Expression pattern of hnRNPK in the developing CNS.

<p>(A) In situ hybridization of hnRNPK mRNA during rat brain development. At embryonic time points (1d), the mRNA of hnRNPK can easily be detected in all areas of the developing brain including the spinal cord. At later stages of maturation (horizontal sections, 3d-adult), the expression levels decrease and become more and more restricted to the cortex (Co), the hippocampal formation (Hc) and the granular layer of the cerebellum (Ce). (B) Immunohistochemical detection of hnRNPK in rat brain sagittal sections. At early time points of brain development (3d), a predominant nuclear labeling of hnRNPK can be detected in nearly all neurons. Again, cortex, hippocampus and cerebellum are most strongly labeled. At later time points, differences in spatial expression become even more prominent and intense staining is especially seen in granule cells of the cerebellum (Ce) and the dentate gyrus as well as in the CA1-4 regions of the hippocampus. In the cortex (Co), the staining intensity diminishes at later stages and only some scattered neurons in deeper cortical layers remain positive for hnRNPK. (C) Analysis of hnRNPK expression in different tissues and organs. hnRNPK is readily detectable in brain, liver and skeletal muscle (SM) while heart as well as lung, kidney, stomach and duodenum are almost devoid of detectable hnRNPK. (D) The analysis of hnRNPK expression in different brain areas (9d). A comparable expression profile of the protein is seen in the prefrontal cortex (PFC), the parietal cortex (Co), hippocampus (Hc), striatum (Str), thalamus (Tha), mesencephalon (Mes), and in the brain stem (Bst) while cerebellum (Ce) shows highest expression levels. Loading control: Actin. (E) Western blot analysis of time dependent hnRNPK expression in selected brain regions. hnRNPK detection in cerebellum, cortex and hippocampus at different stages of maturation shows that in all regions investigated, the strong signal at 8 to 28d becomes slightly weaker at 3 M. Loading control: Actin. Scale bars are as indicated.</p

Downregulation of hnRNPK mimics the “Abi-1 depletion phenotype” in neurons.

<p>(A) hnRNPK-RNAi knockdown in NIH3T3 cells with a construct targeting the 3′UTR-region of hnRNPK. (I) hnRNPK is endogenously expressed in NIH3T3, HeLA and Cos7 cells and is detectable in cultured hippocampal neurons (DIV21). (II) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, the cells were fixed and stained with an antibody against hnRNPK. Only untransfected cells in close proximity to the RNAi-transfected cell in the upper right are immunopositive for hnRNPK, with a predominant distribution of the protein in the nucleus. (III) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, protein expression of hnRNPK is markedly suppressed as confirmed by Western blotting compared to vector control transfected cells. (IV) Double transfections using two different hnRNPK-RNAi constructs, one targeting the 3′UTR of the hnRNPK sequence and one targeting the coding sequence of hnRNPK together with an hnRNPK-Myc-construct which is resistant against RNAi due to 4 nucleotide exchanges in the RNA leading to an unaltered amino acid sequence. The staining against the Myc-tag shows a decreased protein level solely when using the non-resistant construct together with the RNAi targeting the CDS (coding sequence). (B) Neuronal transfection of Abi-1-RNAi and hnRNPK-RNAi constructs. (I) In contrast to the control vector (pSuper), transfection of RNAi constructs resulted in an obvious change of neuronal morphology. The downregulation of hnRNPK as well as Abi-1 is leading to an extended and extremely branched dendritic tree. (II) The number of branching points within the dendritic compartment is significantly upregulated in both RNAi groups compared to the control transfection. (III) The analysis of the dendritic tree shows a significant shift of dendrites towards small, filopodia-like tertiary dendrites. Scale bars are as indicated.</p

Downregulation of hnRNPK reduces the number of mature synaptic contacts in hippocampal neurons.

