genetics

Neuron

Report

Increased L1 Retrotransposition in the Neuronal Genome in Schizophrenia Miki Bundo,1,2 Manabu Toyoshima,3 Yohei Okada,4 Wado Akamatsu,4 Junko Ueda,2 Taeko Nemoto-Miyauchi,2

Fumiko Sunaga,1 Michihiro Toritsuka,5 Daisuke Ikawa,5 Akiyoshi Kakita,6 Motoichiro Kato,7 Kiyoto Kasai,8

Toshifumi Kishimoto,5 Hiroyuki Nawa,9 Hideyuki Okano,4 Takeo Yoshikawa,3 Tadafumi Kato,2,* and Kazuya Iwamoto1,10,* 1Department of Molecular Psychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan 2Laboratory for Molecular Dynamics of Mental Disorders 3Laboratory for Molecular Psychiatry

RIKEN Brain Science Institute, Saitama 351-0198, Japan 4Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan 5Department of Psychiatry, Nara Medical University, Nara 634-8521, Japan 6Department of Pathology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan 7Department of Neuropsychiatry, Keio University School of Medicine, Tokyo 160-8582, Japan 8Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan 9Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan 10PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan

*Correspondence: kato@brain.riken.jp (T.K.), kaziwamoto-tky@umin.ac.jp (K.I.) http://dx.doi.org/10.1016/j.neuron.2013.10.053

SUMMARY

Recent studies indicate that long interspersed nuclear element-1 (L1) are mobilized in the genome of human neural progenitor cells and enhanced in Rett syndrome and ataxia telangiectasia. However, whether aberrant L1 retrotransposition occurs in mental disorders is unknown. Here, we report high L1 copy number in schizophrenia. Increased L1 was demonstrated in neurons from prefrontal cortex of patients and in induced pluripotent stem (iPS) cell-derived neurons containing 22q11 deletions. Whole-genome sequencing revealed brain-specific L1 insertion in patients localized preferentially to synapse- and schizophrenia-related genes. To study the mechanism of L1 transposition, we examined perinatal environmental risk factors for schizo- phrenia in animal models and observed an increased L1 copy number after immune activation by poly-I:C or epidermal growth factor. These findings suggest that hyperactive retrotransposition of L1 in neurons triggered by environmental and/or genetic risk fac- tors may contribute to the susceptibility and patho- physiology of schizophrenia.

INTRODUCTION

Mental disorders including schizophrenia, bipolar disorder, and

major depression affect a large proportion of the global popula-

tion and have a major negative economic impact. Twin, family,

and adoption studies indicate the complex involvement of both

genetic and environmental factors for these diseases (Keshavan

et al., 2011). Despite their apparent heritability, however, causa-

306 Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc.

tive genetic factors are mostly unknown except for rare cases

of schizophrenia associated with chromosomal abnormalities

(Brandon and Sawa, 2011; Cook andScherer, 2008; Karayiorgou

et al., 2010). On the other hand, environmental risk factors

including prenatal infection (Brown, 2006) and obstetric compli-

cations, such as neonatal hypoxia, embryonic ischemia, and

gestational toxicosis (Lewis and Murray, 1987), are well-estab-

lished risk factors for schizophrenia. However, it is not clarified

how these environmental risk factors interact with genomic

factors.

Accumulating evidence indicates that genomic DNA in the

brain contains distinctive somatic genetic variations compared

with nonbrain tissues (Poduri et al., 2013). These genetic signa-

tures include brain-specific somatic mutations (Poduri et al.,

2013), chromosomal aneuploidy (Rehen et al., 2005; Yurov

et al., 2007), chromosomal microdeletion (Shibata et al., 2012),

and the genome dynamics of nonlong terminal repeat (LTR) ret-

rotransposons (Baillie et al., 2011; Evrony et al., 2012;Muotri and

Gage, 2006). These observed somatic variations are hypothe-

sized to contribute to the generation of functionally diversified

brain cells (Muotri and Gage, 2006).

