Current Molecular Medicine 2001, 1, 677-688 677

Molecular Mechanisms of Neuronal Migration Disorders, Quo Vadis?

S. Couillard-Despres1,2, J. Winkler2,3, G. Uyanik2 and L. Aigner*1,2

1Volkswagen Foundation Junior Group, University of Regensburg, Regensburg, Germany

2Department of Neurology, University of Regensburg, Regensburg, Germany
3Department of Neurosciences, University of California at San Diego, La Jolla, USA

Abstract: Following terminal mitosis, neuronal precursor cells leave their site of origin and migrate
towards their definitive site of residency. In order to establish the intricate cytoarchitecture described
in the adult human brain, neuronal migration must be finely regulated. In humans, brain malformations
can result from neuronal migration defects. The spectrum of migration disorder severity extends from
few heterotopic neurons, as observed in periventricular heterotopia, to a complete cortical
disorganization, as observed in cases of lissencephaly. Recently, specific migration disorders have
been linked to mutations/deletions in the doublecortin , filamin-1, LIS1 and reelin genes. These proteins
act at different levels of the signaling cascades transducing extracellular guiding cues into
cytoskeletal reorganization. Here, we summarize the data concerning these four molecules and
speculate on their functions and interaction partners during neuronal development.

INTRODUCTION disorders with genetic defects. Several genes have

The adult human brain is an organ bearing a
fascinating degree of complexity. The establish-
ment of the three-dimensional topography
described in the adult brain requires a tight control
of neural progenitor cell proliferation and
differentiation as well as a precise guidance of the
neuroblasts from their site of origin to their final
destination. The cerebral cortex, with its six layered
structure, is an area of the brain where the
developmental program must be particularly fine
tuned. Following their last mitosis in the ventricular
zone, the future cortical neurons migrate towards
the pial surface, through distances corresponding to
hundreds of times their own size. Extracellular cues
from the surrounding extracellular matrix and
neighboring cells, as well as the internal
cytoskeletal machinery, are required to achieve a
proper migration. Genetic defects in any of these
components can provoke neuronal migration
disorders. These disorders cover a wide spectrum of
severity. In the milder forms, the affected
individuals have normal cognitive functions and
rare episodes of epilepsy, whereas the most severely
affected cases will suffer from intractable epilepsy,
severe mental retardation and shortened life span.
The recent progress in routinely performed
neuroimaging techniques allowed for a more
distinct classification of developmental brain
aberrations and facilitated the linkage of specific
*Address correspondence to this author at the Department of Neurology,
VW-Foundation Junior Group, University of Regensburg, Franz-Josef-
Strauss-Allee 11, D-93053 Regensburg, Germany; Tel: (+49) (941) 944
8950; Fax: (+49) (941) 944 8951;
email: ludwig.aigner@klinik.uni-regensburg.de
been linked over recent years to neuronal migration
disorders, but their function in normal and
pathological conditions is not fully elucidated. In
this review, we will summarize the recent findings
on genes involved in human neuronal migration
disorders in the context of cortical development.
DEVELOPMENT OF THE CEREBRAL CORTEX
Cell Proliferation
Many neurons present in the neocortex originate
from the pseudostratified ventricular epithelium that
lines the embryonic cerebral ventricle wall [1-3].
Prior to neurogenesis, the cells from this epithelium
proliferate and undergo mitosis in a symmetric
mode, resulting in two identical daughter cells that
will continue to proliferate. At the onset of
neurogenesis, on embryonic day 11 in mouse and
approximately the 5th week of gestation in human
[4, 5], some of the mitoses occur in an asymmetric
mode. In the present situation, the process results in
one cell maintaining the "mother cell"
characteristics, the so-called stem cell, and a sister
cell which will not undergo further mitosis, but will
rather migrate towards the pial aspect of the
developing cortex. The proportion of asymmetric
division increases during the entire time of
neurogenesis [2], which ends at approximately
embryonic day 17 in mouse and the 5th month of
gestation in human [4, 6]. Further proliferation of
the stem cells essentially results in an increase in
the glial cell population. The transition from
symmetric to asymmetric division implicates that
the proliferation before the onset of neurogenesis

1566-5240/01 $20.00+.00 2001 Bentham Science Publishers Ltd.

