FOCUSED REVIEW

published: 1 May 2009
doi: 10.3389/neuro.01.004.2009

KF-1 ubiquitin ligase: an anxiety suppressor
Tamotsu Hashimoto-Gotoh1*, Naoyuki Iwabe 2, Atsushi Tsujimura1, Keizo
Takao 3,4,5 and Tsuyoshi Miyakawa3,4,5
Department of Biochemistry and Molecular Genetics, Research Institute for Neurological Diseases and Geriatrics,
Kyoto Prefectural University of Medicine, Japan2
Department of Biophysics, Graduate School of Science, Kyoto University, Japan
3
Institute for Comprehensive Medical Science, Fujita Health University, Japan
4
Frontier Technology Center, Kyoto University Graduate School of Medicine, Japan
5
Japan Science and Technology Agency, BIRD & CREST, Japan
1

Edited by:Carmen Sandi, Ecole PolytechniqueFédérale de Lausanne, Switzerland
Reviewed by:Valerie J. Bolivar, Wadsworth Center,
NYSDOH, USA
Andrew Holmes, National Instituteon Alcohol Abuse and Alcoholism,
NIH, USA* Correspondence:

Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary
danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatricdiseases such as depression. The KF-1, a ubiquitin ligase located on the endoplasmic reticulum(ER), may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior
against light or heights. It is surmised that KF-1 degrades some target proteins, responsible forpromoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson’s
disease (PD). Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic
neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies
have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution
but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice may
be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation,and for screening novel anxiolytic/antidepressant compounds.
Keywords: depression, ERAD pathway, Parkinson’s disease, Alzheimer’s disease, animal evolution

Tamotsu Hashimoto-Gotoh obtained
his DSc from Kyushu University in 1977.
After post-doctoral work at the
Max-Planck Institute for Molecular
Genetics in Berlin with Kenneth Timmis,
he became B1 Wissenschaftler at German
Cancer Research Centre in Heidelberg
with Günther Schütz, and was theninvited to Hoechst AG for establishing
Molecular Biology Laboratory at R&D
Laboratories, Hoechst Japan Limited,in Kawagoe, where he was engaged inresearch fields of Oncology, Hematology,
and Bone Biology. Then, he became
Associate Professor of Biochemistry andMolecular Genetics at Research Institutefor Neurological Diseases and Geriatric,
Kyoto Prefectural University of Medicine,where he obtained DM in 1996.
thg@koto.kpu-m.ac.jp

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INTRODUCTION
Anxiety, or learned fear, is not necessarily harmful to everyday life but, rather, is a natural ability
that may have arose to evade unnecessary dangers. However, excessive anxiety is debilitating or
disadvantageous for life as it reduces behavioral
activities necessary for adaptation. Moreover,anxiety can be a core symptom of various mental/
behavioral disorders, such as major depressive disorders, obsessive-compulsive disorders,panic disorder, adaptive disorder, post-traumaticstress disorder, social withdrawal disorder, andvarious phobias. Patients with anxiety/depression interpret circumstantial incidences, including episodes, comments, and expressions, in anegative way. The interpretation leads individuals to enhanced capture or delusions caused bywww.frontiersin.org

potential signs of danger. This suggests that asystem of negative and positive regulation of theemotional expression may have developed underthe evolutionary constraint. Such a system would
be most apparent in highly social animals withrelatively little reproduction per generation, likehumans (Darwin, 1872). Indeed, there is evidence
that the amygdala is responsible for the expression of anxiety or fear, and the prefrontal cortex plays a role in fear extinction by regulating
the amygdala-mediated expression of fear (seeBishop, 2007). Although the molecular mechanisms underlying negative and positive regulation of the anxiety are not fully understood, many
genes have been reported to affect anxiety or fear
(Chen et al., 1994; Gogos et al., 1998; Heisler et al.,
1998; Holmes et al., 2003; Miyakawa et al., 1994,

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Hashimoto-Gotoh et al.

