Analysis of Gene Expression in Ca2+-Dependent

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

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

Fig. 1. Network model of genes differentially expressed between WT and Cadps2 KO cerebella. A direct interaction network model was constructed by Pathway Architect

analysis of the differentially expressed transcripts between WT and Cadps2 KO cerebella. Up-regulated transcripts are indicated in red whilst down-regulated genes are

shown in green. Arrows link interacting genes and positive (+) and negative (–) associations are marked respectively. Green boxes denote regulation, blue lines with blue

boxes show binding and orange circles indicate phosphorylation.

A2, group IB, pancreas (Pla2g1b), lymphocyte antigen 6 complex, lar signaling pathways. Many MAPK pathway components, such as

locus I (Ly6i), endothelin 2 (Edn2) were down-regulated, whereas Fgf2, Fgf4, Shc1 and Sos1, were evident.

ﬁbroblast growth factor 4 (Fgf4), chemokine-like factor (Cklf), To identify speciﬁc signaling pathways involving MAPK

chemokine (C-C motif) ligand 22 (Ccl22), ectodysplasin-A (Eda) that are affected by Cadps2 KO, we performed graphical map-

were up-regulated. ping of related genes to the KEGG Pathway database. As a

We classiﬁed the 191 genes into functional clusters deﬁned by result, Sos1 (down-regulated, fold change = 2.85, p = 0.0081),

the Gene Ontology (GO) terms in the Mouse Genome Informatics Shc1 (down-regulated, fold change = 4.17, p = 0.0434), Traf6

(MGI) database (Jackson Laboratory) (see Supplemental Table S2 in (down-regulated, fold change = 4.17, p = 0.0492) and Psen2

the online version at DOI: http://dx.doi.org/10.1016/j.neulet.2016. (up-regulated, fold change = 3.73, p = 0.0006) were mapped on

12.068). Genes encoding extracellular proteins were most signiﬁ- the neurotrophin signaling pathway (Fig. 2). Activation of Ras is

cantly affected and genes encoding synaptic proteins, such as Syt6, required for normal neuronal differentiation and also promotes

Sntb1, Sh3kbp1, Grin2b and Gabbr1 were also changed. Interestingly, survival of many neuronal subpopulations [19]. Transient acti-

calcium ion binding proteins were listed. These genes included Syt6, vation of Ras is mediated by the adaptor protein, Shc, which is

F10, Pla2g1b, Grin2b, Calml4, Aoc3, Cdh6, Pla2g2f, Sntb1, Mrc2, Rptn, recruited to phospho-Y490 on Trk receptors via interactions with

Masp1, Tmem37, Plek, Pcdhac1 and Mmp11. Cytokine activity was the Shc PTB domain (Fig. 3) [17]. Trk-mediated phosphorylation of

also listed because Cklf, Spred2, Fgf4, Eda, Fgf2, Il17a and Ccl22 were Shc creates a phosphotyrosine site on Shc that recruits the adaptor,

affected. Grb2, which is bound to the Ras exchange factor, son of seven-

The extent to which these genes bind to or are regulated by other less (SOS). Activated Ras stimulates signaling through c-Raf-Erk,

genes within the data set was determined using the Pathway Archi- p38MAP kinase and class I PI3 kinase pathways [34,35]. However,

tect software, which creates biological interaction networks using the p75NTR receptor binds the unprocessed proneurotrophins

a natural language processing algorithm to extract knowledge from [12]. Several signaling pathways are activated following neu-

known molecular interactions [16]. Of the 191 genes that were ana- rotrophin binding to p75NTR. These are mediated through p75NTR

lyzed, 36 were retained within the network layout, which is shown binding to several adaptor proteins, including Traf6 (Fig. 3). The

in Fig. 1. The application of this approach provided a framework Traf6-associated pathway results in the activation of NF-kappaB,

to examine gene interaction networks from lists of differentially thereby promoting NF-kappaB-dependent neuronal survival [7].

expressed genes that differ between WT and Cadps2 KO cerebella. Trk activation does not inhibit induction of NF-kappaB-mediated

Decreased expression of genes encoding secretory substances in signaling by p75NTR [36]. Consequently, in the presence of Trk

Cadps2 KO mice may result in the differences seen in intracellu- signaling, activation of the NF-kappaB pathway makes a synergistic

T. Sadakata et al. / Neuroscience Letters 639 (2017) 88–93 91

Fig. 2. Neurotrophin signaling pathway as represented by KEGG. Gene expression changes were mapped on the pathways. White text within black boxes indicates signiﬁcantly

up-regulated or down-regulated genes between WT and Cadps2 KO cerebella (see text).

contribution to neuronal survival. Our results suggest decreased Autism is characterized by cerebellar morphological abnor-

expression of proteins that are associated with Trk and p75NTR malities [1,4]. We previously reported that Cadps2 KO mice not

signaling. Decreased neurotrophin (BDNF and NT-3) release has only have deﬁcits in neuronal development and survival but also

been reported in Cadps2 KO cerebellum [23]. The decreased phos- exhibit abnormal behaviors, including impaired social interaction,

phorylation of the Trk receptor and increased apoptosis of granule hyperactivity, an abnormal sleep–wake rhythm, and increased anx-

cells in the Cadps2 KO cerebellum has also been reported [23]. iety in unfamiliar environments [23,27]. Moreover, we identiﬁed