Among the known retrotransposons, only long interspersed

nucleotide element-1 (L1) has autonomous retrotransposition

activity. Full-length L1 elements include a 50 UTR, two open reading frames (ORFs), and a 30 UTR (Figure 1A). Encoded prod- ucts from the ORFs contain activities required for retrotransposi-

tion and are employed in the insertion of new L1 copies as well as

nonautonomous retrotransposons such as Alu and SVA (Cor-

daux and Batzer, 2009). Recent studies indicate that engineered

L1 has retrotransposition activity in neural progenitor cells from

rat hippocampus (Muotri et al., 2005), human fetal brain (Coufal

et al., 2009), and human embryonic stem cells (Coufal et al.,

2009). These in vitro findings were confirmed in human L1 trans-

genic mice in vivo (Muotri et al., 2005). Adult human brain cells

also showed increased L1 copy number compared with non-

brain tissues (Coufal et al., 2009). Moreover, retrotransposition

about 6 kb

ORF1 40 kDa

ORF2 150 kDa

3’ -UTR

RNA binding protein

Reverse Transcriptase/ Endonuclease

5’ -UTR

m5UTR mORF1 mORF2mouse

h5UTR#2

hORF1#5 hORF2#1

hORF2#2 hORF1#1 hORF1#3human

macaque cORF2#4 cORF2#3

CT SZ MD BD hORF2#1 (SATA normalized)

CT SZ MD BD

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p=0.0298 p=0.0035

p=0.0061

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Alexa488_NeuN Merge with PI

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II CT 34 8:26 44.5±7.5 - 0 18:16 6.6±0.3 29.5±13.0

set diagnosis gender (F:M) age onset (yrs) suicide side (R:L) pH PMI (hrs)n

I BD 5:8 41.5±11.2 21.9±8.9 7 7:6 6.1±0.2 31.2±15.213 I MD 5:7 45.2±10.0 33.5±11.8 6 3:9 6.1±0.2 27.8±10.512 I SZ 5:8 44.4±12.9 24.2±8.1 3 6:7 6.2±0.3 34.7±14.613 I CT 5:8 48.2±10.4 - 0 5:8 6.2±0.2 23.6±10.713

II SZ 9:26 42.6±8.5 21.3±6.1 7 18:17 6.5±0.2 31.4±15.335

Figure 1. Increase of Brain L1 Copy Number

in Schizophrenia

(A) Structure of L1 andmap of the primers. Primers

and probes are fromprevious studies (Coufal et al.,

2009; Muotri et al., 2010) or designed for this study

(Table S4). (B) Summary of the demographic vari-

ables of brain samples. (C) L1 copy number in set I.

(D) Neuronal nuclei isolation. Left: example of

NeuN-based nuclei sorting of brain cells from a

patient with schizophrenia. Right: microscopic

confirmation of isolated nuclei. The purity of each

fraction was >95% and 99.9% for NeuN+ and

NeuN�, respectively. (E) Neuronal L1 copy num- ber in set II. In quantitative real-time PCR, L1 copy

number was measured with HERVH or SATA as

internal controls. The ratio of prefrontal cortex to

liver (for set I) or neurons to nonneurons (for set II)

was calculated and then normalized relative to the

average value of control samples. Values were

represented as open or closed diamonds as well

as box plots. The DCt values of L1 and control

probes were not significantly different between

diagnostic groups in set I or set II. p values were

determined by the Mann-Whitney U test. PMI,

postmortem interval; CT, controls; SZ, schizo-

phrenia; MD, major depression; BD, bipolar dis-

order; PI, propidium iodide. See also Tables S1

and S4 and Figures S1 and S2.

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Increased L1 Copy Number in Schizophrenia

is active in MeCP2 mouse models and patients with Rett syn-

drome, indicating a role for this mechanism in thisMendelian dis-

order (Muotri et al., 2010). Together, these findings suggest the

Neuron 81, 306–313

hypothesis that L1 retrotransposition

may also be involved in the pathophysi-

ology of mental disorders.

In this study, we quantified L1 copy

number in genomic DNA derived from

postmortem brains of patients with major

mental disorders.We report significant in-

creases of L1 content in the prefrontal

cortex of patients with schizophrenia. To

confirm this finding, we quantified L1

copy number in neurons and nonneurons

from a second, independent patient

cohort using NeuN-based cell sorting

(Iwamoto et al., 2011; Rehen et al.,

2005; Spalding et al., 2005) and found

that L1 copy number in neurons was

increased in patients with schizophrenia.

We next quantified L1 copy number in

the animal models that are known to

disturb early neural development. These

included maternal polyriboinosinic-poly-

ribocytidilic acid (poly-I:C) injection in

mice (Meyer and Feldon, 2012; Giovanoli

et al., 2013) and chronic epidermal

growth factor (EGF) injection to infant ma-

caques (Nawa et al., 2000). We found that

genomic DNA of brains from both animal

models showed increased L1 copy number, addressing the

importance of environmental factors during perinatal and post-

natal stages. We also found that the increased L1 copy number

, January 22, 2014 ª2014 Elsevier Inc. 307

0

0.5

1.0

1.5

2.0

m5UTR mORF1 mORF2

p=0.0019

p=0.0148

CT (n=8) Poly-I:C (n=8)

p=0.0207

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on te

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0.8

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1.0

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CT (n

=3 )