RING-H2 finger motif
A type of zinc binding domains, similar
to RING finger motif containing
a C3HC4 amino acid motif for binding
to two zinc ions. RING-H2 finger
motif contains two histidine residues
as in C3H2C3 motif.
Many of the RING finger domains
function as ubiquitin ligases.

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Proteasomal degradation and emotional control

2003; Nakajima et al., 2008; Yamasaki et al., 2008).
Among these, the genes related to the serotonergic system are seen to be of special interest. For
example, human genomic studies of anxiety/depression have focused on genes related to themonoaminergic neurotransmission of serotoninreceptors (5-HT1A) and transporters (5-HTT)(see Levinson, 2006; Uher and McGuffin, 2008).
Genetic studies using gene-targeting techniques
have revealed that knockout mice lacking either
5-ht1A or 5-htt exhibit significantly increased anxiety-like behaviors (Heisler et al., 1998; Holmes
et al., 2003). However, proteins that interact
directly with neurotransmitters seem to function downstream, rather than upstream, of theserotonergic pathway.
Anxiety/depression is the most common psychiatric disease seen in patients irrespective of
nations, societies, and religions. Consequently,
pharmaceutical companies have made extensive worldwide efforts to develop anxiolytic/
antidepressant drugs, particularly serotonergic
compounds such as selective serotonin reuptake
inhibitor (SSRI) and serotonin noradrenalin
reuptake inhibitor (SNRI) based on the monoamine hypothesis. The hypothesis assumes thatthe pathogenesis of depression is caused by thedepletion of monoamines such as serotonin in
the brain. This assumption has however not been
proven in the last 60 years. Testing systems havebeen developed for rodents to measure behavioral
despair and to screen serotonergic drugs. These
include the forced swim test and the tail suspension test. Compounds that elevate monoaminelevels in the brain reduce the despair-like behavior or immobility time of animals under fearful
conditions (Porsolt et al., 1978; Steru et al., 1985).
However, approximately one-third of the patientswith anxiety/depression do not respond to theserotonergic drugs, suggesting that despair-likebehavior in rodents does not precisely represent
anxiety/depression in humans, from a pharmacological point of view. Therefore, it is desirable
to have some genetic animal models that display
excessive anxiety-like behavior specifically without affecting despair-like behavior, as observed in
the kf-1−/− mice (Tsujimura et al., 2008), to lookfor novel anxiolytic/antidepressant drugs thatare effective in patients who do not respond toserotonergic drugs.

IDENTIFICATION AND NATURE OF KF-1
The gene for KF-1 was originally discovered
in connection with Alzheimer’s disease (AD).
Genetic studies of familial AD (FAD) made significant progress in 1990s. In this period, three
genes encoding β-amyloid precursor protein,www.frontiersin.org

pressenilin-1 (PS1), and presenilin-2 (PS2)
were identified as the causative genes for FAD(see Hashimoto-Gotoh et al., 2006; Lundkvist andNäslund, 2007), and two other genes were foundexpressed more frequently in the frontal cortex ofan AD patient than a non-AD subject (Yasojima
et al., 1997). One of the later two genes, gfap, wasa known gene encoding glial fibrillary acidic protein, and the other was a novel gene, named kf-1.
The kf-1 gene is expressed most prominently inthe brain and more or less throughout the entire
body, but least, if any, in the liver, which implies
that kf-1 may not be a housekeeping gene. Human
kf-1 has four exons ranging over approximately
30 kb, and is mapped to chromosome 2p11.2.
The protein structure deduced from the cDNA
sequences has revealed that human KF-1 protein(GenBank Acc No. BAA19739) consists of 685
amino acids, and contains a possible leader peptide (amino acid positions 1–19), two membranespanning segments (326–345 and 366–380), anda RING-H2 finger motif (621–662) close to
the C-terminus (Figure 1A). KF-1-like proteins
have been found in other animals including fish
(Danio rerio), lancelet (Blanchistoma floridae), sea
urchin (Strongylocentrotus purpuratus), sea anemone (Nematostella vectensis), and tablet animals(Trichoplax adhaerens) (Figure 1A). Molecularphylogenetic studies suggest that genes for the animal and vertebral proteins may be kf-1 orthologues(Figures 1B,C, respectively). Unexpectedly, KF-1
homologues do not exist in insects (Drosophilamelanogaster) and thread worms (Caenorhabditis
elegans, and C. briggsae), even though it is likelythat the prototype gene appeared in the very earlyphase of animal evolution, before the separationof Placozoa and Eumetazoa (Miller and Ball,
2008; Srivastava et al., 2008). Homologues of the
prototype do not exist in prokaryotes, plants, or
fungi, but are found first in one of the most primitive multicellular animals, such as tablet animals(Schubert, 1993) (Figure 1D). The results may
be consistent with the report based on the EST
analysis that some genes, formerly thought to bevertebrate inventions, must have been present in
the common metazoan ancestor (Kortschak et al.,2003). Choanoflagellates (Monosiga brevicollis,
and M. ovata), supposedly one of the most closely
related unicellular protists to animals (King et al.,2008), do not possess kf-1 (Figure 1D).
The KF-1 protein is an E3 ubiquitin ligasethat may modulate the cellular protein levels of
its unknown substrate(s) (Lorick et al., 1999).
The expression of kf-1 is also increased in thefrontal cortex and hippocampus after chronic
administration of SSRI in rats (Yamada et al.,2000). Furthermore, rat kf-1 expression is elevatedMay 2009 | Volume 3 | Issue 1 | 16

