A sex difference in the composition of the rodent postsynaptic density

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young synGAP+/- mice. Here we show that a highly significant increase in TARP in the PSDs of young synGAP+/- rodents is present only in females and not in males. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP heterozygosity.


Abstract 1 2
SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-3 95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased 4 steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains 5 constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-6 associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was 7 increased in young synGAP +/-mice. Here we show that a highly significant increase in TARP in the PSDs 8 of young synGAP +/-rodents is present only in females and not in males. The data reveal a sex difference in 9 the adaptation of the PSD scaffold to synGAP heterozygosity. 10 SynGAP is a Ras/Rap GTPase Activating Protein that is specifically expressed in neurons and is highly 11 concentrated in the postsynaptic density (PSD) of glutamatergic synapses in the brain (Chen et al., 1998; 12 Kim et al., 1998). Mutations in the human gene synGAP1 that cause heterozygous deletion or dysfunction 13 of synGAP result in a severe form of intellectual disability (synGAP haploinsufficiency, also called 14 Mental Retardation type 5 [MRD5]) often accompanied by autism and/or seizures (Berryer et al., 2013; 15 Hamdan et al., 2011;Hamdan et al., 2009). In mice, heterozygous deletion of synGAP causes similar 16 neurological deficits; homozygous deletion causes death a few days after birth (Komiyama et al., 2002; 17 Vazquez et al., 2004).

18
One function of synGAP is to regulate the balance of active Ras and Rap at the postsynaptic membrane 19 (Walkup et al., 2015), thereby controlling the balance of exocytosis and endocytosis of AMPA-type 20 glutamate receptors (Zhu et al., 2002) and contributing to regulation of the actin cytoskeleton (Tolias et

41
In our original study, we tested the second possibility by determining the ratio of TARPs to PSD-95 in 4 inverse correlation in HET females drives a significant inverse correlation in the pools of all HET animals 62 and of all female animals. The inverse correlation is not found in any subset of animals that contains only 63 males.

66
Creation of rat synGAP KO by the CRISPR-Cas9 method. CRISPR/Cas9 technology was used to 67 establish a SynGAP1 KO rat line that harbors a frameshift mutation in exon8 of SynGAP1 (Fig. 1A), 68 which prevents expression of the protein. SynGAP protein expression level is reduced by 50% in HET 69 knockout rats compared to wild-type (WT) and is absent in homozygous knockouts (Fig. 1B)

87
The ratio of synGAP/PSD-95 and TARP/PSD-95, 88 averaged over all of the rodents, are summarized in the 89 two bars labeled "All" (Fig 2A,B

165
There is not a statistically significant inverse correlation (negative Spearman's r) between the two ratios 166 for the group of all animals (Fig. 3A).

195
We also compared data sets from mice and rats at 7.5 weeks and 12.5 weeks (Fig. 3S2). These data 196 sets were small (9 or 10 animals). Nevertheless, they show a statistically significant inverse correlation 197 between TARP/PSD-95 and synGAP/PSD-95 in HET female mice at both 7.5 and 12.5 weeks. In data 198 from HET rats at 7.5 weeks, the inverse correlation is very close to significance; at 12.5 weeks, it is less 199 significant, but still shows a trend. In the corresponding males, none of the data sets shows a statistically 200 significant inverse correlation. More data would be required to make a definitive conclusion, but the 201 results suggest that competition between synGAP and TARP for binding to PSD-95 in females is more 202 prominent at 7 weeks than at 12 weeks, and more prominent in mice than in rats.

203
Effect of synGAP haploinsufficiency on the relative levels of other PSD proteins. In our previous 204 paper, we examined the levels of neuroligins 1 and 2 (NLG-1, -2), and of the surface protein LRRTM2. In 205 this study, we re-examined the effect of reduction of synGAP on the levels of NLG-1 and 2 in the PSD   does not differ between males and females (Fig. 5). This data shows that synGAP plays a role in 382 localizing GluN2B to the PSD, but that it is not the only protein involved.

383
We

387
The simplest explanation is that NLG's have a higher affinity for PDZ3 than synGAP such that synGAP

505
Each ratio for individual animals (ie. from a single lane) was then adjusted by multiplying by the 506 appropriate correction factor. Eighty percent of the correction factors fell between 0.5 and 1.75. One 507 percent were larger than 2.75 and four percent were less than 0.5. After normalized within cohorts, the 508 ratios were used for the analyses in figures 1 and 3. This procedure allowed us to correct for technical 509 variation within cohorts while preserving true differences in ratios based on species, age, genotype, or 510 sex. Analyses for significance were carried out and graphs were created with Prism 8 (GraphPad 511 Software, San Diego). The D'Agostino-Pearson omnibus test was used to determine the normality of the 512 data sets. The means of groups of data were compared for significant differences with either a one-tailed 513 Mann-Whitney test (when non-normal), or a one-tailed paired T-Test, as indicated. If the variances of the 514 two groups were found to be different using an F test, a one-tailed T-Test with a Welch's correction was 515 applied.
14 To test rigorously for correlations among individual animals between the ratio of synGAP to PSD-95 517 and the ratio of the other target proteins to PSD-95 (Figs. 2, 4, and 5), an additional normalization was 518 applied with the use of Excel. We corrected for differences in intensity of signals between the four 519 cohorts (i.e. 7.5 week old mice, 7.5 week old rats, 12.5 week old mice, and 12.5 week old rats) which had 520 been analyzed separately. (The previous normalization only corrected for technical variation within each   521 cohort.) Data for each cohort was divided into ratios from WT males, WT females, HET males, and HET 522 females. Averages of the ratios for each protein within each of these groups, were calculated. The