Analysis of Gene Expression in Ca2+-Dependent Activator Protein

Neuroscience Letters 639 (2017) 88–93

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Neuroscience Letters

jo urnal homepage: www.elsevier.com/locate/neulet

Research article

Analysis of gene expression in Ca -dependent activator protein for

secretion 2 (Cadps2) knockout cerebellum using GeneChip and KEGG pathways

a,∗ d c b

Tetsushi Sadakata , Yo Shinoda , Yasuki Ishizaki , Teiichi Furuichi

Advanced Scientiﬁc Research Leaders Development Unit, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan

Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan

Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan

Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan

h i g h l i g h t s

•

We analyzed gene expression in the Cadps2 KO cerebellum with a GeneChip microarray.

•

Signiﬁcant differential expression was observed in 5.34% of genes on the GeneChip.

•

Expression of many secretory proteins was changed in the Cadps2 KO cerebellum.

•

The neurotrophin signaling pathway was impaired in the Cadps2 KO cerebellum.

a r t i c l e i n f o a b s t r a c t

Article history: In the mouse cerebellum, Ca -dependent activator protein for secretion 2 (CADPS2, CAPS2) is involved

Received 14 October 2016

in regulated secretion from dense-core vesicles (DCVs), which contain neuropeptides including brain-

Received in revised form 9 December 2016

derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Capds2 knockout (KO) mice show

Accepted 28 December 2016

impaired cerebellar development in addition to autistic-like behavioral phenotypes. To understand the

molecular impact caused by loss of Capds2, we analyzed gene expression proﬁles in the Capds2 KO cerebel-

Keywords:

lum using a GeneChip microarray and the KEGG Pathway database. Signiﬁcant differential expression was

Cadps2

observed in 1211 of 22,690 (5.34%) genes represented on the chip. The expression levels of exocytosis-

GeneChip

KEGG related genes (Stx5a, Syt6), genes encoding secretory (Fgf2, Fgf4, Edn2) and synaptic proteins (Grin2b,

Neurotrophin Gabbr1), neurotrophin signaling-associated genes (Sos1, Shc1, Traf6, Psen2), and a gene for Rett syndrome

Secretion (Mecp2) were signiﬁcantly changed. Taken together, these results suggest that deregulated gene expres-

Autism sion caused by loss of Capds2 may cause developmental deﬁcits and/or pathological symptoms, resulting

in autistic-like phenotypes.

1. Introduction [2,20,33], and two CAPS family members, CAPS1 [2] and CAPS2

[24,30], have been identiﬁed in mammals. Many previous stud-

The Ca -dependent activator protein for secretion (CADPS, ies have suggested that CAPS1 is involved in the secretion of

CAPS) family is involved in dense-core vesicle (DCV) exocytosis 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 exo-

cytosis by binding to phosphatidylinositol 4,5-bisphosphate (PIP2)

Abbreviations: CADPS2 CAPS2, Ca -dependent activator protein for secretion [6,13]. However, recent studies using knockout (KO) or knock-

2; SV, synaptic vesicle; DCV, dense-core vesicle; BDNF, brain-derived neurotrophic down (KD) approaches have led to fresh debates concerning the

factor; NT-3, neurotrophin-3; WT, wild-type; KO, knockout; KD, knockdown; GO,

involvement of CAPS1 in the priming of synaptic vesicle (SV) exocy-

gene ontology; SOS, son of sevenless.

∗ tosis [9], the vesicular loading of catecholamine or serotonin [3,28]

Corresponding author at: Advanced Scientiﬁc Research Leaders Development

and DCV biosynthesis [26]. We previously reported that CAPS2

Unit, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Mae-

bashi, Gunma 371-8511, Japan. is involved in the secretion of brain-derived neurotrophic factor

E-mail address: [email protected] (T. Sadakata).

http://dx.doi.org/10.1016/j.neulet.2016.12.068

T. Sadakata et al. / Neuroscience Letters 639 (2017) 88–93 89

(BDNF) and neurotrophin-3 (NT-3) in the mouse cerebellum [24] Packard, Palo Alto, CA, USA). Gene expression proﬁles were eval-

and that Capds2 KO mice not only have histological abnormali- uated by analyzing the hybridization densities obtained from the

ties similar to those reported in the cerebellum of autistic patients experiments, which were conducted using eight independent tar-

[1,4] but also exhibit autistic-like behaviors, including impaired get cRNAs for each stage.

social interaction, hyperactivity, an abnormal sleep–wake rhythm,

and increased anxiety in unfamiliar environments [23,27]. We also 2.4. Data analysis

identiﬁed increased expression of a rare Capds2 splice variant in

autism patients that speciﬁcally lacks exon 3 and that is not trans- Gene expression data were analyzed using the absolute analysis

ported to axons when exogenously expressed in mouse cortical algorithms of the GeneChip Expression Analysis Software pack-

neurons, suggesting a possible association between Capds2 and sus- age (Affymetrix Microarray Suite 5.0). For data normalization, the

ceptibility to autism [21,27]. We developed a mouse line expressing average difference for each probe was normalized using the global

exon 3-deleted CAPS2, which displayed autistic-like behaviors, normalization and scaling provided with the GeneChip software.

such as increased anxiety in an unfamiliar environment, impaired

social behavior, and a disrupted circadian rhythm [25]. 2.5. Clustering of functional gene groups and gene expression