Ha l (n

=3 )

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CT (n

=3 )

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=3 )

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EG F1

EG F2

EG F1

EG F2

A B

(5s-rRNA normalized) (5s-rRNA normalized)

Figure 2. Increase of Brain L1 Copy Number

in Animal Models

(A) Brain L1 content in thematernal poly-I:Cmodel.

p values were determined by the Mann-Whitney U

test. Values were represented as open or closed

diamonds aswell as box plots. (B) Brain L1 content

in chronic EGF or haloperidol-treated macaque

models. Error bars indicate SDs. The comparative

Ct method, with 5S-rRNA as an internal control,

was used. The ratio of prefrontal cortex to liver (for

poly-I:C model) or prefrontal gray matter to NeuN-

sorted nonneurons in white matter (for macaque

models) was calculated and then normalized

relative to the average value of control samples.

See also Table S4.

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Increased L1 Copy Number in Schizophrenia

in the neurons derived from induced pluripotent stem (iPS) cells

of schizophrenia patients with 22q11 deletion. The 22q11 dele-

tion is a well-defined genetic factor and is one of the highest

risk factors for schizophrenia, affecting about 1%–2% of schizo-

phrenia patients (Karayiorgou et al., 2010). Finally, we performed

whole-genome sequencing (WGS) analysis of brain and liver in

controls and patients. Comparison of brain-specific L1 insertion

sites revealed that brain-specific L1 insertion in patients is

enriched in or near genes related to synaptic function and neuro-

psychiatric diseases. These results suggest that increased retro-

transposition of L1 in neurons, which was triggered by genetic

component and/or environmental factors at the early neural

development, could contribute to the susceptibility and patho-

physiology of schizophrenia.

RESULTS

Increased Brain L1 Content in Schizophrenia We used postmortem prefrontal cortex samples of patients with

schizophrenia, bipolar disorder, and major depression as well as

control subjects for analysis in set I. The demographic variables

are summarized in Figure 1B. We quantified L1 copy number of

postmortem prefrontal cortex and liver in each subject by quan-

titative RT-PCR with two different internal controls, which were

designed for human endogenous retrovirus (HERVH) and

alpha-satellite (SATA). We found a significant increase in the

brain L1ORF2 content in patients with schizophrenia (Figure 1C).

A tendency toward copy number increase was also observed in

mood disorders and in other L1 probes in schizophrenia (Fig-

ure S1 available online).

Somatic L1 retrotransposition was primarily found in neuronal

cells (Kuwabara et al., 2009). To confirm the increased brain L1

copy number in schizophrenia and address whether this copy

number increase is due to alteration of the neuronal genome,

we examined an independent prefrontal cortex sample set (set

II). We separated neuronal and nonneuronal nuclei from frozen

brains using NeuN-based cell sorting (Figure 1D) (Iwamoto

et al., 2011). NeuN is expressed in vertebrate neurons, and its

antibody can be used for labeling neuronal nuclei (Mullen et al.,

1992). We quantified L1ORF2 copy number of genomic DNA

derived from neurons (NeuN-positive nuclei) and nonneurons

(NeuN-negative nuclei) and then calculated the neuron-to-non-

308 Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc.

neuron ratio. We found a significant increase of neuronal

L1ORF2 content in schizophrenia in two different internal con-

trols (Figure 1E). The copy number of the other L1 probes tested

also showed significant increase in schizophrenia compared to

controls in SATA-normalized data, and similar tendency toward

copy number increase was observed in HERVH-normalized

data (Figure S1 and data not shown).

Assessment of Confounding Factors We assessed the effect of confounding factors on L1 content

(Table S1). Among the demographic variables tested, sample

pH showed a weak correlation with L1ORF2 content in set II

but not in set I. Several variables also showed weak correlations,

but none showed consistency across the different internal con-

trol probes or across the two different sample sets.

To consider the possible effect of antipsychotics, we exam-

ined L1 copy number in a human neuroblastoma cell line cultured

with haloperidol or risperidone for 8 days. Both antipsychotics

did not modify the L1 copy number at their low or high concen-

trations (Figure S2). Together with the fact that the lifetime intake

of antipsychotics, which was estimated as fluphenazine milli-

gram equivalents, did not correlate with L1 copy number in

both brain sets (Table S1), medication status did not affect our

results.

