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Introduction

Nearly all mutated genes that have been linked to monogenic

idiopathic epilepsies code for components of ion channels or neuro-

transmitter receptors (Baulac and Baulac, 2009). Leucine-rich,

glioma-inactivated 1 (LGI1), along with Myoclonin1/EFHC1,is

an exception. Mutations in the LGI1 gene are associated

with the autosomal dominant lateral temporal epilepsy

(ADLTE) syndrome (Poza et al., 1999), also known as autosomal

dominant partial epilepsy with auditory features (Winawer et al.,

2000).

ADLTE is an inherited epilepsy syndrome of adolescence onset,

characterized by focal seizures that may generalize. A speciﬁc

feature of the syndrome is the presence of auditory auras. Many

patients hear sounds including singing, ringing, humming or

whistling during seizures, and seizures may also be triggered

by noises or voices. Other less-frequent auras include visual,

psychic, autonomic and other feelings or sensations (Michelucci

et al., 2009).

Interictal electroencephalography shows temporal abnormalities

in 47% of the patients. Magnetic resonance image ﬁndings are

often normal and outcome is usually good, although some pa-

tients may develop pharmacoresistance (Chabrol et al., 2007;

Di Bonaventura et al. , 2009). While the prevalence of ADLTE is

not entirely certain, it may account for up to 19% of familial

idiopathic focal epilepsies (Michelucci et al., 2009).

In 2002, mutations responsible for ADLTE were identiﬁed in the

LGI1 gene by positional cloning (Kalachikov et al., 2002;

Morante-Redolat et al., 2002). A number of ADLTE families and

some sporadic cases with mutations in LGI1 were subsequently

reported (Nobile et al., 2009). Nearly half of known

ADLTE-related LGI1 mutations are nonsense and frameshift

mutations, some of which are predicted to cause a decreased

abundance of mutated mRNA transcripts because of their degrad-

ation by nonsense-mediated decay. Other mutations are typically

missenses. We and others have shown that missense or truncating

mutations impair LGI1 secretion, which also suggests that

LGI1-related epilepsy results from a loss of function (Senechal

et al., 2005; Sirerol-Piquer et al., 2006; Chabrol et al., 2007;

Striano et al., 2008; de Bellescize et al., 2009). It seems likely,

therefore, that ADLTE patients carrying nonsense or missense

mutations express lower levels of extracellular brain LGI1 protein,

causing haploinsufﬁciency. Two recent articles describing seizures

in LGI1-deﬁcient mice conﬁrmed that lack of LGI1 leads to

epilepsy (Fukata et al., 2010; Yu et al., 2010).

LGI1 encodes a neuronal protein (also called epitempin) that is

secreted into the extracellular media by transfected mammalian

cells (Senechal et al., 2005). Expression is highest in the brain

(Chernova et al., 1998; Furlan et al., 2006; Head et al., 2007).

LGI1 has no homology with known ion channel genes. Instead, it

encodes a protein containing three leucine-rich repeats in the

N-terminal half followed by seven epilepsy-associated repeats in

the C-terminal part of the protein. Current evidence suggests that

LGI1 is a multi-functional protein: (i) it suppresses glial tumour cell

progression in vitro (Chernova et al., 1998); (ii) it co-puriﬁes with

the presynaptic voltage-gated Kv1.1 potassium channel and

inhibits fast inactivation of the K

-currents mediated by the

Kvb1 subunit (Schulte et al., 2006); (iii) LGI1-oligomers bind to

postsynaptic disintegrin and metalloproteinase domains 22 and

23 (ADAM22 and ADAM23) (Sagane et al., 2008), and binding

to ADAM22 may enhance AMPA receptor-mediated synaptic

transmission (Fukata et al., 2006); and (iv) LGI1 also contributes

to postnatal dendritic pruning and the maturation of glutamatergic

synapses in the hippocampal dentate gyrus (Zhou et al., 2009).

Since mutations may result in LGI1 haploinsufﬁciency in patients

with ADLTE, we attempted to model the human genetic disorder

by disrupting the LGI1 gene. We found that adult heterozygous

mice have reduced seizure thresholds, and homozygous mice dis-

play early-life spontaneous seizures associated with neuronal loss

in the hippocampus.

Materials and methods

Targeted disruption of the

LGI1

gene

A mouse line harbouring a ‘ﬂoxed’ (loxP-ﬂanked encompassing

exons 6 and 7) conditional allele of LGI1 was established at the

Mouse Clinical Institute (Illkirch, France). The targeting vector was

constructed as follows. A 1.1 kb fragment encompassing LGI1

exons 6 and 7 was ampliﬁed by polymerase chain reaction on

129S2/SvPas mouse embryonic stem cells genomic DNA and sub-

cloned in a Mouse Clinical Institute proprietary vector, resulting in a

step 1 plasmid. This Mouse Clinical Institute vector has a ﬂoxed neo-

mycin resistance cassette. A 4.4 kb 5

homologous arm encompassing

part of intron 4, exon 5 and part of intron 5 was ampliﬁed by poly-

merase chain reaction and subcloned in step1 plasmid to generate the

step2 plasmid and ﬁnally a 3.4 kb 3

homologous arm was subcloned

in a step2 plasmid to generate the ﬁnal targeting construct. The line-

arized construct was electroporated in 129S2/SvPas mouse embryonic

stem cells. After selection, targeted clones were identiﬁed by polymer-

ase chain reaction using external primers and further conﬁrmed by

Southern blot with 5

and 3

external probes. Two positive embryonic

stem clones were injected into C57BL/6J blastocystes, and the derived

male chimeras gave germline transmission. Crossing LGI1

loxP/+

males

with PGK-Cre females (C57BL/6J) yielded heterozygous LGI1

+/

ani-

mals. Polymerase chain reaction analysis of DNA extracted from

mouse tails with Purelink

Genomic DNA puriﬁcation (Invitrogen)

revealed a Cre-dependent LGI1 allele excision. LGI1

+/

animals were

then intercrossed to obtain LGI1

/

, LGI1

+/

and LGI1

+/+

littermates,

derived from 75% C57BL/6 and 25% 129S2Sv/pas hybrid back-

ground. LGI1

+/+

(wild-type) mice harbour 2 LGI1 wild-type alleles

(not ﬂoxed) and serve as controls. Animals were treated according

to the guidelines of the European Community (authorization number

75-1622) and our protocol was approved by the Local Ethical

Committee for animal experimentation. All efforts were made to min-

imize the number of animals and their suffering.

Western blots

Mice were decapitated; whole brains and organs were quickly

removed and lysed in 5 M urea, 2.5% sodium dodecyl sulphate,

50 mM Tris, 30 mM NaCl buffer. Total protein concentrations were

determined by the Bradford method. Of each sample, 25 mg was sepa-

rated on 10% Tris–glycine polyacrylamide gels, analysed by Western

blot using the following antibodies: rabbit polyclonal anti-LGI1

2750 | Brain 2010: 133; 2749–2762 E. Chabrol et al.

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