It is possible that decreased phosphorylation leads to decreased increased expression of a rare CADPS2 splice variant in autism

expression of proteins that are related to the neurotropin sig- patients that speciﬁcally lacks exon 3 and that is not transported

naling pathway, which results in increased apoptosis, as shown to axons when exogenously expressed in mouse cortical neurons,

previously [23]. On the other hand, our GeneChip analysis did suggesting a possible association between CAPS2 and susceptibility

not show any signiﬁcant change in the mRNA levels of BDNF or to autism [21,27]. In the mouse cerebellum, CAPS2 protein is exclu-

NT-3 between WT and Cadps2 KO cerebella. However, disparity sively localized in granule cells [22,24]. We previously reported

between BDNF protein and mRNA levels has been reported [15]. that the primary defect in the Cadps2 KO mouse cerebellum was

The highly sensitive two-site enzyme immunoassay [11] showed a deﬁcient release of neurotrophin from granule cells, which con-

signiﬁcant difference in BDNF protein levels in the P21 cerebellum sequently leads to decreased lobulation between lobules VI and

between WT and Cadps2 KO mice (1.32 ± 0.05 ng/g in WT versus VII and impaired parallel ﬁber–Purkinje cell synapse function [23].

1.09 ± 0.19 ng/g in Cadps2 KO, mean ± SD; P < 0.05; Student’s t Abnormal reduction in the size of lobules VI and VII of the cere-

test). In contrast, there was no signiﬁcant change in NT-3 protein bellar vermis is a characteristic morphological deﬁcit reported in

levels in the P21 cerebellum (10.59 ± 0.76 ng/g in WT versus the brains of autistic patients [4], and hypoplasia of these lobules

11.00 ± 1.19 ng/g in Cadps2 KO, mean ± SD). Detailed changes in was suggested to be related to the abnormal exploratory behav-

the neurotrophin signaling pathway of Cadps2 KO mice remain to ior of autism [18]. Deﬁcits in pursuit eye movement [32] and

be determined. motor coordination [14], which are both likely to be dependent

on impaired parallel ﬁber-Purkinje cell synapse function, have also

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

Fig. 3. Neurotrophin signaling. This ﬁgure depicts the interactions of neurotrophin with Trk and p75NTR receptors and major intracellular signaling pathways (modiﬁed

from [19]). Activation of Ras results in activation of the MAP kinase-signaling cascade, which promotes neuronal differentiation and neurite outgrowth. Activation of PI3

kinase through Ras or Gab1 promotes survival and growth of neurons and other cells. However, NF-kappaB activation results in transcription of multiple genes, including

several that promote neuronal survival.

Table 1

Autism-related genes.

Gene name Gene Symbol Expression in p-value Absolute fold Regulation

cerebellum change (WTvsKO)

oxytocin receptor Oxtr GC,Golgi 0.781 1.078 down

cadherin 10 Cdh10 GC,PC 0.558 1.039 up

cadherin 9 Cdh9 GC 0.346 1.655 up

semaphorin 5A Sema5a weak 0.229 2.495 up

glutamate receptor, ionotropic, kainate 2 (beta 2) Grik2 GC 0.853 1.044 up

Abelson helper integration site Ahi1 GC,PC,st,bs 0.209 2.493 down

reelin Reln GC 0.418 1.071 up

met proto-oncogene Met weak 0.817 1.166 down

engrailed 2 En2 GC 0.364 1.079 down

contactin associated protein-like 2 Cntnap2 GC,PC 0.084 1.297 down

tuberous sclerosis 1 Tsc1 GC 0.181 1.244 up

phosphatase and tensin homolog (pten) Pten GC 0.058 1.167 down

7-dehydrocholesterol reductase Dhcr7 GC,PC 0.125 1.370 up

calcium channel, voltage-dependent, L type, alpha 1C subunit Cacna1c 0.995 1.001 up

arginine vasopressin receptor 1A Avpr1a weak 0.497 1.604 up

cytoplasmic FMR1 interacting protein 1 Cyﬁp1 weak 0.623 1.051 down

Ubiquitin protein ligase E3A Ube3a PC 0.136 1.399 up

gamma-aminobutyric acid (GABA-A) receptor, subunit beta 3 Gabrb3 0.165 1.278 up

protein kinase C, beta 1 Prkcb1 GC 0.220 1.089 down

tuberous sclerosis 2 Tsc2 GC 0.097 1.127 up

ataxin 2 binding protein 1 A2bp1 PC,Golgi 0.138 1.114 down

solute carrier family 6 (serotonin transporter), member 4 Slc6a4 none 0.832 1.195 down

integrin beta 3 Itgb3 weak 0.331 1.714 down

plasminogen activator, urokinase receptor Plaur weak 0.466 1.084 up

SH3/ankyrin domain gene 3 Shank3 GC 0.061 1.187 down

fragile X mental retardation syndrome 1 homolog Fmr1 GC? 0.299 1.090 up

methyl CpG binding protein 2 Mecp2 GC,PC 0.031 1.115 down

Ca -dependent activator protein for secretion 2 Cadps2 GC 3.31E-08 77.362 down

GC, granule cell; PC, Purkinje cell; st, stellate cell; bs, basket cell.

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

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