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

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

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

Table_1_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.xlsx

<p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

Image_2_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.TIF

<p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

<i>SHANK</i> variants in patients with ASD and controls.

<p>Coding-sequence variants identified only in patients with ASD (upper panel), shared by patients and controls (lower panel and underlined), and present only in controls (lower panel). Truncating variants are indicated in red. The variants predicted as deleterious or benign are indicated in orange and green, respectively. Coding-sequence variants with a proven <i>in vitro</i> functional impact are indicated with black stars. Conserved domains are represented in color: SPN (yellow), Ankyrin (red), SH3 (orange), PDZ (blue) and SAM (green).</p

Scatter plots of the intellectual quotient and the Autism Diagnostic Interview-Revised (ADI-R) scores of the patients with ASD screened for <i>SHANK1-3</i> mutations.

<p>Mutations in <i>SHANK1-3</i> are associated with a gradient of severity in cognitive impairment. <i>SHANK1</i> mutations were reported in patients without ID (green dots). <i>SHANK2</i> mutations were reported in patients with mild ID (orange dots). <i>SHANK3</i> mutations were found in patients with moderate to severe deficit (red dots). Black dots correspond to the patients enrolled in the PARIS cohort screened for deleterious <i>SHANK1-3</i> mutations (n = 498). In addition to the PARIS cohort <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Pinto1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>, three patients with a <i>SHANK1</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a> and two patients with a <i>SHANK2</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a> were included in the scatter plot. A high score of the ADI-R is associated with a more severe profile. The threshold of the “Social”, “Verbal”, “Non-Verbal” and “Repetitive Behavior” Scores are 10, 8, 7 and 3, respectively.</p

Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.

<p>The frequency of mutation in patients and control individuals was calculated from the total cohort (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen-1004580-t001" target="_blank">Table 1</a>). The frequency of mutation in patients with normal IQ (IQ>70) and low IQ (IQ<70) were calculated for the patients with available IQ scores (copy-number variants for all SHANK: nASD with IQ>70 = 1 638 & nASD with IQ<70 = 917; SHANK1 coding-sequence variants: nASD with IQ>70 = 354 and nASD with IQ<70 = 278; SHANK2 coding-sequence variants: nASD with IQ>70 = 335 & nASD with IQ<70 = 344; SHANK3 coding-sequence variants: nASD with IQ>70 = 667 & nASD with IQ<70 = 611). The mean IQ and standard deviation was given only for patients carrying truncating or <i>de novo</i> mutations. The black star indicates that Schmeisser et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Bonaglia1" target="_blank">[21]</a> found an increase in NMDA currents, while Won et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Betancur2" target="_blank">[22]</a> found a decrease in NMDA currents in two independent SHANK2 knock-out mice.</p><p>Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.</p

Prevalence and meta-analysis of coding-sequence variant studies in ASD.

<p>A. The prevalence and the confidence interval from a set of single coding-sequence variant studies, and the pooled prevalence and the confidence interval of the meta-analysis. The prevalence is indicated by circles in red, pink, purple and black for “ASD all” (all ASD patients), “ASD IQ<70” (patients with ID; IQ<70), “ASD IQ>70” (patients with normal IQ), and “CTRL” (controls), respectively. Three categories are used to study the prevalence of coding-sequence variants in ASD and controls: all or “A” (all mutation), Damaging or “D” (damaging missense mutation; score obtained from polyphen-2), and Truncating or “T” (mutation altering SHANK protein). The plotted circles are proportional to the corresponding sample size. B. Meta-analysis of coding-sequence variant studies altering <i>SHANK</i> genes. For each study, the Odds ratio and confidence interval is given. Each meta-analysis is calculated using inverse variance method for fixed (IV-FEM) and random effects (IV-REM). The statistics measuring heterogeneity (Q, I² and Tau²) are indicated. The number under the scatter plot correspond to independent studies: 1 = “This study”, 2 = “ Sato et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a>”, 3 = “Berkel et al. (2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a>”, 4 = “Leblond et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>”, 5 = “Boccuto et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Boccuto1" target="_blank">[17]</a>”, and 6 = “[This Study and Durand et al. 2007 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>]”, 7 = “[Gauthier et al. (2009–2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier2" target="_blank">[47]</a>]”, 8 = “Moessner et al. (2007) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Moessner1" target="_blank">[13]</a>”, 9 = “Schaff et al. (2011) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Schaaf1" target="_blank">[35]</a>”. IV, Inverse Variance; FEM, Fixed Effect Method; REM, Random Effect Method; OR, Odds Ratio; CI, Confidence Interval; IQ, Intellectual Quotient; CNV, Copy Number Variant.</p