L1 Quantification in Animal Models To assess the potential roles of environmental factors on

increased L1 copy number, we employed two different animal

models that mimic environmental risk factors that affect early

neural development. They included maternal poly-I:C injection

in mice and chronic EGF injection to neonatal macaques. The

poly-I:C, which mimics viral double-stranded RNA, injection to

pregnant mice induces elevated maternal immune activation,

and the offspring is known to show schizophrenia-like behavioral

alterations such as impairments of prepulse inhibition and social

behavior at the later stage (Meyer and Feldon, 2012). Pregnant

mice received a single intraperitoneal injection of poly-I:C. L1

copy number in the prefrontal cortex of offspring was tested at

postnatal day 21. We found that significant elevation of L1 copy

number at all the tested probes compared to controls (Figure 2A).

We then examined the L1 copy number in macaques treated

with EGF during neonatal period. Perinatal and postnatal

0.9

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9

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SZ _a

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e

Figure 3. L1 Content in Neurons Derived from iPS Cells of Schizophrenia Patients with 22q11 Deletions

The comparative Ct method, with SATA as an internal control, was used. The ratio of NeuN-sorted neurons to nonneurons was calculated and then normalized

relative to average value of control samples. Error bars indicate SDs. See also Table S4 and Figure S3.

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Increased L1 Copy Number in Schizophrenia

perturbation of EGF is known to evoke schizophrenia-like phe-

notypes, including deficits in prepulse inhibition, latent inhibition,

social interaction, and working memory, in adulthood (Nawa

et al., 2009, 2000).The neonatal macaques (n = 2) subcutane-

ously received EGF for seven times over 11 days. After 4 and 7

years from treatment, L1 copy number in the prefrontal cortex

was tested. In addition, chronic haloperidol-treated macaques

(n = 3) were also tested. Due to unavailability of other tissues,

we isolated nonneuronal nuclei from frozen white matter and

calculated the grey matter-to-nonneuron ratio in each subject.

Although statistical approach could not be applied, we observed

increase of L1 copy number in EGF-treatedmacaques, but not in

the haloperidol-treated macaques, compared to controls (Fig-

ure 2B). Taken together, these results suggest that early environ-

mental factors play important roles in the L1 content in the brain.

We further confirmed that chronic haloperidol treatment did not

influence L1 copy number in this model.

L1 Quantification in the iPS Cells of Schizophrenia Patients with 22q11 Deletion We next assessed the importance of genetic risk factor on the L1

copy number in brain. We quantified L1 copy number in the neu-

rons derived from iPS cells of schizophrenia patients with 22q11

deletion (n = 2) as well as controls (n = 2) (Figure S3). The iPS cells

were established from the fibroblasts according to the previously

developed method (Imaizumi et al., 2012; Takahashi et al., 2007;

M.T., unpublished data). To estimate the L1 copy number, we

used two independently established iPS cell lines per patient.

After induction of neuronal cells (Imaizumi et al., 2012), we iso-

lated neuronal nuclei by NeuN-based sorting (Figure S3). We

then examined L1 copy number and calculated the neuron-to-

nonneuron ratio. Compared to controls, we observed consistent

increase of L1 copy number in iPS cell-derived neurons of

patients with schizophrenia with 22q11 deletion (Figure 3). These

results suggest that the well-defined strong genetic risk factor

also plays an important role in the L1 content in the brain.

Identification and Comparison of Brain-Specific L1 Transposition We next performed WGS of brain and liver DNA from same sub-

jects by self-assembling DNA nanoarray technology (Drmanac

et al., 2010). For this experiment, schizophrenia patients (n = 3)

and control subjects (n = 3) were selected to match age, PMI,

gender, brain pH, and race from set I. Selected patients ex-

hibited increased L1 content by quantitative RT-PCR assay,

compared to average L1 content of the controls and selected

control subjects. The WGS metrics and identified variations

were summarized in Table S2. Distribution of the detected mo-

bile elements was almost equal between the tissues and across

subjects, and over the half of the identified elements was related

Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc. 309

Control Term Count p value FE height 4 0.0132 7.7 scoliosis 3 0.0316 10.3

Schizophrenia Term Count p value FE schizophrenia;

bipolar disorder schizoaffective disorder; 5 0.0125 5.2

schizophrenia 29 0.0135 1.6 hypertension 20 0.0194 1.7 bipolar disorder 13 0.0373 1.9

A Control Term Count p value GO:0005856~cytoskeleton 74 5.92E-04 GO:0005509~calcium ion binding 56 0.0031 GO:0005930~axoneme 9 0.0095 GO:0035085~cilium axoneme 7 0.0289 GO:0003779~actin binding 26 0.0322 GO:0044425~membrane part 240 0.0387 GO:0016010~dystrophin-associated glycoprotein complex 6 0.0405