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Figure 1 | Molecular phylogenetic studies of the kf-1 gene. (A) The alignmentof KF-1 protein sequences. The sequence identities (%) given at the ends wereobtained against human sequences minus the regions after the RING-H2 finger
motif, namely after amino acid position 621 in human KF-1. Asterisks show thealignment sites used for inferring the molecular phylogenetic tree in (B). The
two transmembrane regions of human KF-1 are indicated by ‘=’ and the RING-H2
finger motif for zinc-binding is indicated by ‘#’. The number given at the right side
indicates the amino acid position of the C-terminal amino acid in each line.

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The alignment positions, including gaps, are given on the top right of each row.
Hs, Dr, Bf, Sp, Nv, and Ta denote Homo sapiens, Danio rerio, Blanchistoma
floridae, Strongylocentrotus purpuratus, Nematostella vectensis and Trichoplax
adhaerens, respectively. The gene identification (GI) numbers are 1945615,
52219054, 210102052, 115660659, 156406687, and 196009364 in the respective
species. Blast Expect values (blastp with BLOSUM62 for Matrix) of Dr, Bf, Sp, Nv,
and Ta against Hs are E < 10−130, E = 2 × 10−130, E = 3 × 10−117, E = 3 × 10−104,
and E = 3 × 10−57, respectively. CS denotes consensus amino acid. (continued)

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Figure 1 | Molecular phylogenetic studies of the kf-1 gene. (B) The molecular phylogenetic tree for animal KF-1.
The tree is inferred by the genetic algorithm-based maximum likelihood method (Katoh et al., 2001). The evolutionaryrate heterogeneity among the sites was taken into account by assuming Yang’s discrete gamma model (Yang, 1994)
and an optimized shape parameter alpha of 1.93. Highly homologous sites without gap sites (404 amino acid sites)
were selected for the comparison as indicated by asterisks in (A). The numbers at each branching node represent
the bootstrap probability. The branching orders within the ellipse are not significant. The shaded region is expanded in (C)for vertebral KF-1. GI numbers are given in (A). (C) The maximum likelihood tree for vertebral KF-1, which corresponds
to the shaded region in (B). Highly homologous sites without gap sites within the vertebrate sequences (580 amino acidsites; not shown) were used for tree inference. The optimized shape parameter alpha is 0.43. The numbers at thebranching nodes are the bootstrap probabilities. GI numbers are given in (A) for H. sapiens and D. rerio, and 2058263,
126305140, 71896439, and 20126691 for M. musculus, M. domestica, G. gallus, and X. laevis, respectively. (continued)

Molecular phylogenetic tree
A diagram showing the evolutionary
relationship or history of organisms,
genes or proteins. It is inferred
by a phylogentic method such asneighbor-joining, maximum
parsimony, or maximum likelihood,based on the nucleotide or aminoacid sequences of various species.