678 Current Molecular Medicine, 2001, Vol. 1, No. 6 Couillard-Despres et al.

enlarges the surface of the future cortex, whereas
the proliferation during the neurogenic period
influences the thickness of the future cortex [7, 8]. A
recent study using retroviral infection for clonal
lineage analysis demonstrated that radial glia
constitute the neural stem cell population during
the period of corticogenesis [9]. These bipolar cells
have their somata in the ventricular zone and
extend two fibers reaching the ventricular wall and
the pial surface. During mitosis, the somata of the
radial glia migrate to the luminal surface where
cells divide. The somata of the two daughter cells
then return to more basal regions of the ventricular
zone, according to a process called interkinetic
nuclear migration. Surprisingly, the radial glia do
not need to retract their processes in order to
undergo mitosis [9]. In cases of asymmetric division,
uneven distribution of certain molecules along the
basal-apical axis of radial glia, such as Notch1 [10]
or Numb [11], is thought to be responsible for the
differential fate of the daughter cells.
their final site of residency. During corticogenesis,
one mode of neuroblasts migration is the so-called
"radial migration" [4, 12, 13]. This process is
characterized by the use of radial glia as guiding
tracks for migrating neurons. In contrast, the
tangential migration does not rely on radial glia
guidance. This type of migration is responsible for
the lateral dispersion of clonally related neurons
within the developing cortex and also for the
colonization of the latter by neurons originating
from the ganglionic eminence [14-18]. In this
review, we will mainly address issues related to
radial migration. In humans, neuroblast migration
mainly occurs between the 8th and the 24th week
of gestation [4]. During this process, the neuroblasts
migrate along their "mother" radial glia towards the
pia [9, 12, 19]. Extracellular molecules, such as
laminin, fibronectin, astrotactin and cerebroglycan,
are thought to be particularly important for the
interaction between neuroblasts and radial glia [20-
23].

The first step in cortical development is the

Neuronal Cell Migration establishment of a pre-plate (Fig. (1)). This early
structure, located above the ventricular zone, can
Following terminal mitosis, neuroblasts must be observed during the 5th to 8th week of gestation.
migrate considerable distances in order to reach It consists of the first wave of post-mitotic

Figure (1). Development of the cerebral cortex.

Neuroepithelial cells constitute the pseudostratified ventricular epithelium lining the wall of the neural tube. The radial
oriented progenitor cells (blue) divide according to a mode typical for pseudostratified epithelia termed interkinetic nuclear
migration. The cell cycle is closely associated with the repetitive migration of the nuclei between the basal (facing the
future pial surface) and the apical (facing the lumen of the ventricle) part of the ventricular zone. Symmetric division
produces identical progeny and increases the number of neural progenitor cells resulting in an expansion of the surface
area of the ventricular zone. At the fifth week of gestation, cells start to divide asymmetrically and produce postmitotic
neuronal precursor cells (orange) that will migrate along the radial process of their mother cells (yellow) towards the pial
surface. The first waves of neuronal precursors migrate to the surface to form the preplate (PP). Subsequently, migrating
neuronal precursors form the cortical plate by separating the pre-plate into the marginal zone (MZ) (future layer I) and the
subplate (SP). Additional neural precursors migrate into the cortical plate and generate the layers II to VI. In the adult, the
ventricle is lined by the ependymal cell layer (E). Radial glial cells have differentiated into astrocytes (not shown) and a
band of white matter containing axons and oligodendrocytes (W) separates the cortex from the ventricle.
Neuronal Migration Disorders, Quo Vadis Current Molecular Medicine, 2001, Vol. 1, No. 6 679

