ARTICLES https://doi.org/10.1038/s41593-019-0380-9 Corrected: Publisher Correction Neuronal deletion of Gtf2i, associated with Williams syndrome, causes behavioral and myelin alterations rescuable by a remyelinating drug Boaz Barak 1,2,3*, Zicong Zhang4, Yuanyuan Liu4, Ariel Nir3, Sari S. Trangle2, Michaela Ennis 1, Kirsten M. Levandowski5, Dongqing Wang1, Kathleen Quast1, Gabriella L. Boulting6, Yi Li4, Dashzeveg Bayarsaihan7, Zhigang He 4* and Guoping Feng 1,5* Williams syndrome (WS), caused by a heterozygous microdeletion on chromosome 7q11.23, is a neurodevelopmental disor- der characterized by hypersociability and neurocognitive abnormalities. Of the deleted genes, general transcription factor IIi (Gtf2i) has been linked to hypersociability in WS, although the underlying mechanisms are poorly understood. We show that selective deletion of Gtf2i in the excitatory neurons of the forebrain caused neuroanatomical defects, fine motor deficits, increased sociability and anxiety. Unexpectedly, 70% of the genes with significantly decreased messenger RNA levels in the mutant mouse cortex are involved in myelination, and mutant mice had reduced mature oligodendrocyte cell numbers, reduced myelin thickness and impaired axonal conductivity. Restoring myelination properties with clemastine or increasing axonal con- ductivity rescued the behavioral deficits. The frontal cortex from patients with WS similarly showed reduced myelin thickness, mature oligodendrocyte cell numbers and mRNA levels of myelination-related genes. Our study provides molecular and cellular evidence for myelination deficits in WS linked to neuronal deletion of Gtf2i. illiams syndrome (WS) is a multisystemic neurodevelop- WS-relevant behavioral phenotypes, including hypersociability and mental disorder characterized by hypersociability with social dishabituation. Homozygous Gtf2i knockout mice are embry- Wunique cognitive and personality profiles1,2. These include onically lethal, mainly due to failed neural tube closure12–14. Thus, intellectual disability, distinct overfriendliness, increased empathy the neuronal function of the Gtf2i gene and its role in the molecular and elevated anxiety derived from fear and specific phobias2. WS is and cellular mechanisms underlying WS behavioral and neurode- caused by haploinsufficiency of about 26 genes from the WS chromo- velopmental phenotypes remain completely unknown. In this study, some region (WSCR) at chromosome 7q11.23. While approximately we selectively deleted Gtf2i in the excitatory neurons of the fore- 95% of patients with WS have a hemizygous deletion of the entire brain and characterized molecular, cellular and behavioral changes WSCR, the other approximately 5% of patients have atypical micro- in the brain. We found that forebrain excitatory neuron-specific deletions that may only affect a few genes of the WSCR3–6 and thus are Gtf2i knockout mice show WS-relevant abnormalities, including helpful in defining genetic links to certain symptoms. In particular, neuroanatomical defects, fine motor deficits, increased sociabil- atypical microdeletions that overlap with GTF2I and GTF2I repeat ity and anxiety. Surprisingly, RNA sequencing (RNA-Seq) analysis domain containing 1 (GTF2IRD1), which are members of the general revealed that 70% of the genes with significantly decreased mRNA transcription factor 2I gene family, affect the cognitive phenotype in level were involved in myelination. Furthermore, we found reduced patients with WS3,4,6. Rare cases of individuals with microdeletions that numbers of mature oligodendrocytes, reduced myelin thickness affect either gene, but not both, strongly indicated that GTF2IRD1 dis- and impaired axonal conductivity. We further showed that nor- ruption is associated with WS craniofacial dysmorphism and cognitive malization of impaired myelination properties or axonal conduc- deficits, whereas GTF2I deletion may contribute to WS social behav- tivity rescues behavioral deficits. Importantly, similar myelination iors and mental impairment4,5. defects were also found in the postmortem human frontal cortex of GTF2I encodes the general transcription factor II-I (TFII-I) patients with WS. Together, these data implicate myelination, linked protein, a highly conserved and ubiquitously expressed multifunc- to neuronal loss of Gtf2i, as an important pathophysiology in WS tional transcription factor that regulates gene expression through and point to new paths for exploring potential therapeutic targets. interactions with tissue-specific transcription factors and com- plexes related to chromatin remodeling7. Previous work on mouse Results models for WS8 studied the role of Gtf2i deletion by using either Gtf2i deletion in the excitatory neurons of the forebrain causes heterozygous mice with the majority of the WSCR9 deleted, partial neuroanatomical and behavioral alterations. To dissect the func- WSCR deletions10 or single-gene deletion models11–13. These studies tion of Gtf2i in neurons, we crossed Gtf2i loxP mice15 with NEX-Cre showed correlations between Gtf2i haploinsufficiency and certain mice16, a Cre line that expresses Cre recombinase selectively in the 1McGovern Institute for Brain Research and Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA. 2The School of Psychological Sciences, Faculty of Social Sciences, Tel Aviv University, Tel Aviv, Israel. 