Upregulation of SYNGAP1 expression in mice and human neurons by redirecting alternative splicing

Abstract

The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency.

Keywords: AS-NMD; PTBP1; SynGAP; antisense oligonucleotide; autism; cerebral organoid; epileptic encephalopathy; haploinsufficiency; intellectual disability; poison exon.

Figure 1.. Alternative 3’ splice site of mouse Syngap1 inton10 induces nonsense-mediated mRNA decay.

A) Sashimi plots of isolated cortical neurons and apical neural progenitors showing Syngap1 A3SS in embryonic day 14.5 (E14.5) mouse dorsal forebrain. The A3SS exon inclusion ratios are indicated. B) RNA-Seq results showing that Syngap1 A3SS was enriched in early brain development. C) RT-PCR results showing that the Syngap1 A3SS was higher in the developing forebrain (76% at E12.5) and remained detectable in adulthood (5% at P40). PSI represents Percent Spliced In. One biological sample per lane. D) Syngap1 A3SS introduces in-frame stop codons that truncate the protein and/or induce nonsense-mediated mRNA decay. E) The predicted Syngap1 short protein isoform was not detectable in mouse brain lysates. F) The Syngap1 A3SS transcripts were enriched in Neuro2a cells treated with cycloheximide (CHX, two biological replicates per condition, p<0.001, unpaired t-test). G) The Syngap1 A3SS-NMD was upregulated in Neuro2a treated with two siRNAs against Upf1 (adj.p<0.05 for siRNA-1, adj.p<0.01 for siRNA-2, one-way ANOVA). Two biological replicates per condition. See also Figure S1.

Figure 2.. Human SYNGAP1 A3SS induces nonsense-mediated mRNA decay in neural development.

A) RNA-Seq results showing SYNGAP1 A3SS in the laser microdissected cortical plate (CP) and ventricular zone (VZ) of gestational week 16 (GW16) fetal brains. B) RT-PCR results showing that SYNGAP1 A3SS was enriched in fetal cortical development. C) RT-PCR results showing that SYNGAP1 A3SS levels significantly decreased in iPSCs during NGN1/2-induced neuronal differentiation. Two biological replicates per condition, p<0.001, one-way ANOVA. D) SYNGAP1 A3SS was enriched in iPSCs after CHX treatment. p<0.001 by t-test, three biological replicates. E) SYNGAP1 A3SS ratio was increased in iPSC-derived neurons after CHX treatment. p<0.001 by t-test, three biological replicates. F) Sequence alignment showing that the premature stop codon (TGA) in SYNGAP1 A3SS is conserved in mammals while the splice acceptor sites (AG) are variable. The AG sites were annotated according to RNA-Seq results of corresponding species in NIH Genome Data Viewer. G-H) RT-PCR results of SYNGAP1 mini-gene constructs (G) transfected in Neuro2a cells showing that human SYNGAP1 intronic mutations identified in autism and ID patients led to intron 10 retention (c.1676 +5 G>A, NM_006772.2) or abnormal A3SS inclusion (c.1677 −2_1685del, p<0.001 by t-test, three biological replicates in H) . See also Figure S2.

Figure 3.. SYNGAP1 A3SS-NMD is regulated by PTBP proteins.

A) Western blot results showing shRNA knockdown of Ptbpl and Ptbp2 in Neuro2a cells. Two different shRNAs were used for each of Ptbpl and Ptbp2. B) RT-PCR results showing that loss of Ptbp1/2 promoted splicing of the canonical/productive Syngap1 isoform. C) Immunofluorescence staining of primary cortical neurons (DIV5) showing that ectopic expression of PTBP1 (red, mCherry) decreased Syngap1 protein level (green). D) CLIP-Seq analyses showing Ptbp1, Ptbp2 and U2af65 binding peaks in the A3SS region. E) Mini-gene constructs of the human SYNGAP1 showing deletion of predicted splicing elements, and RT-PCR results showing their effects on A3SS insertion. Noticeably, the intronic element #1 and predicted upstream U2AF65-PTBP binding site #1 were required for A3SS inclusion; the predicted U2AF65-PTBP binding site #2 was required for canonical splicing and A3SS skipping. Two biological replicates for each condition. F) EMSA assay showing that PTBP1 protein had a higher affinity to the PTBP binding site #2 RNA probe. G) Current working model: in neural progenitors and differentiating neurons, PTBP proteins bind to site#2 in A3SS (red) and suppress the canonical/neuronal 3’ splice site; in neurons, PTBP proteins are turned down/off, site #2 is exposed for splicing machinery (U2AF65) recognition and promotes neuronal isoform expression. See also Figure S3.