neuroblasts [5]. The following wave of neuroblasts
migrating into the neocortex will intercalate in this
pre-plate to form a structure called the cortical
plate. The pre-plate is thereby bisected in two
entities, the marginal zone and the sub-plate (Fig.
(1)). Pial to the cortical plate, the marginal zone
contains the Cajal-Retzius neurons that will form the
future layer 1 of the mature neocortex. As
neuroblasts initiate their migration in the ventricular
zone, the radial glia fibers on which they migrate
are arranged into fascicles. However, the radial glia
processes defasciculate at the level of the sub-plate
and penetrate into the cortical plate as single fibers
[24]. Interestingly, this level is an important site of
neuronal heterotopia in neuronal migration
disorders.
As more neuroblasts migrate into the cortical
plate, the latter undergoes an important increase in
thickness. The addition of new neurons into the
cortical plate proceeds according to an inside-
outside sequence. Each new wave of neuroblasts
migrates through the previous cell generation to
occupy a more superficial position (Fig. (1)). The
need for a greater surface area in the outer cortical
layers, as compared to the inner layers, is thought to
provoke a compressive force involved in the
formation of gyri [25]. These gyri develop
significantly between the 26th and 28th week of
gestation, but continue to be further elaborated up
to the early postnatal period [26].
NEURONAL MIGRATION DISORDERS
Neuronal migration can be affected at different
levels, each defect producing a characteristic
pathological condition. In lissencephaly, for
example, the majority of neurons fail to reach their
proper cortical layers and interrupt prematurely their
migration. The resulting cortex is therefore
thickened and typically lacks or bears a significant
reduction of gyri (Fig. (2d)). In type 1 lissencephaly,
also named classical lissencephaly, the cerebral
wall architecture is similar to the one observed in a
12 week-old foetus [25]. Instead of the characteristic
six cortical layers, only four layers can be
described. The outermost layer, the molecular
layer, consists of the former marginal zone, which is
settled on a layer of neurons, resembling those
normally found in the deepest layers of the
neocortex. Underneath is a cell-sparse layer, and
the deepest layer consists in columns of
heterotropic neurons [27]. A fine ribbon of white
matter separates the gray matter from the
ventricular region. The neurons forming the
heterotopia are likely to have arrested during their
migration and never migrated through the previous
layers of neurons. The clinical symptoms associated
with lissencephaly comprise epileptic seizures,
cognitive impairment and focal neurological
deficits, resulting in most cases in premature death
[28, 29].
Lissencephaly constitutes one of the most severe
forms of impaired migration described in patients.
On the other hand, several milder defects can now
be detected using standard magnetic resonance
imaging (MRI) techniques. It is estimated that
neuronal heterotopia could be one of the major
cause of idiopathic epileptic seizures. Two of the
milder neuronal migration defects that have
recently attracted attention are periventricular
nodular heterotopia (PNH) and subcortical laminar
heterotopia (SCLH), also termed subcortical band
heterotopia or double cortex syndrome. In these
disorders, only a fraction of neurons experiences
migration defects, while on the contrary unaffected
neurons establish a characteristic cortical structure.
The underlying causes of these disorders are
described below in more detail.

Figure (2). Coronal Magnetic Resonance Images (MRI) of different neuronal migration disorders.

