A simple cell-based assay reveals that diverse neuropsychiatric risk genes converge on primary cilia

Abstract

Human genetic studies are beginning to identify a large number of genes linked to neuropsychiatric disorders. It is increasingly evident that different genes contribute to risk for similar syndromes and, conversely, the same genes or even the same alleles cross over traditional diagnostic categories. A current challenge is to understand the cellular biology of identified risk genes. However, most genes associated with complex neuropsychiatric phenotypes are not related through a known biochemical pathway, and many have an entirely unknown cellular function. One possibility is that diverse disease-linked genes converge at a higher-level cellular structure. The synapse is already known to be one such convergence, and emerging evidence suggests the primary cilium as another. Because many genes associated with neuropsychiatric illness are expressed also outside the nervous system, as are cilia, we tested the hypothesis that such genes affect conserved features of the primary cilium. Using RNA interference to test 41 broadly expressed candidate genes associated with schizophrenia, bipolar affective disorder, autism spectrum disorder and intellectual disability, we found 20 candidates that reduce ciliation in NIH3T3 cells when knocked down, and three whose manipulation increases cilia length. Three of the candidate genes were previously implicated in cilia formation and, altogether, approximately half of the candidates tested produced a ciliary phenotype. Our results support the hypothesis that primary cilia indeed represent a conserved cellular structure at which the effects of diverse neuropsychiatric risk genes converge. More broadly, they suggest a relatively simple cell-based approach that may be useful for exploring the complex biological underpinnings of neuropsychiatric disease.

Figure 1. Schematic of the siRNA screening strategy.

The work flow was organized into two rounds of screening. If both duplexes produced cilia loss and knocked down gene expression as assessed by qRT-PCR, the gene was scored as a hit. If only one duplex was effective, and only this duplex knocked down expression, a second round was pursued as indicated. All other outcomes were scored as a negative. A similar strategy was pursued for increased ciliation and, for genes whose manipulation produced this phenotype, additional analysis of ciliary length was conducted as described in the text.

Figure 2. Screening candidate genes encoding DISC1 interactors.

(A) Representative example fluorescence micrographs showing the analysis of ciliation. NIH3T3 cells were fixed 72 hours after RNA transfection, nuclei were identified by DAPI stain (top row, shown in blue) and primary cilia were detected by acetylated tubulin immunoreactivity (middle row, shown in red). Arrow indicates an example. Lower row shows merged images. The inset shows the example of a typical cilium, as indicated by arrow, at higher magnification. Scale bar, 10 um. (B) Compiled results from the screen of candidate genes. Normalized percent ciliation was determined for cells transfected with each RNA duplex and compiled across multiple experiments (n = 5) and specimens (>150 cells/condition/experiment), as described in Materials and Methods. First three bars from the left (coded in gray) indicate negative (scrambled duplex) and positive controls (IFT88 and PCM-1 knockdown). The degree of cilia depletion produced by PCM-1 depletion was chosen as the threshold for scoring a positive ciliation defect for the unknowns (blue line). (C) Relative transcript level observed in siRNA-transfected cells was determined by qRT-PCR analysis and normalized to transcript level in cells transfected with control (scrambled) duplex. Each RNA duplex is indicated in the abscissa, and the mean and standard deviation from triplicate determinations of transcript level are plotted on the ordinate as ‘% RNA remaining’. Blue bars indicate duplexes that met criteria both for reduced ciliation and produced gene knockdown. Duplexes indicated in red text represent ‘splits’ that were carried into a second round for screening with two additional duplexes. SYNE1 passed the second round and CEP170 did not. (D) Summary list of genes scored positive for ciliation defect.

Figure 3. Screening rare variants.

(A) Compiled ciliation results for the candidates listed in the abscissa, carried out and indicated in the figure as described in Fig. 2 B. (B) Endogenous expression and candidate gene knockdown assessed by qRT-PCR analysis. All of the siRNA duplexes that produced a ciliation defect were tested for knockdown (blue bars). Green bars indicate duplexes that increased % ciliation, and these were also tested by qRT-PCR. Correlative qRT-PCR analysis was also carried out on all ‘splits’ to determine if the duplex not producing a ciliation defect also failed to knock down expression. Those splits for which this was true (black bars with duplex identity indicated in red) were carried into the second round (the same strategy as described in Fig. 2). (C) Summary list of hits characterized by decreased % ciliation indicated in blue, and hits characterized by increased % ciliation indicated in green.

Figure 4. Screening common variants.

(A) Compiled ciliation results for the candidates listed in the abscissa, carried out and indicated in the figure as described in Fig. 2 B. (B) Endogenous expression and candidate gene knockdown assessed by qRT-PCR analysis. The color coding scheme for scoring of duplexes is the same as in previous figures. All duplexes supporting a hit were tested by qRT-PCR and a subset of duplexes targeting genes scored as negatives were spot-checked as indicated. (C) Summary list of hits characterized by decreased % ciliation indicated in blue, and hits characterized by increased % ciliation indicated in green.

Figure 5. Hits that increase % ciliation also increase cilia length in NIH3T3 cells.

(A) Example of ciliary lengthening produced by MIR137 mimic. Cells were transfected with scrambled siRNA control (‘siControl’, left column) or MIR137 mimic (right column). Example images of acetylated tubulin immunoreactivity (top row) and DAPI fluorescence (second row from top) are shown. Merged images are displayed in the third row and, the indicated areas at 7× higher magnification in the fourth row. Scale bar, 10 µm. (B) Cilia length was measured in randomly selected fields as described in Materials and Methods. Average cilium length for the indicated experimental conditions is indicated by the bar, and error bar indicate S.E.M. (scrambled siRNA Control, N = 45; MIR137 Mimic, N = 43; siCCDC18-1, N = 31; siCCDC18-2, N = 46; siFOXP1-1, N = 38; siFOXP1-2, N = 27). ***, P<0.0001 by two-tailed unpaired Student’s t-test.