<p>(AI,II) Decrease in mature synaptic contacts after Abi-1- or hnRNPK-RNAi knockdown. There is a significant decrease in the number of mature synapses with bassoon-positive presynaptic counterparts in the neurons transfected with Abi-1- or hnRNPK-RNAi constructs compared to vector control. At the same time, a more filopodia-like phenotype of the dendritic tree can be observed after RNAi knockdown of Abi-1 or hnRNPK. The RNAi resistant construct is able to rescue the observed reduction. Scale bars are as indicated.(BI,II) Analysis of the reduction of excitatory synapses by using ProSAP2/Shank3 antibodies that label postsynaptic densities (PSDs) reveals a comparable reduction of postsynaptic specializations that is also rescued by the RNAi resistant construct.</p

hnRNPK interacts with Abi-1 via its KH2 domain.

<p>(A) Yeast two-hybrid screen. The full length Abi-1 cDNA was cloned as bait to screen a human fetal brain cDNA-library for putative interaction partners. 9 independent partial C-terminal hnRNPK clones were identified and retested for interaction by a yeast two-hybrid assay. Results are shown for the longest (aa248–464) and shortest (aa267–464) prey clone. hnRNPK is a 464 aa long protein that codes for several specific domains: N-terminal NLS, <i>nuclear localization signal</i>, KH1-KH3, <i>K homology domains 1–3 (light grey)</i>; KI, <i>K interaction domain (black)</i>; KNS, <i>K nuclear shuttling signal</i>. Abi-1 (476 aa) codes for the following domains: WAB, <i>WAVE binding domain</i>; SNARE, HHR, <i>homeobox homology region</i>; PP, <i>proline rich domain</i>; SH3 <i>src homology 3 domain</i>. (B) Schematic illustration of the Abi-1 and hnRNPK clones (and abbreviations) that have been used for further experiments. (C) The hnRNPK KH2 domain colocalizes with Abi-1.Several partial GFP- or Myc-tagged hnRNPK and Abi-1 clones were coexpressed in Cos7 cells to identify the interacting subdomains of the two proteins. In single transfection experiments, hnRNPK full length protein predominantly localizes to the nucleus, whereas Abi-1 shows a typical cytoplasmic staining pattern. When coexpressed, both proteins are localized in identical dotted structures (I). In contrast, the K1-recombinant protein alone is restricted to the nucleus and does not colocalize with Abi-1 after cotransfection (II). K2 fusion protein readily colocalizes with full-size Abi-1 in the cytoplasm (III). As shown for K1, K3 also shows no colocalization with Abi-1 (IV). The cotransfection of hnRNPK K2 with Abi-1 missing the SH3 domain (AbiΔSH3) results in no colocalization (V), the expression of Abi-1 SH3 domain alone (AbiSH3), however, gives rise to a perfect overlay in the perinuclear region (VI). (D) Coimmunoprecipitation experiments with overexpressed and endogenous Abi-1 and hnRNPK proteins. Plasmids encoding full-size hnRNPK-GFP and Abi-1-Myc were cotransfected in Cos7 cells and Abi-1-Myc was immobilized using anti-Myc microbeads loaded on a column. Protein-complexes then were eluted, separated by SDS-Page and hnRNPK-GFP (size 95 kDA) was detected by immunoblot using a specific anti-GFP antibody (I). As controls, beads loaded with lysate only (ctrl) and the input lysate were used. (II) Cos7 cells were transfected with partial hnRNPK-coding constructs K1-GFP (KH1 domain), K2-GFP (KH2 and KI domain), K3-GFP (KH3 domain) and K-full-GFP (full length) as GFP-fusion proteins. The correct expression of the hnRNPK constructs was controlled by using an anti-GFP antibody and a commercial anti-hnRNPK antibody that could detect the GFP fusion protein as well as the endogenous hnRNPK in the lysate (95 kDA and 65 kDA). Moreover, the commercial antibody detects the K2 construct. The correct expression and antibody specificity of the Abi-1-Myc construct was tested by cotransfection with truncated hnRNPK constructs and subsequent immunoblotting with an anti-Myc antibody. Afterwards, precipitation was performed with GFP-tagged microbeads after cotransfection of Abi-1-Myc and hnRNPK constructs. The precipitates were subjected to immunostaining with an anti-Myc antibody. The Abi-1-Myc protein could only be detected within hnRNPK-K2-GFP precipitate but not within K1-GFP and K3-GFP precipitate or within the GFP-only and/or negative controls. (III) <i>Vice versa</i> experiments were done by coimmunoprecipitations using lysates of Cos7 cells cotransfected with a combination of full length hnRNPK-Myc (K-Myc) and AbiΔSH3-GFP or AbiSH3-GFP, respectively. The immunoprecipitation was performed using antibodies directed against GFP and immunoblot-detection was performed using anti-hnRNPK antibodies showing that expression of the Abi-1 SH3 domain is a prerequisite for protein binding. (IV) The hnRNPK antibody was used to precipitate the protein complex from brain lysate as well as from the synaptosomal fraction. In the Western blot, an antibody against Abi-1 could readily detect its antigen in the precipitate. As positive control, brain lysate or synaptosomal material was used (Input lane: 4% of the total lysate used for immunoprecipitation), a negative control was performed with unspecific IgG (ctrl IgG). Scale bars are as indicated.</p