In contrast to a normal brain a) the brain of individuals suffering of PNH contains nodules or bands of gray matter lining
the ventricle (arrows) b). In cases of subcortical band heterotopia (double cortex) a band of heterotopic neurons can be
detected between the normal cortex and the ventricles (arrows) c) and in lissencephaly d) a significant reduction of the gyri
and a thickening of the cortical gray matter can be observed. Mutations in some genes, mentioned underneath each panel,
have been linked to these disorders.
680 Current Molecular Medicine, 2001, Vol. 1, No. 6
MOLECULAR ASPECTS OF MIGRATION
DISORDERS
Filamin-1 and Periventricular Nodular Heterotopia
Following terminal mitosis, newly formed
neurons of the ventricular zone leave their site of
origin and migrate towards the marginal zone at the
surface of the developing cerebrum. In cases where
neurons fail to initiate the migratory process, they
accumulate in the ventricular wall as nodules,
causing a pathological condition referred to as
periventricular nodular heterotopia (PNH) (Fig. (2b)).
Histological examination of individuals affected by
PNH revealed the presence of differentiated
neurons inside the nodules and normal cortical
layering [30, 31]. Interestingly, heterotopic neurons
seem to be incorporated in the motor circuitry. A
recent study using positron emission tomography
(PET) and oxygen-15-labeled water revealed an
increased cerebral blood flow during a finger
tapping task in the nodular heterotopic gray matter
located in the white matter below the frontal
neocortex, suggesting that neuronal activation
occurs in subcortical gray matter heterotopia. [32].
Using an identical paradigm during MRI scanning,
we observed an increased blood flow in the gray
matter lining the ventricular surface in patients
suffering of PNH [33]. These preliminary functional
neuroimaging studies suggest that heterotopic
pyramidal neurons, located in the subcortical white
matter and along the ventricular wall, project to the
spinal cord and could therefore be functionally
integrated in motor circuits required during motor
task performance.
In studying various pedigrees, some cases of
PNH were linked to mutations in the filamin-1 (FLN1)
gene [34]. The mutations identified are likely to
provoke a loss of function of filamin-1 proteins.
Filamin-1 is an actin-binding protein producing
orthogonal branching on the actin filaments and
can induce filament bundles similar to those
observed in filopodia [35, 36]. The function of
filamin-1 is, however, not restricted to the nervous
system. A role in the development of the immune
and vascular system is supported by recent studies
[37-39]. In males, mutations in filamin-1 are
normally embryonically lethal, but in cases where
the prenatal development reached advanced
stages, deficient myeloid and erythroid
differentiations were reported, together with
systemic bleeding [34]. In females, the phenotype is
significantly milder. The reason for this discrepancy
is the localization of the filamin-1 gene on the X-
chromosome (Xq28). Therefore, as a result of
random X-chromosome inactivation, females
bearing a mutation in filamin-1 express the mutant
gene in a mosaic pattern. Yet, these females also
show some vascular anomalies as revealed by a
higher incidence of patent ductus arteriosus, viz.
the shunt between the aorta and the pulmonary
artery does not close properly after birth, thus
Couillard-Despres et al.
impairing the establishment of a proper postnatal
blood circulation [34].
Filamin-1 is a large protein of 280 kDa, encoded
by a gene composed of 47 exons spanning 26 kbp
of genomic sequences [40]. An actin-binding
domain similar to -actinin and dystrophin is
localized at the amino-terminus of filamin-1,
followed by 24 repeats of a 96 amino acids
sequence forming Ig-like -sheet motifs. The last
repeat is truncated and contains sequences
allowing for the dimerization of filamin-1
monomers. The 24 repeats are also interrupted
between the repeats 15-16 and 23-24 by sequences
creating hinges in the rod domain [41].
Previous reports demonstrated that filamin-1 can
bind the intracellular domain of transmembrane
receptors, such as the 1 and 2 integrins [42-44].
Therefore, filamin-1 can constitute a link
transducing extracellular signals into actin network
reorganization. The function of filamin-1 is
regulated by phosphorylation, which alters its
subcellular localization and binding affinity to
membrane receptors and actin filaments [41].
Interestingly, phosphorylation consensus sequence
analysis revealed that filamin-1 is very likely to be a
substrate for Cdk5 phosphorylation [34]. Null
mutation of the Cdk5 is known to provoke profound
neuronal migration disorder, in a manner related,
but not identical, to the reeler phenotype [45]. The
reeler phenotype has been linked to a null mutation
of the reelin gene (see below) [46]. The reelin
protein is secreted into the extracellular matrix by a
subpopulation of neurons and binds to the 31
integrins. Thus, reelin and filamin-1 could be part
of the same signaling pathway via their common
interaction partner, the 1 integrin.
A role of filamin-1 in the formation of filopodia
