant difference between WT and Src^thl/thl females in total number of USVs produced in the first and second minutes (n = 11 WT, n = 10 Src^thl/thl). C, There is a significant difference in the number of flat (Type 1) USVs produced by WT versus Src^thl/thl females at minute 1 (n = 11 WT, n = 10 Src^thl/thl). D, There was no significant difference in the number of broken (Type 2) USVs produced by WT versus Src^thl/thl females across time (n = 11 WT, n = 10 Src^thl/thl). E, There is a significant difference in the number of frequency modulated (Type 3) USVs produced by WT versus Src^thl/thl females at minutes 1 and 2 (n = 11 WT, n = 10 Src^thl/thl). Data are presented as mean ± SEM for each time point. *p < 0.05 between genotypes.

Figure 4.

Src^thl/thl mutant mice show hyperactivity in the open field. A, Src^thl/thl mutant mice showed an increase in total distance travelled in the open field compared with WT mice (n = 10 WT (5 males, 5 females), n = 16 Src^thl/thl (9 males, 7 females)). B, Percentage of time spent in the center of the arena versus the entire open field was decreased in Src^thl/thl (n = 10 WT (5 males, 5 females), n = 16 Src^thl/thl (9 males, 7 females)). *p < 0.05 between genotypes. C, Percentage time spent in the open arms versus the closed arms of the elevated plus maze showed no significant differences between genotypes (n = 13 WT (7 males, 6 females), n = 18 Src^thl/thl (9 males, 9 females)). Data are presented as mean ± SEM.

Figure 5.

Balance and motor strength deficiency in Src^thl/thl mice and the inability to learn motor skills. Quantitative analysis of raised balance beam, grip strength, and rotarod task in WT and Src^thl/thl mice. A, Src^thl/thl mice needed significantly more time to cross the balance beam than did WT mice (n = 13 WT (7 males, 6 females), n = 12 Src^thl/thl (6 males, 6 females)). B, Src^thl/thl mice had significantly more hindfoot slips than did WT mice. C, Grip strength analysis revealed significantly reduced forelimb grip strength in Src^thl/thl mice compared with WT animals (n = 16 WT (9 males, 7 females), n = 12 Src^thl/thl (6 males, 6 females)). D, Grip strength analysis revealed significantly reduced combined forelimb and hindlimb grip strength in Src^thl/thl mice compared with WT animals (n = 16 WT (9 males, 7 females), n = 12 Src^thl/thl (6 males, 6 females)). E, Mice were tested on an accelerating rotarod with the same speed for 4 consecutive days. The latencies from rotation onset until the mice fell off the rod were measured. WT mice managed to stay significantly longer on the accelerating rotarod than Src^thl/thl mice on days 3 and 4. The falling latencies were similar in both groups on days 1 and 2. Falling latencies were compared within genotype between days 1 and 4 (n = 12 WT (6 males, 6 females), n = 12 Src^thl/thl (6 males, 6 females)). Data are presented as mean ± SEM. *p < 0.05.

Figure 6.

Acoustic startle in response to high startling stimulus is increased in Src^thl/thl mice. Both the WT and Src^thl/thl mice exhibited similar startle amplitude responses to the low-intensity acoustic stimuli (70-105 dB). Src^thl/thl mice showed higher responses to the high-intensity stimuli (110-115 dB) (n = 8 WT (8 males), n = 8 Src^thl/thl (18 males)). *p < 0.05.

Figure 7.

Impaired memory in Src^thl/thl mutant mice. A, Mice were assessed in the Morris water maze procedure (n = 12 WT (12 males), n = 12 Src^thl/thl (12 males)). Mean latency to reach a target platform in a visible platform session (days 1 and 2), and in a hidden-platform acquisition phase (days 3-8). B, Spatial memory retention was assessed in the probe trials administered 2 h after day 8 (n = 12 WT (12 males), n = 12 Src^thl/thl (12 males)). Mean number of platform crosses during probe trial 1 are shown. *p < 0.05. C, Freezing (as a percentage of total time) in a novel conditioning chamber (n = 24 WT (13 males, 11 females), n = 22 Src^thl/thl (12 males, 10 females)). After recording baseline freezing for 120 s, a tone was presented for 30 s (novel tone), after which mice were given a footshock and left inside the chamber for another 30 s (after shock). A subtle increase in freezing was observed during baseline and novel tone presentation in Src^thl/thl mice. *p < 0.05. D, Cued fear memory 24 h after pairing (n = 24 WT (13 males, 11 females), n = 22 Src^thl/thl (12 males, 10 females)). Pre-CS is freezing in the absence of the tone in a novel context, while CS is percentage freezing during tone presentation. Src^thl/thl mice showed impaired auditory fear conditioning, however, context was not affected. Data are presented as mean ± SEM, *p < 0.05.

Figure 8.

Src^thl/thl mice have a deficit in short term object recognition. A, Mean time (s) spent exploring the displaced versus nondisplaced objects in the spatial change session (n = 12 WT (6 males, 6 females), n = 15 Src^thl/thl (8 males, 7 females)). B, Mean time spent exploring the novel item versus three familiar objects in the nonspatial change session (n = 12 WT (6 males, 6 females), n = 14 Src^thl/thl (7 males, 7 females). Data are expressed as mean ± SEM, *p < 0.05.

Figure 9.

Altered cellular localization of TFII-I and TRPC3 in Src^{thl /thl} mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Src^thl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Src^thl/thl). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.

Figure 10.

Altered cellular localization of TRPC3 and TFII-I in cultured cortical neurons. TFII-I and TRPC3 fluorescence intensity was calculated from pooled data derived from two independent experiments, each using 10 pups from two different litters (n = 60). A, DIV6 cortical neurons were labeled for TFII-I in red, TRPC3 in green, and NeuN in blue. Scale bar is shown in the first panel, WT TFII-I, 40 μm. B, TFII-I, TRPC3, and NeuN fluorescence intensity was measured in the nucleus (I_N), cytosol (I_Cyt) and plasma membrane (I_Pm). Fluorescence intensity was measured in a single section in the middle of the entire Z-stack for every neuron imaged. Scale bar, 5 μm. C, Src^thl/thl mice had higher levels of TFII-I in the membrane than WT littermates. D, There was no significant differential expression of TFII-I in the cytosol. E, WT mice showed higher levels of TFII-I in the nucleus compared to Src^thl/thl littermates. F, The 30% difference in TFII-I levels between the cytoplasm and nucleus in Src^thl/thl mice and 17% difference in WT mice shows that TFII-I was more evenly distributed throughout the cell in WT mice. G, Src^thl/thl mice had higher levels of TRPC3 in the membrane than WT littermates. H, Src^thl/thl mice had less TRPC3 in the cytosol than WT littermates. I, Src^thl/thl mice had less TRPC3 in the nucleus compared to WT littermates. J, There was a 36% difference in TRPC3 levels between the cytosol and the nucleus in Src^thl/thl mice. In WT mice, there was only a 17% difference of fluorescence, which means that TRPC3 was more evenly distributed throughout the cell in WT mice. Data are expressed as mean ± SEM, *p < 0.05.