Characterization of the functional impact of <i>SHANK2</i> mutations in cultured neuronal cells.

<p>A. The colocalization of <i>ProSAP1A/SHANK2</i>-EGFP (postsynaptic marker) and Bassoon (presynaptic marker) indicated that the mutations did not disturb the formation of SHANK2 clusters at excitatory synapses along the dendrites. B. The quantification of synapse density was performed on 20 transfected hippocampal neurons per construct from at least three independent experiments. The majority of the <i>ProSAP1A</i> variants affecting a conserved amino acid among SHANK proteins reduced significantly the synaptic density compared with the variants that affect amino acid non conserved among SHANK proteins (Mann-Whitney U-test: n_WT = 20, n_mut = 20; U_S557N = 82.5, p_S557N = 0.001; U_R569H = 124, p_R569H = 0.04; U_L629P = 149, p_L629P = 0.17; U_V717F = 114, p_V717F = 0.02; U_A729T = 73, p_A729T = 0.000; U_K780Q = 154, p_K780Q = 0.221; U_R818H = 108, p_R818H = 0.012; U_A822T = 154.5, p_A822T = 0.224; U_V823M = 129, p_V823M = 0.056; U_Y967C = 134, p_Y967C = 0.076; U_G1170R = 78, p_G1170R = 0.001; U_R1290W = 142, p_R1290W = 0.121; U_Q1308R = 162, p_Q1308R = 0.314; U_D1535N = 97, p_D1535N = 0.005; U_P1586L = 137, p_P1586L = 0.910; U_L1722P = 79, p_L1722P = 0.001, *p<0.05, **p<0.01, ***p<0.001). <b>C.</b> Effect of the variants on synaptic density. The y-axis represents −log P compared to WT (P obtained with Mann-Whitney test). After Bonferroni correction for 16 tests, only P values<0.003 were considered as significant. Variants represented in red were specific to ASD, in orange were shared by ASD and controls, and in green were specific to the controls. Open circles and filled circles represent non conserved and conserved amino acids, respectively. Prim, primary; second, secondary.</p

Characterization of CNVs in three patients carrying a <i>de novo</i> deletion of <i>SHANK2</i>.