was recently reported [47]. Filamin-1 was found to
bind the activated RalA protein, a small GTPase,
with its carboxy-terminal region. The microinjection
of constitutively active RalA in cells causes the
extension of filopodia. Taking advantage of a
human melanoma cell line lacking filamin-1, Ohta
and colleagues [47] were able to demonstrate that
the ability of RalA, and other proteins upstream of
the RalA pathway, to promote filipodia extension
was dependent on the presence of filamin-1. In
vitro, the presence of RalA does not affect the
binding or cross-linking activity of filamin-1 on actin
filaments. In vivo, however, activated RalA is able
to recruit filamin-1 beneath the cellular membrane.
This concentration of filamin-1 could result in the
transition of its actin filament branching activity
observed at low concentrations to the filament
bundling activity reported for high concentrations of
filamin-1, thereby promoting the formation of
filopodia [35, 36]. Similarly, in individuals lacking
filamin-1 protein, the neurons, having completed
their last mitosis, would be impaired in their
capacity to extend filopodia. The filopodia are an
Neuronal Migration Disorders, Quo Vadis
essential element of the cell migration machinery,
probing the environment and transducing guiding
cues to the soma. In the absence of appropriate
guiding cues, the newly born neurons could remain
anchored at their site of origin, forming such
nodules as observed in PNH. Moreover,
modification of filamin-1 subcellular distribution via
protein-protein interaction, with RalA and 1 for
example, or via phosphorylation through kinases
such as Cdk5, could also be at the origin of
migration disorders.
Doublecortin and LIS1, Molecules Involved in
Lissencephaly and Subcortical Laminar
Heterotopia
LIS1 haplo-insufficiency is the first specific gene
defect demonstrated to be causative of
lissencephaly [48]. Microdeletions of chromosome
17p13.3 were already reported to be present in
more than 90% of individuals suffering of Miller-
Dieker syndrome, a type I lissencephaly associated
with dysmorphic facial appearance [49]. Although
the LIS1 gene is ubiquitously expressed, the levels
of expression are particularly high in the brain,
heart, muscle and kidney [48]. In humans, the ~80
kb gene is organized into 11 exons and two major
transcripts of 5.5 kb and 7.5 kb can be detected
[48]. The use of two different polyadenylation
signals is likely to be at the origin of the two
transcripts. The 45 kDa protein resulting from LIS1
expression has been identified as a non-catalytic
subunit of the platelet-activating factor
acetylhydrolase isoform 1b (PAFAH1B), a brain
specific isoform of PAFAH [50]. The main structural
feature of the protein is the presence of seven
WD40 repeats, often found in G-protein -subunits
[51]. However, the degree of similarity between
LIS1 and the other conventional G-protein -
subunits is relatively low (55%) [48]. The WD40
repeats in LIS1 suggest that the latter is involved in
multiple protein-protein interactions.
Detection of LIS1 proteins by immunohisto-
chemistry revealed a subcellular distribution that
closely resembled the microtubule network [51].
Further investigation demonstrated that LIS1 could
bind microtubules with an affinity similar to typical
microtubule-associated proteins (MAPs). In
addition, the use of amino-truncated LIS1 protein
indicates that this segment participates directly in
microtubule binding [51]. The influence of LIS1
protein on the microtubule dynamic was therefore
investigated. In vivo, microtubules constantly
oscillate between a polymerization phase and a
depolymerization phase. The duration of each
phase determines the average polymer length of
the microtubule pool. Using differential interference
contrast microscopy (DIC), Sapir and colleagues
[51] reported that LIS1 proteins could reduce the
frequency of polymerization to depolymerization
transition, referred to as the "catastrophe event",
Current Molecular Medicine, 2001, Vol. 1, No. 6 681
and thereby contribute to an increase of the
maximal polymer length measured in the
microtubules pool.
The LIS1 protein is extremely conserved
between mouse and human, with only one amino
acid difference [52]. LIS1 was also reported to be
serine-phosphorylated [53]. Phosphorylation is likely
to modulate the binding properties of LIS1 to
microtubules. The presence of multiple LIS1
phosphorylation isoforms was described when the
fraction of protein tightly bound to microtubules was
enriched. Therefore, the phosphorylation of LIS1 by
protein kinases could constitute a rapid mechanism
to control locally the polymerization dynamic of the
microtubules.
As mentioned above, the LIS1 protein has been
identified as a -subunit for the PAFAH1B. Although
the purification of microtubules allows for the
enrichment of LIS1 protein, the resulting fraction is
devoid of PAFAH activity [51]. Hence, LIS1 proteins
seem to be involved in two distinct pathways: first as
a subunit of the PAFAH1B, leading to the
inactivation of the platelet-activating factor (PAF),
and second as a microtubule-associated protein
(MAP), promoting the stabilization and elongation