Schizophrenia Term Count p value GO:0045202~synapse 57 3.09E-09 GO:0030054~cell junction 64 8.57E-06 GO:0044459~plasma membrane part 187 1.49E-05 GO:0004674~protein serine/threonine kinase activity 58 2.27E-05 GO:0044456~synapse part 38 4.44E-05 GO:0004672~protein kinase activity 72 7.45E-05 GO:0030554~adenyl nucleotide binding 147 1.09E-04 GO:0005856~cytoskeleton 126 1.19E-04 GO:0005488~binding 820 1.25E-04 GO:0006468~protein amino acid phosphorylation 76 1.75E-04 GO:0006796~phosphate metabolic process 100 1.98E-04 GO:0006793~phosphorus metabolic process 100 1.98E-04 GO:0016773~phosphotransferase activity, alcohol group as acceptor 80 2.31E-04 GO:0001882~nucleoside binding 148 2.52E-04 GO:0005524~ATP binding 138 2.63E-04 GO:0001883~purine nucleoside binding 147 2.77E-04 GO:0032559~adenyl ribonucleotide binding 139 3.43E-04 GO:0017076~purine nucleotide binding 169 4.19E-04 GO:0000166~nucleotide binding 190 0.0011 GO:0014069~postsynaptic density 17 0.0011 GO:0032553~ribonucleotide binding 161 0.0012 GO:0032555~purine ribonucleotide binding 161 0.0012 GO:0043167~ion binding 323 0.0015 GO:0016043~cellular component organization 204 0.0018 GO:0043169~cation binding 318 0.0021 GO:0016310~phosphorylation 83 0.0021 GO:0046872~metal ion binding 315 0.0025 GO:0016301~kinase activity 85 0.0032 GO:0005737~cytoplasm 489 0.0037 GO:0008092~cytoskeletal protein binding 58 0.0045 GO:0007155~cell adhesion 74 0.0049 GO:0022610~biological adhesion 74 0.0052 GO:0019898~extrinsic to membrane 54 0.0053 GO:0043687~post-translational protein modification 108 0.0169 GO:0030030~cell projection organization 45 0.0219 GO:0005509~calcium ion binding 88 0.0234 GO:0015629~actin cytoskeleton 33 0.0439

B

insertion site CT1 CT2 CT3 average ratio intergenic (low) 0.64 0.76 0.69 0.69 intragenic (low) 0.36 0.24 0.31 0.31 intron (low) 0.90 1.00 0.99 0.96 exon (low) 0.10 0.00 0.01 0.04 intergenic (high) 0.65 0.81 0.69 0.72 intragenic (high) 0.35 0.19 0.31 0.28 intron (high) 0.88 1.00 0.99 0.96 exon (high) 0.12 0.00 0.01 0.04

C

90 0.0473 GO:0045211~postsynaptic membrane 21 0.0487

insertion site SZ1 SZ2 SZ3 average ratio intergenic (low) 0.72 0.63 0.58 0.64 intragenic (low) 0.28 0.37 0.42 0.36 intron (low) 0.99 0.98 0.98 0.98 exon (low) 0.01 0.02 0.02 0.02 intergenic (high) 0.74 0.63 0.57 0.65 intragenic (high) 0.26 0.37 0.43 0.35 intron (high) 0.99 0.98 0.98 0.98 exon (high) 0.01 0.02 0.02 0.02

Control

Schizophrenia

GO:0016772~transferase activity, transferring phosphorus-containing groups

Figure 4. Insertion Site, Gene Ontology, and Disease Association Analyses

(A) L1-insertion site analysis. Proportion of intergenic and intragenic L1 insertion and that of intronic and exonic L1 insertion are given. The low and high mean

estimated proportions based on both less and stringent criteria are given. Note that ratios are not significantly different between patients and controls. (B) Gene

ontology analysis. p values indicate Bonferroni-corrected modified Fisher’s exact test p value. The terms showing p < 0.05 are shown for both groups. (C)

Disease-association analysis. p values indicate noncorrected modified Fisher’s exact test p value. FE, fold enrichment. In both analyses, gene lists generated by

the stringent criteria were used. See also Tables S2 and S3 and Figure S4.

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Increased L1 Copy Number in Schizophrenia

to the L1-Hs (Figure S4). Among the detected mobile element

insertion sites in each sequenced sample, we first identified

brain-specific L1 insertions in each subject (Tables S2 and S3).

