Research articleAnalysis of gene expression in Ca2+-dependent activator protein for secretion 2 (Cadps2) knockout cerebellum using GeneChip and KEGG pathways
Introduction
The Ca2+-dependent activator protein for secretion (CADPS, CAPS) family is involved in dense-core vesicle (DCV) exocytosis [2], [20], [33], and two CAPS family members, CAPS1 [2] and CAPS2 [24], [30], have been identified in mammals. Many previous studies have suggested that CAPS1 is involved in the secretion of catecholamines (e.g., norepinephrine) [2], [33], neuropeptides (e.g., neuropeptide Y) [5], and peptide hormones (e.g., insulin) [29]. CAPS1 has also been shown to play a role in the priming of DCV exocytosis by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) [6], [13]. However, recent studies using knockout (KO) or knockdown (KD) approaches have led to fresh debates concerning the involvement of CAPS1 in the priming of synaptic vesicle (SV) exocytosis [9], the vesicular loading of catecholamine or serotonin [3], [28] and DCV biosynthesis [26]. We previously reported that CAPS2 is involved in the secretion of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the mouse cerebellum [24] and that Capds2 KO mice not only have histological abnormalities similar to those reported in the cerebellum of autistic patients [1], [4] but also exhibit autistic-like behaviors, including impaired social interaction, hyperactivity, an abnormal sleep–wake rhythm, and increased anxiety in unfamiliar environments [23], [27]. We also identified increased expression of a rare Capds2 splice variant in autism patients that specifically lacks exon 3 and that is not transported to axons when exogenously expressed in mouse cortical neurons, suggesting a possible association between Capds2 and susceptibility to autism [21], [27]. We developed a mouse line expressing exon 3-deleted CAPS2, which displayed autistic-like behaviors, such as increased anxiety in an unfamiliar environment, impaired social behavior, and a disrupted circadian rhythm [25].
Autistic patients exhibit several abnormalities in cerebellar morphology and function, including hypoplasia of the cerebellar lobules [4], [18], and impaired eye movement [32] and motor coordination [14]. In this study, to elucidate whether the loss of CAPS2 function in mice leads to abnormal cerebellar gene expression, we examined the Capds2 KO mouse cerebellum using a GeneChip microarray. The results revealed a gross impairment in the neurotrophin signaling pathway in Capds2 KO mice.
Section snippets
Animals
All experimental protocols were approved by the Institutional Animal Care and Use Committee by Gunma University and by the Tokyo University of Science. All efforts were made to minimize the number of animals used and their suffering.
RNA and cDNA preparation
Cerebella were dissected from post-natal day 21 (P21) wild-type (WT) and Cadps2 KO mice after anesthesia with diethyl ether. Total RNA samples from each genotype were prepared from pooled cerebella using an RNeasy RNA extraction kit (Qiagen, Chatsworth, CA, USA).
Results and discussion
To examine gene expression in wild-type (WT) and Cadps2 KO mouse cerebella, we performed a genome-wide parallel monitoring of gene expression in P21 mice using an oligonucleotide-based microarray system, the Affymetrix U74 subA GeneChip, on which 12,422 genes were represented. Significant differential expression (p < 0.05) was observed in 1211 of 22,690 (5.34%) genes represented on the chip. Neurons constitute approximately 82% of cerebellar cells, and approximately 99% of cerebellar neurons in
Conclusions
GeneChip analysis of Cadps2 KO cerebellum showed that expression of many secretory protein genes was affected by Capds2 deficiency. In addition, the expression of some genes involved in neurotrophin signaling was significantly decreased in the Capds2 KO mouse cerebellum. These results suggest that deregulated gene expression caused by loss of Capds2 may cause developmental deficits and/or pathological symptoms, resulting in autistic-like phenotypes.
Acknowledgements
This study was supported by Grants-in-Aid for Scientific Research from the Japan Intractable Diseases Research Foundation, the SENSHIN Medical Research Foundation, the Takeda Science Foundation, the Sumitomo Foundation, the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT; grant number, 23110524), the Japan Society for the Promotion of Science (JSPS; grant number, 25430061), the Program to Disseminate Tenure Tracking System of MEXT granted to Gunma University, and
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