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after physical antidepressant treatments, such as
electroconvulsive therapy (Nishioka et al., 2003),
and repetitive transcranial magnetic stimulation(Kudo et al., 2005). This implies that the upregulation of kf-1 expression is associated withsome physiological responses to antidepressanttreatments rather than being caused by a chemical reaction to serotonergic compounds such as
SSRIs. It was, however, not clear whether this wasa result, cause, or coincidental side effect of antidepressive processes.
As a possible correlation between KF-1 and AD
has been suggested (Yasojima et al., 1997), and as
depression can occur early in the course of AD
in ps1 mutation carriers unaware of their genetic
status (Ringman et al., 2004), the first working
hypothesis was that presenilins could be KF-1 substrates in the endoplasmic reticulum (ER) associated degradation (ERAD) pathway (see Carvalho
et al., 2006; Lorick et al., 2006). Therefore, theintracellular localization of KF-1 was examinedin comparison to that of presenilins known to be

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located on ER (Kovacs et al., 1996). The results
have revealed that KF-1 is co-localized with both
PS1 and PS2 (Figures 2A,B and Tsujimura et al.,2008), which implies that KF-1 may in fact be an
ER-ubiquitin ligase. The co-localization of KF-1
with other ER markers, such as Der-1 and VCP,
has also been observed (Maruyama et al., 2008).

SELECTIVE EXPRESSION OF INCREASEDANXIETY-LIKE BEHAVIORS IN kf-1 –/– MICE
Although mouse kf-1 is expressed in many tissues, particularly in the brain, the lack of kf-1 does
not lead to any abnormalities in appearance or
behaviors, including body weight, reproductive
capability, exploratory locomotion, nociception,social behavior, motor coordination, behavioral
despair, spatial working memory, and context
memory (Table 1).
The exception is that kf-1−/− mice display
a pronounced increase in anxiety-like behavior in the light/dark transition test (stay timein light compartment: p = 0.0004; number of

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Proteasomal degradation and emotional control

D

Monkey (M. fascicularis)
MacacaArtiodactyla

CetaceaPongo

LagomorphaPan

ProsimiMouse (M. musculus)
Rodentia

Human (H. sapiens)

Catarrhini

Cetartiodactyla

Opossum (M. domestica)

Homo
GorillaPrimateMetatheria

Chimpanzee (P. troglodytes)

Mammalian adaptive radiation

Platyrrhini(80 mys. ago)

Eutheria
Bird (G. gallus)
Aves

Monotremata

Crocodylia

Frog (X. laevis)

Mammalia

Amphibia

Anapsida

Hemichordata

Dipneustei

Sea urchin (S. purpuratus)

Lancelet (B. floridae)

Coelacanthiformes

Echinodermata

Cephalochordata

Zebrafish (D. rerio)

Fruit fly (D. melanogaster)Sea squirt (C. intestinalis)

Actinopterygii

Arthoropoda

Urochordata
Chondrichthyes

Thread worm
(C. elegans, C. briggsae)

VertebrataSea anemone (N. vectensis)

Cambrian explosion?
Chordata

Nematoda
Mollusca

Cnidaria

Tablet animal (T. adhaerens)

Triploblastica

Diploblastica
D

Placozoa

Platyhelminthes(540 mys. ago)

Eumetazoa

Yeast (S. pombe)
Porifera?

Thale cress (A. thaliana)
Dicotyledonopsida
Rice (0. sativa)
Monocotyledonopsida

Archiascomycetes

Anthophyta

Hemiascomycetes
Neurospora (N. crassa)
Pyrenomycetes

Metazoa
Choanoflagellates (M. brevicollis,
M. ovata)

Yeast (S. cerevisiae)

Ascomycota

Kingdom Animalia

Basidiomycota
Kingdom Fungi

Zoomastigophorea

Microsporididea

Kingdom Plantae

Eukaryotic diversification
Kingdom Protoctista

Kinetoplastida

Dictyosteliomycota
Gymnamoebia

Domain Eucarya

Methanococcus jannaschii + etc(1.0-1.2 bys. ago)