Although the total number of brain-specific L1 insertion tended

to be higher in schizophrenia patients, this was not statistically

significant, most likely due to the limited sample size and high

interindividual variation. We then compared genomic locations

of the insertion sites of brain-specific L1 between patients and

controls (Figure 4A). The inter-to-intragenic L1 insertion ratio as

well as exonic-to-intronic L1 insertion ratio did not differ between

patients and controls. We then compared the affected genes by

brain-specific L1 insertion by gene ontology approach. This

310 Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc.

analysis revealed that the number of enriched terms is higher

in schizophrenia than controls, in spite that the number of

brain-specific L1 insertions did not significantly differ. We found

that neuronal function-related terms such as synapse and

protein phosphorylation are clearly overrepresented in schizo-

phrenia compared to controls (Figure 4B). In addition, disease-

association analysis revealed that affected genes in patients

are specifically enriched in terms related to schizophrenia and bi-

polar disorder, while those in controls are enriched in nonneur-

opsychiatric terms such as height and scoliosis (Figure 4C).

These results were consistently confirmed when we used less

stringent definition of brain-specific L1 insertion (Figure S4). In

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Increased L1 Copy Number in Schizophrenia

addition, enrichment of the L1-inserted genes to the terms

related to neuropsychiatric disorders in schizophrenia was also

detected by the ingenuity pathway analysis (IPA) (Figure S4).

DISCUSSION

We report that the neuronal genome of schizophrenia contains

higher copy number of a retrotransposon, L1. To validate this

finding, we utilized iPS cells from patients with schizophrenia

carrying the 22q11 deletion and observed an increase in L1

copy number in iPS cell-derived neurons. Moreover, using

WGS, we found that L1 preferentially inserted into genes related

to synaptic functions and schizophrenia. Animal model studies

showed that environmental factors related to infection or inflam-

mation that disturbs early neurodevelopmental processes

increase L1 copy number in the brain. Collectively, these results

suggest that hyperactive L1 retrotransposition into critical

genes during neural development, triggered by genetic and/or

environmental factors, contribute to the pathophysiology of

schizophrenia. Our results significantly expand the range of

neuropsychiatric illnesses linked to aberrant L1 retrotransposi-

tion, from Mendelian disease patients with MECP2 mutations

in Rett syndrome (Muotri et al., 2010) and ATM mutations in

ataxia telangiectasia (Coufal et al., 2011) to schizophrenia, a

complex mental disorder.

The observed increase of L1 content in schizophrenia was not

due to, or modulated by, biological or experimental artifacts,

because changes were measured in two independent patient

cohorts and each result was confirmed with two different internal

controls. Although the L1 region showing significant increases

differed between the two brain sets, this is attributable to cohort

differences amplified by the strict threshold we employed. Actu-

ally, a significant increase of L1 content was widely observed in

all probes in the SATA-normalized data in set II, where neuronal

L1 copy number was directly examined (Figure S1). In addition,

from the data analysis utilizing lifetime intake of antipsychotics

of patients, and from the cell culture and macaque experiments,

we conclude that antipsychotics do not affect L1 copy number in

the brain. A significant increase was also observed in patients

with mood disorders in one internal control in set I (Figure 1C).

Future work will clarify whether there are L1 content increases

in other mental disorders using larger and/or stratified patient

cohorts.

L1 retrotransposition has been detected during adult neuro-

genesis in the rat hippocampus, indicating that neural progenitor

cells retain retrotransposition activity even in adult stages (Muotri

et al., 2009). However, we analyzed potential confounding fac-

tors, including age, age of onset, and duration of illness, and

did not observe any significant correlation with L1 copy number

in the brain. The transcript level of L1 in adult brain sample was

also increased in patients compared to controls (data not

shown). However, elevated expression is unlikely to contribute

to increase of L1 copy number in patients, as significant increase

of L1 transcripts was detected only in the 50 region of L1 such as 50 UTR and ORF1. These results suggest that L1 copy number does not globally increase with aging and that the variation of

L1 copy number in patients is probably confined to early neuro-

developmental stages, at least in the prefrontal cortex. This

prediction would be consistent with the neurodevelopmental hy-

pothesis of schizophrenia, where abnormalities during critical

early periods of brain development may trigger the later appear-

ance of clinical symptoms (Bloom, 1993; Murray et al., 1992;

Weinberger, 1987).

In Rett syndrome, increased L1 copy number in human brain

was linked to mutations in MECP2 (Muotri et al., 2010) and

MeCP2 knockout mice also showed increased L1 content

(Muotri et al., 2010). It has also been suggested that SOX2 and

MECP2 regulate L1 transcription in neurons (Muotri et al.,

2005; Yu et al., 2001). However, we did not observe a significant

correlation between MECP2 or SOX2 expression and brain L1

content, by using the previously performed gene expression

analyses on the same sample sets (Iwamoto et al., 2004, 2005)

(data not shown). In addition, patients with high levels of L1

copy number (two schizophrenia and one major depression in

set I, and two schizophrenia patients in set II) did not show

altered MECP2 or SOX2 expression levels (data not shown).