<p>Paternally or maternally inherited CNVs are indicated by squares and circles, respectively. <i>De novo</i> CNVs are indicated by stars. Deletions and duplications are indicated in red and blue, respectively. CNVs hitting exons or only introns are filled with grey and white, respectively. Squares and circles within star represent <i>de novo</i> CNV of paternal or maternal origin; circles within squares represent CNV inherited by father or mother. ABCC6, ATP-binding cassette, sub-family C, member 6 pseudogene 2; ADAM, ADAM metallopeptidase; AMY1, amylase (salivary); AMY2A, amylase (pancreatic); ARHGAP11B, Rho GTPase activating protein 11B; CAMSAP1L1, calmodulin regulated spectrin-associated protein 1-like 1; CHRNA7, cholinergic receptor, nicotinic, alpha 7; CNTN4, contactin 4; CTNNA3, catenin (cadherin-associated protein), alpha 3; CYFIP1, cytoplasmic FMR1 interacting protein 1; DUSP22, dual specificity phosphatase 22; GALM, galactose mutarotase; GCNT2, glucosaminyl (N-acetyl) transferase 2; GOLGA, golgi autoantigen, golgin subfamily a; GSTT1, glutathione S-transferase theta 1; HLA-DRB, major histocompatibility complex, class II, DR beta; LAMA4, laminin, alpha 4; NIPA, non imprinted in Prader-Willi/Angelman syndrome; NLGN1, neuroligin 1; NME7, non-metastatic cells 7; OR, olfactory receptor; PCDHA, protocadherin alpha; RFPL4B, ret finger protein-like 4B; RHD, Rh blood group, D antigen; SFMBT1, Scm-like with four mbt domains 1; SHANK2, SH3 and multiple ankyrin repeat domains 2; SMC2, structural maintenance of chromosomes 2; TNS3, tensin 3; TUBGCP5, tubulin, gamma complex associated protein 5; UGT2B17, UDP glucuronosyltransferase 2 family, polypeptide B17.</p

Genetic alterations identified in the control subject SWE_Q56_508.

<p>A. <i>SHANK2</i> splice mutation (IVS22+1G>T) detected in a Swedish female control, SWE_Q56_508. The mutation altered the donor splicing site of exon 22 and led to a premature stop in all <i>SHANK2</i> isoforms except for the <i>AF1411901</i> isoform, where it altered the protein sequence (G263V). B. CNVs in the same individual altering <i>LOC339822</i>, <i>SNTG2</i>, <i>PXDN</i> and <i>MYT1L</i>. The two close duplications span 264 kb and 245 kb on chromosome 2 and altered <i>LOC339822</i> and <i>SNTG2</i>, and <i>PXDN</i> and <i>MYT1L</i>, respectively. Dots show the B allele frequency (BAF; in green), Log R ratio (LRR; in red), and QuantiSNP score (in blue). Lower panel: all CNVs listed in the Database of Genomic Variants (DGV) are represented: loss (in red), gain (in blue), gain or loss (in brown). H, homer binding site; D, dynamin binding site; C, cortactin binding site.</p

<i>SHANK2</i> mutations in patients with ASD.

<p>A. A heterozygous deletion of <i>SHANK2</i> was identified with the Illumina Human 1M-Duo SNP array in a patient with autism (AU038_3). The deletion spans 421 kb on chromosome 11q13.3, covers twelve exons of the human <i>SHANK2</i> and is not present in the parents. Each dot shows Log R Ratio (LRR; in red) and B allele frequency (BAF; in green). QuantiSNP score is represented with a blue line and indicates the deletion size. B. Location of the CNV and sequence variants (from this study and Berkel <i>et al.</i> 2010) along the SHANK2 protein: in red the variations specific to ASD, in orange the variations shared by ASD and controls and in green the variations specific to controls <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-Berkel1" target="_blank">[26]</a>. The breakpoints of the <i>SHANK2</i> deletion in AU038_3 are represented with a dotted line on the protein. Stars indicate the variants affecting conserved amino acids. C. A total of 40 variants were identified and variants affecting conserved amino acids in other SHANK proteins are enriched in patients with ASD (n_conserved = 12 and n_{non-conserved} = 3) compared with controls (n_conserved = 6 and n_{non-conserved} = 11) (Fisher's exact test 1-sided, P = 0.013, OR = 6.83, 95% IC = 1.19–53.40). D. The percentage of carriers of <i>SHANK2</i> variants in patients with ASD and Controls. Variants affecting a conserved amino acid among the SHANK proteins are enriched in patients with ASD (n_conserved = 29 and n_{non-conserved} = 822) compared with controls (n_conserved = 16 and n_{non-conserved} = 1074) (Fisher's exact test 1-sided, P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). Open squares and filled squares represent the non-conserved and conserved amino acids, respectively. ANK, Ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, postsynaptic density 95/Discs large/zona occludens-1 homology domain; SAM, sterile alpha motif domain; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The proline-rich region is represented as a horizontal gray line.</p