3The Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel. 4F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Department of Neurology, Harvard Medical School, Boston, MA, USA. 5Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA. 6Department of Neurobiology, Harvard Medical School, Boston, MA, USA. 7Department of Reconstructive Sciences, University of Connecticut, Farmington, CT, USA. *e-mail: [email protected]; [email protected]; [email protected] 700 NatURE NEUROscIENCE | VOL 22 | MAY 2019 | 700–708 | www.nature.com/natureneuroscience NATURE NEUROSCIENCE ARTICLES ab cd ef Control cKO 150 kDa TFII-I * 20 Control 600 1,600 175 Tubulin 18 * * 250 * cKO 1,400 150 16 500 *** 1,200 14 m) 125 200 400 µ 150 12 ( 1,000 100 150 10 300 800 100 75 8 Events 600 Time (s) 100 Weight (g) 200 6 Weight (mg) 50 50 Thickness 400 4 100 50 (arbitrary units) 200 25 Expression level 2 0 0 0 0 0 0 P2 P4 P6 P8 cKO P10 P30 cKO cKO cKO cKO Control Control Control Control Control ghControl cKO ijk 500 80 * 1,200 * 320 ** 125 * 450 ** **** 300 400 1,000 * * 100 60 * * ) 350 * 280 ) ) 800 300 260 75 250 40 600 240 200 50 Time (s 400 Duration (s 150 220 Duration (s 20 Distance (cm) 100 Percentage wins 200 Control 200 Control 25 50 cKO 200 cKO 0 0 0 0 0 10 20 30 40 50 60 10 20 30 40 50 60 cKO Time (min) cKO Object Object Time (min) Stranger Stranger Control Control Fig. 1 | Neuroanatomical and behavioral deficits in Gtf2i cKO mice. a, Significantly reduced expression of TFII-I in whole cortex of 1-month-old cKO mice compared to controls, as measured by western blot. The image has been cropped from a full western blot (P = 0.001). b, cKO mice have similar body weight compared to controls. c,d, One-month-old cKO mice have significantly reduced brain weight (P = 0.0162) (c) and cortical thickness (P = 0.0159) (d) compared to controls. e,f, Increased levels of social behavior in cKO mice compared to controls as demonstrated by significantly longer duration (P = 0.03) (e, dyadic interaction time) and higher frequency of close social interaction events (P = 0.0476) (f, dyadic social events) during the dyadic social interaction test. g, In the three-chambered social interaction test, when looking at social preference duration, cKO mice showed significantly higher social preference compared to controls, interacting significantly longer with a stranger mouse compared to controls (*P = 0.04, **P = 0.0086, ****P < 0.0001). h, Decreased levels of social dominance in cKO mice, demonstrated by a significantly lower percentage of wins in the tube test compared to controls (P = 0.0392). i–k, cKO mice showed significantly increased levels of anxiety-like behaviors compared to controls as demonstrated by significantly shorter total exploration distance in the open field exploration test (P = 0.0108) (i), significantly longer duration in the margins of the open arena (P = 0.0024) (j) and significantly less time in the open arms of an elevated zero maze (P = 0.043) (k) compared to controls. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001, two-tailed t-test (a,c–h,k), two-way repeated-measures ANOVA with Bonferroni post hoc test (b,i,j). Data are shown as mean ± s.e.m. a–d, n = 8 control, n = 10 cKO. e–g, n = 8 control pairs, n = 8 cKO pairs. h–k, n = 10 control, n = 10 cKO. See Supplementary Fig. 2A for gel source data. excitatory neurons of the forebrain (Supplementary Fig.1) starting ture of WS1, we tested mice for anxiety-like behaviors in an open around embryonic day (E) 11.5 (ref. 16). The resulting mice, referred field exploration test (Fig. 1i,j and Supplementary Fig. 6A–C) and herein as cKO (Gtf2if/f:NEX-Cre+/−), had homozygous deletion of elevated zero maze test (Fig. 1k). We found significantly higher Gtf2i selectively in the excitatory neurons of the forebrain (Fig. 1a levels of non-social anxiety-like behaviors in cKO mice compared and Supplementary Figs. 2 and 3). to controls. No other significant behavioral differences were found Patients with WS show altered brain volume and cortical ana- in cKO mice compared to controls (Supplementary Fig.7). These tomical properties17–19. Therefore, we examined whether deletion of data suggest that Gtf2i deletion in the excitatory neurons of the Gtf2i from the excitatory neurons in the forebrain is sufficient to forebrain is sufficient to induce hypersociability and increased cause neuroanatomical abnormalities. While cKOs showed normal levels of non-social anxiety, resembling some core phenotypes body weight compared to control (Gtf2if/f:NEX-Cre−/−) littermates found in patients with WS. (Fig. 1b), 1-month-old cKO mice showed significantly reduced brain weight (Fig. 1c) and cortical thickness compared to controls Gtf2i neuronal deletion affects myelination-related transcrip- (Fig. 1d and Supplementary Fig.