These findings suggest that the molecular mechanism of

increased L1 in schizophrenia is different from Rett syndrome.

In this study, we found that both early environmental and well-

defined strong genetic factors for schizophrenia are involved in

the increase of L1 copy number in the brain. A recent study using

the poly-I:C model indicated that the offspring of this model

had exacerbated schizophrenia-like phenotypes, if they were

exposed to environmental stress during puberty, suggesting

that early environmental factors can lower the threshold for onset

of schizophrenia (Giovanoli et al., 2013). Therefore, increased L1

insertions induced by environmental factors may increase the

susceptibility to schizophrenia by disrupting synaptic and

schizophrenia-related genes in neurons, rather than being a

direct cause of the disease. On the other hand, the pathological

consequences of increased L1 content in neurons derived from

iPS cells of schizophrenia patients with 22q11 deletions remain

unclear. We chose patients with 22q11 deletions to examine

L1 dynamics where there is a well-defined strong genetic risk

for schizophrenia. InMeCP2-knockoutmice, Rett-like behavioral

abnormalities could be rescued by the re-expression of wild-

type MeCP2 at both young and adult stages (Cobb et al.,

2010; Ehninger et al., 2008), suggesting that L1 content itself

may not be directly causal to disease phenotypes but instead

modulate phenotypic variability among patients (Muotri et al.,

2010). Similarly, we speculate that the L1 increase in schizo-

phrenia patients with 22q11 deletions is likely to modulate phe-

notypes of schizophrenia rather than a direct cause, because

many genes related to schizophrenia, such as TBX-1, SEPT5,

COMT, and PRODH, are located within the deletion (Hiroi

et al., 2013; Karayiorgou et al., 2010). Nevertheless, our findings

will facilitate further studies of the mechanism of increased L1

retrotransposition associated with schizophrenia.

Our WGS analysis could not detect increased brain-specific

L1 insertions in schizophrenia; however, we found that L1 inser-

tions were more frequent in genes for synaptic function and

schizophrenia relative to controls. Evrony et al. cloned one L1

insertion event from 300 single neurons and showed that 2 of

83 cortical neurons from an individual had this insertion, but

detection of such a low level mosaic insertion in bulk brain tissue

of the same individual was difficult and needed optimization

Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc. 311

Neuron

Increased L1 Copy Number in Schizophrenia

(Evrony et al., 2012). Thus, rare L1 insertion events could be

missed in our WGS analysis. Apart from L1, nonautonomous ret-

rotransposons such as Alu and SVA also show an increased

copy number in the brain, possibly via the aid of L1ORF products

(Baillie et al., 2011) and their copy number might also be

increased in patients. Further studies on the neuronal genome

of patients with mental disorders, and supporting mechanistic

evidence from animal and cellular models, may establish a

broader role for instability of neural genome in the pathophysi-

ology of schizophrenia. We expect that our findings will promote

the further study of genomic instability in disease etiology due to

L1 retrotransposition in brain development.

EXPERIMENTAL PROCEDURES

Postmortem Samples

Postmortem brain and liver samples were obtained from the Stanley Medical

Research Institute. The demographics are summarized in Figure 1B and are

described at the web site (http://www.stanleyresearch.org/). Ethics commit-

tees of RIKEN and the University of Tokyo Faculty of Medicine approved the

study.

Animal Models

Animal experiments were performed in accordancewith the NIHGuidelines for

the Care and Use of Laboratory Animals and guidelines of relevant facilities.

For poly I:C model, pregnant mice (C57BL/6) received either a single intraper-

itoneal injection of poly-I:C (2 mg/ml, Sigma-Aldrich) dissolved in PBS

(20 mg/kg) or an equivalent volume of PBS at embryonic day 9.5. At postnatal

day 21, tissues were dissected from pups. For macaque models, cynomolo-

gusmonkeys (Macaca fascicularis) (4 years old; all males) were given oral halo-

peridol (0.25–0.5 mg/kg; Wako Pure Chemical Industries) or vehicle for

2 months (Shibuya et al., 2010). After transiently separating two male monkey

neonates (2 weeks old) from dams, neonates received subcutaneous admin-

istration of human recombinant EGF (0.3 mg/kg, Funakoshi) for seven times

over 11 days and then quickly returned to their dams. Preliminary behavioral

assessment of the EGF-treated monkeys was performed at ages of 4 and 6

years and reported (Nawa et al., 2009). These monkeys were sacrificed at

the age of 4 and 7 years with the overdose of pentobarbital (26 mg/kg;