Genomic structure, isoforms, and expression of human <i>SHANK2</i>.

<p>A. Genomic structure of the human <i>SHANK2</i> gene. Transcription of <i>SHANK2</i> produces four main mRNA from three distinct promoters: <i>SHANK2E</i> (<i>AB208025</i>), <i>ProSAP1A</i> (<i>AB208026</i>), <i>ProSAP1</i> (<i>AB208027</i>) and <i>AF141901</i>. There are three translation starts: in exon 2 for <i>SHANK2E</i>, in exon1b for <i>ProSAP1A</i>, and in exon1c for <i>ProSAP1</i> and <i>AF141901</i>; and two independent stop codons: in exon 22b for <i>AF141901</i> and in exon 25 for <i>SHANK2E</i>, <i>ProSAP1A</i> and <i>ProSAP1</i>. Conserved domains of protein interaction or protein binding site are represented in color: ANK (red), SH3 (orange), PDZ (blue) and SAM (green), H (pink), D, (dark blue) and C (purple). Black stars identify the alternative spliced exons (‘brain-specific exons’ in turquoise: 19, 20 and 23). B. RT-PCRs of <i>SHANK2</i> isoforms on RNA from different human control tissues (Clontech), and different brain regions of four controls (2 males and 2 females). The amplified regions specific to each isoform of <i>SHANK2</i> are indicated by gray boxes. C. Alternative splicing of human <i>SHANK2</i>; exons 19, 20 and 23 are specific to the brain. ANK, ankyrin; SH3, Src homology 3; PDZ, PSD95/DLG/ZO1; SAM, sterile alpha motif; He, heart; Li, liver; B, brain; SM, skeletal muscle; Pl, placenta; K, kidney; Lu, lung; Pa, pancreas; FC, frontal cortex; Hi, hippocampus; TC, temporal cortex; T, thalamus; OC, occipital cortex; Ce, cerebellum; Cx, whole cortex; BLCL, B lymphoblastoid cell lines; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The ages of the two males and the two females studied were 74, 42, 55, and 36 years with a post-mortem interval of 10, 21, 24, and 2 h, respectively.</p

Inherited 15q11–q13 CNVs identified in three ASD patients carrier of a <i>de novo</i> SHANK2 deletion.

<p>Deletions (del) and duplications (dup) are indicated in red and blue, respectively. Paternally and maternally imprinted genes are indicated in yellow and pink, respectively. Genes altered by the CNVs are indicated in blue or red. The bottom part of the figure indicates the location of the deletions/duplications previously associated with neuropsychiatric disorders <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-Miller1" target="_blank">[43]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-deKovel1" target="_blank">[61]</a>. BP, breakpoint; Inh_M, inherited by mother; Inh_F, inherited by father; AS, Angelman syndrome; ASD, Autism spectrum disorders; ADHD, attention deficit-hyperactivity disorder; BP, bipolar disorder; DD: developmental delay; DBD, disruptive behavior disorder; EPI, epilepsy; GAD, generalized anxiety disorder; OCD, obsessive-compulsive disorder; ID, intellectual disability; PWS, Prader-Willi syndrome; SCZ, schizophrenia.</p