Neuropsychological Deficits in Mice Depleted of the Schizophrenia Susceptibility Gene <i>CSMD1</i>

<div><p>Recent meta-analyses of schizophrenia genome-wide association studies (GWASs) have identified the <i>CUB and SUSHI multiple domains 1</i> (<i>CSMD1</i>) gene as a statistically strong risk factor. CSMD1 is a complement control-related protein suggested to inhibit the classical complement pathway, being expressed in developing neurons. However, expression of <i>CSMD1</i> is largely uncharacterized and relevance for behavioral phenotypes is not previously demonstrated. Here, we assess neuropsychological behaviors of a <i>Csmd1</i> knockout (KO) mouse in a selection of standard behavioral tests. Deregulation of neuropsychological responses were observed in both the open field and the elevated plus maze tests, in which KO mice spent 55% and 33% less time than WT littermate mice in open areas, respectively. Altered behaviors were also observed in tail suspension and to higher acoustic stimuli, for which <i>Csmd1</i> KO mice showed helplessness and moderate increase in startle amplitude, respectively. Furthermore, <i>Csmd1</i> KO mice also displayed increased weight-gain and glucose tolerance, similar to a major phenotype of the metabolic syndrome that also has been associated to the human <i>CSMD1</i> locus. Consistent with a role in the control of behaviors, <i>Csmd1</i> was found highly expressed in the central nervous system (CNS), and with some expression in visceral fat and ovary, under tissue-specific control by a novel promoter-associated lncRNA. In summary, disruption of <i>Csmd1</i> induces behaviors reminiscent of blunted emotional responses, anxiety and depression. These observations suggest an influence of the <i>CSMD1</i> schizophrenia susceptibility gene on psychopathology and endophenotypes of the negative symptom spectra.</p></div

Behavior of <i>Csmd1</i> KO and WT mice to acoustic stimuli and tail suspension.

<p>(A) Startle responses of <i>Csmd1</i> KO mice in response to acoustic stimuli in the range of 80–120 dB, as compared to WT mice. The startle baseline was similar in male and female WT mice. A marginal increase in startle responses could be observed for higher acoustic stimuli in both genders of <i>Csmd1</i> KO mice, reaching statistical significance when analyzing all mice together (genotype-group interaction <i>P</i>-value<0.05). (B) There was no difference in amplitude response between KO and WT mice in the degree of inhibition after pre-pulse inhibition. (C) Tail suspension test demonstrated longer accumulated time immobile for KO mice as compared WT mice (P-value<0.05). Borderline statistical significance was observed for male <i>Csmd1</i> KO mice as compared to WT mice (P-value = 0.1).</p

<i>Csmd1</i> RNA and protein expression in <i>Csmd1</i> knock-out and wild-type mice.

<p>(A) Schematic representation of the KO-strategy. A 1 kb genomic region (white lines) of exon1/intron1 was replaced with a selection cassette (grey box). (B) Expression of <i>Csmd1</i> mRNA measured by QPCR in an adult mouse tissue panel. <i>Csmd1</i> is predominantly expressed in brain tissues as compared to peripheral tissues. The highest expression level was identified in areas of the cortex. (C) Depletion of <i>Csmd1</i> mRNA in the cortex was documented by two exon-exon specific QPCR assays. Transcription of exon 1–2 was depleted, while about 20% residual expression could be observed when amplifying exon 32–33. KO mice lacked a protein band of expected size (389 KDa, arrow), as demonstrated by immunoblotting. Signals of lower molecular weight are indicated (a and b). (D) Mapping of RNA-seq reads to the <i>Csmd1</i> locus. RNA sequencing of cortex is shown for 4wild-type (green) and 4 <i>Csmd1</i> KO (red) mice (transcript scale: 0–150 reads). Coverage signals of modified nucleosomes (H3K4me3, H3K4me1 and H3K27Ac) and polymerase-2 binding profiles are shown for the mouse cortex. The 1 kb deleted sequence of <i>Csmd1</i> is highlighted in yellow (upper panel) and blue (lower panel). No RNA reads were mapped to the deleted genomic region in the KO mice. Abbreviations: Cx, cortex; VCx, visual cortex; FCx, frontal cortex; Hipp, hippocampus; Hyp, hypothalamus; Ob, olfactory bulb; Cer, cerebellum; Visc. Fat, visceral fat.</p

Behavior of <i>Csmd1</i> KO and WT mice in the open field arena.