Domain Archaebacteria
Escherichia coli + etc
Domain EubacteriaFirst cell ancestors, Kingdoms/Domains;, Phyla/Divisions;, Orders;
Figure 1 | Molecular phylogenetic studies of the kf-1 gene.
(D) Presence/absence, generation, and transmission of kf-1 on
the evolutionary tree of life. The tree is essentially the same as the one,that was previously presented (Hashimoto-Gotoh et al., 2003), with minor
modifications and corrections except for the genes concerned. ‘?’with bi-arrows and two open stars or crosses denotes uncertaintyof the precise position of generation or loss of kf-1, respectively.

Frontiers in Neuroscience, Classes;, Genera.
Circles or crosses represent species in which the presence or absenceof kf-1 has been confirmed in the Gen Bank database on January 29, 2009.
In the case of sea squirt (Ciona intestinalis), the presence of kf-1
is represented by a triangle as only fragmented amino acid sequences
have been found similar to those of KF-1. This is probably due toan incomplete determination of genomic sequences. Detailed dataon which this illustration is essentially based are given in (A), (B), and (C).

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Figure 2 | Immunostaining of Xenopus KF-1 tagged with the Myc epitopeand PSα or PSβ co-expressed in COS-1 cells. (A) Proteins were stainedwith anti-Myc antibody probed with rhodamin conjugated anti-mouse IgG
for KF-1 (red) and anti-PSα (Xenopus PS1 homologue) probed with fluorescein

isothiocyanate (FITC) conjugated to anti-rabbit IgG for PSα (green). Imagesof ‘anti Myc (KF-1)’ and ‘anti PSα’ are merged (‘merge’). (B) The same as(A) except PSβ (Xenopus PS2 homologue) was used instead of PSα. KF-1
is co-localized with either PSα or PSβ as indicated in yellow or orange (‘merge’).

Table 1 | Behavioral phenotypes of kf-1–/– mice compared to the behavioral phenotypes of kf-1+/+ littermates.
Tests

Measurements

Phenotypesa

General health examination

Whisker, coat, reflexes=

Physical test

Body temperature=

Body weight=

Wire hanging time↑

Grip strength=

Auditory capacity=

Light/dark transition test

Anxiety↑Open field test

Exploratory locomotion=

Elevated plus maze test

Anxiety↑

Hot plate test

Pain sensitivity (latency time)=

Social interaction test

Total duration of contacts=

Number of contacts=

Total duration of active contacts=

Mean duration/contact=

Distance traveled=

Rotarod test

Motor coordination=

Prepulse inhibition (PPI) test

Sensorimotor gating↑

Porsol forced swimming test

Immobility time (behavioral despair)=T-maze test

Spatial working memory=

Immediate freezing during conditioning phase↑

Cued and contextual fear conditioning test

Tail suspension test

Contextual testing conducted after conditioning=

Cued test with altered context=

Immobility time (behavioral despair)=

The testing order is from the top to the bottom of this table. Further details are presented in Tsujimura et al. (2008). a =, no significant difference; ↑, increased in
kf-1−/− compared to kf-1+/+. The raw data of the results are available at https://behav.hmro.med.kyoto-u.ac.jp/.

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SNP
DNA sequence variation, called single
nucleotide polymorphism, occurring
on a single nucleotide in the genome
among individual members of a species
or between paired chromosomes within
an individual. ‘SNP (pronounced snip)’
was defined initially as that witha minor allele frequency of ≥1%, but
this definition is rather artificial and notmeaningful.
Ubiquitination
Ubiquitination (or ubiquitylation)is the post-translational tagging
reaction mediated by E3 ubiquitin
ligase to a protein with one or more
highly conserved peptide molecules
called ‘ubiquitin’ consisting of 76
amino acids. Ubiquitination targets
the substrate protein for proteasomal
degradation.