Figure 4.. Genetic deletion of Syngap1 A3SS-NMD increases Syngap1 protein in the neocortex.

A) CRISPR deletion of Syngap1 A3SS-NMD in mice to generate the Syngap1 NISO (N) allele (chr17:26959184-26959451, mm10) and the short NISO allele (S, chr17:26959185-26959353, mm10). B-C) RT-PCR results showing that Syngap1 A3SS-NMD exon11 was included in wild-type controls (+/+), decreased in Syngap1-NISO heterozygotes (N/+), and excluded in Syngap1-NISO homozygotes (N/N) in P1 (B) and P10 (C) dorsal cortices. D) Western-blot results showing Syngap1 protein levels in P2 cortices. E) Quantification of Syngap1 Western bot signals in (D) relative to Gapdh showing that Syngap1 levels were significantly increased in Syngap1 N/+ (32±9%, p<0.005) and Syngap1 N/N (58±6%, p<0.001, one-way ANOVA, Dunnett’s multiple comparison test) when compared with wild-type (+/+). F) fEPSP recordings of adult Syngap1 N/+ and N/N mice. Numbers of animals and slices for each genotype were: +/+ (n=6 animals, 11 slices), N/+ (n=6, 13), N/N (n=5, 11). *, adj.p = 0.0223. G-I) Barnes maze test results showing similar performance between wild-type (+/+, n=6) and mutants (N/+, n=7; N/N, n=6). (G) All genotypes show improvement in the primary latency to the exit zone over the course of training. H) Comparisons of primary latency to the exit zone during the probe trial shows that Syngap1 mutant mice performed similarly to wild-type mice. (I) Heat maps and a box plot showing entry probability to the exit zone I and each false exit during the probe trial. Syngap1 N/+ and Syngap1 N/N animals were not significantly different in behavior from wild-type (+/+) mice (adj.p > 0.05). The greatest entry probability for each genotype corresponds to the exit zone. See also Figure S4.

Figure 5.. Genetic deletion of Syngap1 A3SS-NMD alleviates LTP and membrane excitability deficits caused by a conditional Syngap1 knockout allele.

A) fEPSP recordings showing that the N allele alleviates the LTP deficit in P35-40 Syngap1 cKO mice. Numbers of animals and slices for each genotypes are: WT (replotted from Figure 4F), cKO/+ (n=5, 12), and cKO/N (n=6, 12). ****adj.p < 0.0001. B) Typical traces from L2/3 pyramidal neurons in S1 cortex in WT (top row), cKO/+ (middle row) and cKO/N mice (bottom row). Left to right: traces at current steps ~200pA (left), ~400pA (middle), and ~600pA (right). C) Spike count (number of evoked spikes) per current step for WT (n=8), cKO/+ (n=8) and cKO/N mice (n=7). The average ± SD is shown for all data points. P=0.0003, F (2, 177)=8.420 for genotype, two-way ANOVA. D) Dot plots showing the maximal spike frequency obtained from the same recordings. p=0.0007 by one-way ANOVA. Each dot represents one animal. The average ± SD is shown for all data points. See also Figure S5.

Figure 6.. The lead SSO upregulates SYNGAP1 expression in human iPSCs and iPSC-derived neurons.

A) Schematic illustration of the SSO design targeting the SYNGAP1 A3SS. B) RT-PCR results showing the screening of SSOs in iPSCs (PGP1-iNGN). One biological sample per lane. C-E) Identification of the lead SSO in iPSC-derived neurons. RT-PCR results (C) and quantification (D) showing that CH937 suppresses SYNGAP1 A3SS in iPSC-derived neurons. Q-PCR results (E) showing that the productive SYNGAP1 transcript was upregulated in CH937-treated human iPSC-derived neurons. F-K) The lead SSO suppresses SYNGAP1 A3SS in two additional human iPSC lines. RT-PCR results (F, I) and quantification (G, J) showing that CH937 suppressed SYNGAP1 A3SS in human iPSCs (NA19101 and 28126). Q-PCR results (H, K) showing that the CH937 significantly increased the productive SYNGAP1 transcript levels in human iPSC lines. L-N) The lead SSO suppresses SYNGAP1 A3SS-NMD in SYNGAP1 patient-derived iPSCs. RT-PCR results (L) and quantification (M) showing that CH937 suppressed SYNGAP1 A3SS in SYNGAP1 patient-derived iPSCs (333del, Lys114SerfsX20). Q-PCR results (N) showing that the CH937 significantly increased the productive SYNGAP1 mRNA level. O-Q) Application of CH937 to human iPSC-derived cerebral organoids (O) led to increased SYNGAP1 protein expression (83%±28%, P-Q). p<0.05 by unpaired t-test. See also Figure S6.