of microtubules.
The presence of functional PAFAH1B is
essential during development as revealed by the
post-implantation embryonic lethality of mice
bearing a null mutation for the PAFAH1B -subunit,
i.e. the LIS1 protein [54]. Mice heterozygous for the
LIS1 gene present a blurred cortical layering, as
compared to the normal mice, and a trabeculated
neuronal distribution [54]. Nevertheless, the cortex,
marginal and white matter zones were not
significantly affected with regard to size and
cellularity. Since the mouse is a priori an animal
with lissencephaly, the study of the cortex is not
very informative in this respect. However, another
region of the human brain severely affected by
mutation of the LIS1 gene is the hippocampus. The
inactivation of one allele of LIS1 in mice leads to a
disorganization of the hippocampus, in which the
pyramidal cells do not pack properly and appear as
discontinuous split layers, especially in the CA1
region [54]. These histological abnormalities are
dosage dependent, since mating using different
LIS1 mutant alleles showed that the cerebral
defects are proportional to the decrease of LIS1
expression.
The hemidepletion of LIS1 does not lead to an
inversion of the cortical layering as is observed in
the Reeler mouse (see below). Labeling the neurons
born at specific time points using bromo-
deoxyuridine (BrdU) revealed a typical inside-out
construction of the cortical layers. However, the
waves of migrating neurons appear broader when
examined in newborn animals lacking one allele of
LIS1. In one month-old animals, however, the
682 Current Molecular Medicine, 2001, Vol. 1, No. 6
neurons reached their appropriate residing
positions, suggesting that migration was only slowed
down [54]. To investigate this issue, an in vitro assay
was performed using unilateral migration of
reaggregated cerebellar cells on a laminin
substrate [55]. In this setting, the migration of
neurons hemizigous for the LIS1 gene was severely
impaired when monitored 24h after plating.
Moreover, chimeric reaggregates demonstrated that
the LIS1 deficiency affects neurons in a cell-
autonomous fashion [54].
The mechanism by which LIS1 deficiency can
lead to migration disorders is not yet understood. It
was reported that extracellular application of PAF
provokes the collapse of the growth cones [56].
Therefore, an insufficient activity of the PAFAH1B
complex, caused by a reduction in LIS1 protein,
would result in an accumulation of PAF and
thereby to an impaired migration. The fact,
however, that LIS1 reduction affected neuronal
migration in a cell-autonomous fashion in the in
vitro migration assay does not support this
hypothesis. However, accumulation of PAF cannot
be ruled out as an aggravating factor leading to
neuronal migration disorder in vivo.
On the other hand, some lines of evidence point
to perturbation in mitosis and nucleokinesis of
proliferating neuronal precursors as a result of
reduction in LIS1 protein content. As mentioned
above, the timing of terminal mitosis and the
migration of neuronal nuclei are crucial aspects of
the cerebral development. In Drosophila, deletion
of one allele of LIS1 leads to inappropriate nucleus
positioning in the developing embryo [57].
Moreover, immunoprecipitation experiments
revealed that LIS1 proteins can interact with
dynein, a microtubule-anchored motor complex
[58, 59]. The interaction of LIS1 with the dynein
heavy-chain stimulated the activity of the dynein
motor complex, thereby modifying the distribution
of different organelles and also influencing the
microtubule-organizing center (MTOC) [59]. Since
the migration/positioning of the nucleus is proposed
to be a MTOC-dependent process [60], hindered
nuclear positioning could be at the origin of the
impeded migration reported. Furthermore, LIS1 was
shown to influence different aspects of mitosis [58].
For instance, cells with increased or decreased
LIS1 protein content display a significantly longer
duration of mitosis and problems with the proper
alignment of chromosomes on the metaphase
plate. Moreover, the overexpression of LIS1 clearly
perturb the mitotic spindle, leading to a
randomization of its orientation [58]. Together,
these observations indicate that aberrant levels of
LIS1 can perturbed the timing of the last neuronal
mitosis, its plane of symmetry and the rate of
migration towards the superior aspects of the
developing cortex. Although, the mechanism by
which LIS1 mutation leads to lissencephaly remains
Couillard-Despres et al.
to be elucidated, the protein is clearly involved in
the fundamental aspects of neuronal cell biology.
Recently, a new gene, doublecortin, linked with
another type of lissencephaly has been recently
reported. This type of lissencephaly is characterized
by the presence within the same pedigree of males
affected by classical lissencephaly and females
affected by a milder condition called subcortical
laminar heterotopia (SCLH). The females affected
by SCLH present a heterotopic band of neurons
located between the ventricles and the cortex,
producing a second band of gray matter, the so-
called double cortex (Fig. (2c)). Although the real