<p>(A) Total time spent in the center of the arena is shown for KO and WT mice, respectively. (B) Sequential time bin analysis demonstrates that WT mice adapt to the test arena after the first time bin, while KO mice avoid the center throughout the test period. (C and D) Comparison of total path length and sequential bin analysis of path length demonstrate no statistically significant difference between KO and WT mice. Abbreviations: n.s., not significant; asterisk, statistically significant <i>P</i>-value<0.05; s, seconds.</p

Behavior of <i>Csmd1</i> KO and WT mice in the elevated plus maze.

<p>(A) Analysis of total time spent on open arms demonstrate significant less time for KO mice (N = 13) as compared to WT mice (N = 8). (B) Compiled tracks from all mice show that <i>Csmd1</i> KO mice avoid entering open arms, as opposed to WT mice traveling over the entire test arena. Abbreviations: asterisk, statistically significant (<i>P</i>-value<0.05); s, seconds.</p

Expression of <i>Csmd1</i> promoter-associated long non-coding RNA (<i>pas-lncRNA</i>) in mouse tissues.

<p>(A) Map of RNA sequencing reads aligned to the promoter region of <i>Csmd1.</i> Data from individual <i>Csmd1</i> KO (N = 4; red lines) and WT (N = 4; green lines) mice are shown. The novel <i>pas-lncRNA</i> RNA (black thick line) is expressed antisense to the <i>Csmd1</i> promoter sequence (transcript scale: 0–30 reads). (B) Heat map representing the relative expression level of <i>pas-lncRNA</i> and <i>Csmd1</i> mRNA in peripheral and CNS tissues of <i>Csmd1</i> KO and WT mice, respectively. Expression values of each transcript are calculated relative to their respective expression level in cortex of WT mice. <i>pas-lncRNA</i> expression was induced in the CNS but not in peripheral tissues of <i>Csmd1</i> KO mice. Fold change values (FC) and statistical significances are listed for each tissue. (C) Co-regulated expression of <i>Csmd1</i> and pas-lncRNA in the developing cortex and cerebellum (<i>Csmd1</i>:<i>pas-lncRNA</i> expression correlation coefficient, cortex: r² = 0.92). Y-axis represents relative expression level of RNA. X-axis indicates the postnatal day. Abbreviations: asterisk, t-test <i>P</i>-value<0.05; n.s.; not significant.</p

Analysis of object recognition memory in Csmd1 KO and WT mice.

<p>(A and B) Object contacts in the first and second trial of exposure to novel and familiar objects demonstrated increased contact counts for <i>Csmd1</i> KO mice (P-value<0.05). (B and C) Discrimination and preference analysis demonstrated no effect of <i>Csmd1</i> on object recognition. Abbreviation: n.s., not significant; asterisk, statistically significant (<i>P</i>-value<0.05).</p

Effect of subchronic olanzapine administration on appetite-regulating neuropeptides.

<p>Expression levels of the appetite-regulating neuropeptides in the arcuate nucleus following 5 days of treatment (b.i.d) with vehicle (ctrl), olanzapine with food restriction (olanz pair-fed) or olanzapine with free access to food (olanz ad libitum). Calculations are based on results from groups of rats (n = 8) from each treatment group fasted over night and killed in the morning on day 6. Representative images demonstrating the calculated differences were selected. Delineated areas are shown at higher magnification at the bottom. * P≤0.05 <i>vs.</i> vehicle. ** P≤0.01 <i>vs.</i> vehicle. *** P≤0.001 <i>vs.</i> vehicle.</p

Leptin, adiponectin and insulin plasma levels in control and olanzapine (<i>ad libitum</i> and pair-fed) treated rats.

<p>Data are expressed as mean±SEM.</p

Description of the samples.

<p> BPD, bipolar disorder; SCZ, schizophrenia; NCNG, Norwegian Cognitive NeuroGenetics; TOP, Norwegian Thematically Organized Psychosis; WTCCC, British Wellcome Trust Case Control Consortium; Danish, Danish sub-sample of the Scandinavian Collaboration on Psychiatric Etiology; PGC, Psychiatric Genomics Consortium.</p><p> indicates the cases and controls in the single-centre samples that are also included in the PGC multi-centre sample.</p