Autistic patients exhibit several abnormalities in cerebellar patterns

morphology and function, including hypoplasia of the cerebellar

lobules [4,18], and impaired eye movement [32] and motor coordi- We took into account the functional classiﬁcation terms used

nation [14]. In this study, to elucidate whether the loss of CAPS2 in public databases such as Gene Ontology of NCBI (http://www.

function in mice leads to abnormal cerebellar gene expression, geneontology.org/), Mouse Genome Informatics (http://www.

we examined the Capds2 KO mouse cerebellum using a GeneChip informatics.jax.org/searches/GO form.shtml), and the NetAffyx

microarray. The results revealed a gross impairment in the neu- database. Hierarchical clustering analysis of the gene expression

rotrophin signaling pathway in Capds2 KO mice. patterns for each functional gene cluster was performed using the

microarray informatics software package, Acuity 3.1 (Axon Instru-

ments, Union City, CA, USA).

2. Materials and methods

2.6. Molecular pathway analysis and visualization of gene

2.1. Animals

expression data

All experimental protocols were approved by the Institutional

We used the KEGG Pathway database to investigate the molecu-

Animal Care and Use Committee by Gunma University and by the

lar reactions and pathways that showed signiﬁcant gene expression

Tokyo University of Science. All efforts were made to minimize the

changes [10]. The KEGG Pathway is a database of biological

number of animals used and their suffering.

systems, consisting of over 4252 genes and 204 molecular pathway-

wiring diagrams of interaction and reaction networks (http://www.

2.2. RNA and cDNA preparation

genome.jp/kegg/pathway.html). Prior to the pathway analysis, we

selected probe sets that were differentially expressed (fold change

Cerebella were dissected from post-natal day 21 (P21) wild-

greater than 2) between WT and Cadps2 KO mice.

type (WT) and Cadps2 KO mice after anesthesia with diethyl ether.

Total RNA samples from each genotype were prepared from pooled

3. Results and discussion

cerebella using an RNeasy RNA extraction kit (Qiagen, Chatsworth,

CA, USA). Four total RNA samples were prepared from four differ-

To examine gene expression in wild-type (WT) and Cadps2 KO

ent brain pools. PolyA RNAs were puriﬁed from total RNAs with

mouse cerebella, we performed a genome-wide parallel monitor-

an Oligotex-dT30 kit (Takara Bio, Shiga, Japan) and were used for

ing of gene expression in P21 mice using an oligonucleotide-based

cDNA synthesis. Double-stranded cDNA samples for the GeneChip

microarray system, the Affymetrix U74 subA GeneChip, on which

analysis were prepared as follows. Single-stranded cDNAs were

12,422 genes were represented. Signiﬁcant differential expression

synthesized using the polyA RNAs, a T7-(dT)24 primer (Amer-

(p < 0.05) was observed in 1211 of 22,690 (5.34%) genes repre-

sham Biosciences, Piscataway, NJ, USA), and Superscript II reverse

sented on the chip. Neurons constitute approximately 82% of

transcriptase (Invitrogen, Carlsbad, CA, USA). Double-stranded (ds)

cerebellar cells, and approximately 99% of cerebellar neurons in

cDNA samples were synthesized according to the instructions pro-

rodents are granule cells [8,31]. It is probable that the high pro-

vided by Invitrogen. Then, four independent ds-cDNA templates

portion of granule cells in the cerebellum resulted in our gene

were prepared from each of the different RNA sources that were

chip analysis detecting a high percentage of genes with changed

puriﬁed from each of the four different brain pools.

expression in Cadps2 KO mice. To reliably determine character-

istic differences in gene expression between WT and Cadps2 KO

2.3. GeneChip expression data collection mice, the ﬁrst cutoff point of the selection was set using the fol-

lowing criteria: the P value was lower than 0.05 and the fold

Target cRNAs were prepared using T7 RNA polymerase change was higher than 2.0. A total of 191 genes were selected

ampliﬁcation with biotin-labeled UTP and biotin-labeled CTP for further analysis as genes thought to be differentially expressed

(Enzo Diagnostics, Farmingdale, NY, USA) and four independent between WT and Cadps2 KO cerebella (see Supplemental Table S1 in

ds-cDNA templates. The hybridization of biotinylated targets the online version at DOI: http://dx.doi.org/10.1016/j.neulet.2016.

to oligonucleotide-based mouse cDNA probes immobilized on 12.068). Trafﬁcking-associated genes were affected. For example,

GeneChip U74 subA glass slides (Affymetrix, Santa Clara, CA) was kinesin family member 12 (Kif12), synaptotagmin VI (Syt6), myosin

performed according to the protocol provided by the manufac- VIIb (Myo7b), pleckstrin homology, Sec7 and coiled-coil domains,

turer. After washing the chips, hybridization signals were detected binding protein (Cytip) were down-regulated, whereas kinesin

using a streptavidin–phycoerythrin conjugate in a GeneChip ﬂu- light chain 3 (Klc3), myosin IG (Myo1g), syntaxin 5A (Stx5a) were

idics station (Affymetrix). Expression of genes represented on the up-regulated. In addition, many secretory proteins were also dif-

GeneChips was read using a GeneArray scanner (excitation and ferentially regulated: ﬁbroblast growth factor 2 (Fgf2), interleukin

emission wavelengths at 488 and 570 nm, respectively) (Hewlett 17A (Il17a), phospholipase A2, group IIF (Pla2g2f), phospholipase