65 mg/ml). Experiments were subjected to review by the Ethical Committee

of Shinn Nippon Biomedical Lab.

iPS Cells

All procedures for skin biopsy and iPS cell production were approved by the

Keio University School of Medicine ethics committee and RIKEN ethics com-

mittee. The 201B7 iPS cells were kindly provided by Dr. Yamanaka (Takahashi

et al., 2007). For the control WD39, a skin-punch biopsy from a healthy

16-year-old Japanese female obtained after written informed consent was

used to generate iPS cells (Imaizumi et al., 2012). 22q11.2 deletion syndrome

iPS cells (SA001 and KO001) were generated from a 37-year-old Japanese

female patient (Toyosima et al., 2011) and a 30-year-old Japanese female

patient, respectively, using the same method used to generate the WD39

(M.T., unpublished data). 22q11 deletion was characterized by the CGH array

analysis (Figure S3). Production and maintenance of iPS cells were performed

according to the previous studies (Imaizumi et al., 2012; Takahashi et al.,

2007). All the iPS cells and differentiated neuronal cell lines were characterized

with immunofluorescence staining and their morphologies (Figure S3).

L1 Copy Number Estimation

We performed either Taqman-based quantitative real-time PCR according to

Coufal et al. (2009) with minor modifications (100 or 500 pg DNA as starting

material and single amplicon analysis) or SYBR-Green-based quantitative

real-time PCR according to Muotri et al. (2010). SYBR-Green assay was

performed using 500 pg DNA and Power SYBR Green PCR Master Mix (Life

Technologies). Primers, probe location, and reaction chemistry are listed in

Figure 1A and Table S4. Quantification was performed in triplicate. A nonpara-

312 Neuron 81, 306–313, January 22, 2014 ª2014 Elsevier Inc.

metric Mann-Whitney U test was employed for two group comparison and

p < 0.05 was considered significant.

Whole-Genome Sequencing

WGS of brain and liver samples from controls and schizophrenia patients was

performed by Complete Genomics, with the paired-end library preparation

and sequencing-by-ligation using self-assembling DNA nanoball (DNB)

(Drmanac et al., 2010). Data process, mapping, and detection of variations

were performed using the software developed by the Complete Genomics

(version 2.2.0.26 and format version 2.2). Among the detectedmobile insertion

elements, we compared the genomic location of L1 insertion between brain

and liver within an individual and identified brain-specific L1 insertions.

Further experimental details are available in the Supplemental Experimental

Procedures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and four tables and can be found with this article online at

http://dx.doi.org/10.1016/j.neuron.2013.10.053.

ACKNOWLEDGMENTS

This work was supported in part by the Grant-in-Aid for Scientific Research on

Innovative Areas (Unraveling the microendophenotypes of psychiatric disor-

ders at the molecular, cellular, and circuit levels) from the Ministry of Educa-

tion, Culture, Sports, Science and Technology (MEXT) to T.Y., H.N., T.K.,

and K.I., and a Grant-in-Aid from Ministry of Health, Labour and Welfare to

T.K. This work was also supported by JST, CREST to T.K. and by JST,

PRESTO to K.I. This work was also supported in part by Leading Project for

Realization of Regenerative Medicine from MEXT and ‘‘Funding Program for

World-Leading Innovative R&D on Science and Technology’’ to H.O., and by

the ‘‘Development of biomarker candidates for social behavior’’ carried out un-

der the Strategic Research Program for Brain Sciences fromMEXT to T.Y. and

K.K. This work was also supported in part by the Collaborative Research Proj-

ect of the Brain Research Institute, Niigata University. Postmortem samples

were donated by the Stanley Medical Research Institute, courtesy of

Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H.

Yolken. We thank Tomoko Toyota and Atsuko Komori-Kokubo at RIKEN BSI

for their technical assistance. We also thank Kenji Ohtawa at Research Re-

sources Center at the RIKEN BSI for the cell-sorting analysis. M.B., F.S.,

and K.I. belong to the Department of Molecular Psychiatry, which is endowed

by Dainippon Sumitomo Pharma and Yoshitomiyakuhin. H.O. is a scientific

consultant for San Bio, Eisai, and Daiichi Sankyo. T.K. received a grant from

Takeda Pharmaceutical. These companies had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript.

Accepted: October 18, 2013

Published: January 2, 2014

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