Proteasomal degradation and emotional control

transitions: p = 0.0392) (Tsujimura et al., 2008).
Consistently, the mice also show ‘timidity-like’
responses under stressful situations, such as
prolonged wire-hanging time, enhanced immediate freezing with an aversive foot shock, anddecreased locomotor activity at heights (Table 1
and Tsujimura et al., 2008). As significant differences are not observed in body weight and grip
strength, and in general exploratory locomotion,the ‘timidity-like’ responses are likely to be related
to psychological but not physical alterations in
kf-1−/− mice. This implies that KF-1 plays a role inemotional control by suppressing anxiety at leastin mice. In this context, it is of particular interest to note that a number of SNPs are reported
in the coding and non-coding exonic regions ofhuman kf-1 according to the NCBI’s SNP database, and some are supposed to substantiallymodulate KF-1 activity in homozygous or dual
heterozygous carriers; for example, rs35921467
resulting in a frame shift at amino acid position
180, rs17857046 and rs17853383 in critical aminoacid substitutions (S to P and P to H) at 251 and
502, respectively, and rs11695337 in a termination
codon at 626.
As KF-1 is an E3 ubiquitin ligase located on
ER, KF-1 may be responsible for conducting the
ERAD pathway, in which some factors promoting

Figure 3 | Schematic representation of the anxiety suppressor modelthrough the ERAD pathway. (A) When KF-1 is absent or decreases, theputative anxiety promoter will be increased on the ER, resulting in ‘enhanced
anxiety’. (B) When KF-1 is present or increases, the putative anxiety

Frontiers in Neuroscienceanxiety may be targeted. Therefore, the absence or
reduction of KF-1 activity may result in ‘enhanced
anxiety’ by increasing putative anxiety promoters (Figure 3A). Regarding serotonergic metabolisms, it is not clear at the moment whether or how
the ERAD pathway directed by KF-1 is associated with the serotonergic pathway, and it has tobe elucidated in future. However, KF-1’s targets
for proteasomal degradation should be neither
the serotonin receptor (5-HT1A) nor the transporter (5-HTT), because a lack of these proteins
increases anxiety-like behaviors (Heisler et al.,
1998; Holmes et al., 2003).

KF-1 AND PARKIN AS ER-BASED E3UBIQUITIN LIGASES
Ubiquitination plays an essential regulatoryrole in all critical eukaryotic cellular processes.
Proteasomal degradation through the ERAD
pathway is not an exception. It has been well established that these processes play an important rolein a variety of human somatic diseases, ranging
from cancer, viral infection, diabetes, and inflammation to muscle wastage and neurodegenerative disorders (see Kostova et al., 2007; Petroski,2008). However, there are no reports on the animal specific ubiquitin ligase, which is responsible for emotional control or mental disorders.

promoter will be degraded or lost due to 26S proteasomal degradation throughthe KF-1 mediating ERAD pathway, resulting in ‘anxiolysis’. ‘Ub’ represents
the ubiquitin molecule. ‘E1’, ‘E2’, and ‘E3’ represent the ubiquitin-activating
enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase, respectively.

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Hashimoto-Gotoh et al.

PPI
Prepulse inhibition (PPI) is aneurological phenomenon wherea weaker pre-stimulus inhibits
the reaction of animals to a subsequent
strong startling stimulus.
Sensorimotor gating
The brain’s ability to filter
out the unnecessary information.

Proteasomal degradation and emotional control

Concerning neurodegenerative disorders, Parkin,
another ER-ubiquitin ligase, plays a key role inpreventing the degeneration of dopaminergic
neurons in Parkinson’s disease (PD). A recessive mutation in parkin is responsible for theneurodegenerative disorder known as the autosomal recessive juvenile form of Parkinsonism,
the most common form of familial PD (Kitada
et al., 1998). Human parkin with 12 exons ranging
over approximately 1.5 Mb is located on chromosome 6q25.2.27, and encodes an E3 ubiquitin
ligase, consisting of 465 amino acids, with two
RING finger motifs. Parkin substrates have been
identified being degraded through the ERAD
pathway, such as Pael-R, CDCrel-1, α-Syn, andsynphilin-1 (Yang et al., 2003). The accumulation of these proteins in dopaminergic neurons
leads to ER stress-induced apoptosis, resulting
in PD (see von Coelln et al., 2004). Promoting
their degradation would aid PD patients by preventing neurodegeneration. In fact, the neuronal
synthesis of the Parkin substrate Pael-R has beenshown to cause a loss of dopaminergic cells intransgenic Drosophila models. The co-expressionof parkin results in the degradation of Pael-R
and nearly completely blocks neurodegeneration, whereas interfering with the function ofendogenous Drosophila Parkin promotes Pael-R
accumulation and augments its toxicity (Yanget al., 2003). Hence, the over-expression of parkinhas emerged as a powerful approach to PD withcomplementary effects to approaches described
for the use of neurotrophic factors against PD(Ulusoy and Kirik, 2008). An analogy could bemade by having such Drosophila models to study
the role of KF-1 in anxiety/depression. However,
the analogy may not be feasible as no evidence
is available for the involvement of neuronal cell
death in anxiety/depression, and kf-1 is absent in
Drosophila (Figure 1D). It should be noted thatkf-1−/− mice have increased PPI values (Table 1),
which implies that at least one of the KF-1 target
proteins is involved in regulation of sensorimotor gating. In such cases, over-expression of kf-1
might cause serious side effects as the PPI values
are usually reduced in schizophrenic patients.
Despite this, one cannot rule out the possibilityof kf-1 up-regulation as an anxiolytic/antidepressant treatment.
Instead, kf-1−/− mice provide a powerful tool
for some pharmacological approaches to anxiety/depression. For example, by using the mice, one
can identify the putative anxiety promoters thatare degraded through the ERAD pathway mediated by KF-1 and are responsible for the sensitivity to potential signs of danger or to stressful
situations (Figures 3A,B). KF-1 substrates acting
as anxiety promoter may be found by the computed differential screening in 2-dimensional gel
electrophoresis (see Marengo et al., 2008) of cellhomogenates derived from the frontal cortex or
hippocampus of kf-1−/− and kf-1+/+ mice. As the

Figure 4 | A circuitry presentation of the anxiety suppressor model by the up-regulation of kf-1 expression.
The circuitry model was constructed by considering the fear extinction neural model (Sotres-Bayon et al., 2006).
The anxiolytic pathway after kf-1 up-regulation is incorporated into the model.

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expression of kf-1 is up-regulated in the frontal
cortex and hippocampus after antidepressanttreatments, it is likely that the anxiolytic/antidepressive effect of KF-1 functions primarily
in these tissues and that their inhibitory signals
are transmitted to the amygdala to prevent the
manifestation of anxiety or fear (Figure 4). This
proposal may be highly hypothetical and hence
has to be elucidated in future studies. To conclude,
examining kf-1−/− mice in a simple light/darktransition test is an effective method of screening
novel anxiolytic/antidepressant drugs that inhibit
the putative anxiety promoters.

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ACKNOWLEDGEMENTS
We are grateful to Drs. M. Matsuki, and K. Yamanishi
for their collaborations during this work, andMessrs. N. Matsumoto and T. Aramaki, andDrs. H. Yamagishi, S. Clapcote, and M. Nakagawa
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THG thanks Drs. T. Mizuno, K. Yasojima, Y. Ariga,
Y. Fukumaki, C. Mitchell and M. Miyata for valuable discussions and comments. NI is a recipient of Grant-in-Aid for Scientific Research on
Priority Area “Comparative Genomics” from the
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Conflict of Interest Statement: The
authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.
Received: 29 January 2009; paper pending published: 17 February 2009; accepted:
27 February 2009; published: 01 May 2009.
Citation: Front. Neurosci. (2009) 3,1:
15–24. doi: 10.3389/neuro.01.004.2009
Copyright © 2009 Hashimoto-Gotoh,
Iwabe, Tsujimura, Takao and Miyakawa.
This is an open-access article subject toan exclusive license agreement between
the authors and the Frontiers Research
Foundation, which permits unrestricted
use, distribution, and reproduction in any
medium, provided the original authors andsource are credited.

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