cortex appears normal, the neurons within the
supplementary band of gray matter appear
disorganized, with their apical dendrites pointing
either towards the cortex or towards the ventricles
[61]. Moreover, in the lower aspect of the
heterotopic band, the neurons are disposed in a
columnar organization, suggesting an arrest during
radial migration towards the cortex. Finally, at the
lower edge of the heterotopic neuron band, some
heterotopic neurons forming small nodules are
frequently found [61].
The pattern of inheritance of this type of
lissencephaly is characteristic for a X-linked genetic
defect and is referred to as SCLH/X-LIS. In 1998,
two groups reported simultaneously that mutations
in the doublecortin gene associated with inherited
and sporadic SCLH/X-LIS cases [62, 63]. Together,
mutations/deletions in the LIS1 and the
doublecortin genes account for approximately 76%
of all the sporadic cases of classical lissencephally
[64]. This new gene is localized on chromosome X
at the position X922.3-23, where it covers
approximately 85 kbp of genomic sequences. The
milder pathology observed in females bearing
doublecortin mutations is believed to be the
consequence of random X-chromosomal
inactivation. The neuroblasts in which the X-
chromosome bearing the wild type allele of the
gene is active will migrate properly to the cortex,
whereas the neuroblasts expressing the mutated
copy will display aberrant migration and form the
additional band of gray matter observed between
the cortex and the ventricles. This explanation is
based on a cell autonomous effect of the
mutations.
The doublecortin transcription product of 9.5 kb
originates from the alternative splicing of 9 exons
[62, 63]. Three exons (1A, 1B or 1C) can form the 5'
end of the mRNA. The second exon possesses the
initial ATG of a 1080 bp long open reading frame,
i.e. encoding a polypeptide of 360 amino acids.
Another potential ATG is located in the exon 1C.
Although not yet reported, the use of this codon as
translation start point would produce a polypeptide
bearing an additional 42 amino acids [62]. Two
other polymorphisms have also been observed in
doublecortin cDNAs. The first is an insertion that
Neuronal Migration Disorders, Quo Vadis
would result in 6 additional residues, Gly-Asn-Asp-
Glu-Asp-Ala, at the amino acid position 311,
whereas an alternative use of the splicing junctions
between exons 8 and 9 could result in the presence
of an additional valine residue [63]. One
particularity of the doublecortin transcript is its very
long 3' untranslated region. This sequence of
approximately 7.9 kb is encoded by a single exon
and contains two AU-rich elements (AREs). The
ARE sequences are often found within the 3' region
of labile mRNAs and are likely to influence their
stability [62].
In situ hybridizations and northern blot analysis
revealed that the doublecortin transcript is specific
for young neurons, and it is found to be particularly
abundant in neurons forming the cortical plate and
the subventricular region [62]. In mouse embryos,
the expression of the doublecortin transcript is first
detectable in the neuroepithelium at day E10.5
and can later be detected in various regions of the
CNS and PNS [65]. The fact that doublecortin
mutations predominantly affect the cortex, as
compared to the rest of the CNS and PNS, is
intriguing considering the widespread expression of
doublecortin in migrating neurons.
A polypeptide of 40 kDa is predicted by the
analysis of the doublecortin open reading frame.
The protein sequence does not bear significant
homology with known functional domains. It is,
however, highly homologous to another protein
designated as KIAA0369 [62, 63]. The amino-
terminal of this protein shares 75% similarity at the
amino acids level with the doublecortin
polypeptide. Interestingly the carboxy-terminal of
KIAA0369 encodes a calcium calmodulin-
dependent kinase (CaM kinase) domain.
Doublecortin sequence analysis also revealed
phosphorylation target sequences for MAP kinases
and Abl [62, 63]. In brains of embryonic day 12.5
and older mouse embryos, a fraction of the
doublecortin proteins has a slower electrophoretic
mobility on SDS-PAGE. The presence of this
fraction, forming a band-doublet, can be
Figure (3). Doublecortin affects the microtubular network.
COS-7 cells transiently expressing a doublecortin / red fluorescent (RFP) fusion protein. The same microscopic field is
shown for a) phase contrast, b) doublecortin-RFP, c) -tubulin and d) and overlay of doublecortin-RFP and -tubulin.
Doublecortin colocalizes with and bundles microtubules.
Current Molecular Medicine, 2001, Vol. 1, No. 6 683
eliminated by the pre-treatment of the protein
extract with alkaline phosphatase [65]. The sites
and kinases responsible for this in vivo
phosphorylation have not been identified to date.
Interestingly, the level of doublecortin
phosphorylation appears to be related to its
interaction with the microtubules. For example, in
vitro the depolymerization of the microtubule
network using nocodazole led to the disappearance
of the phosphorylated doublecortin protein doublet
[65].
Reports on the intra-cellular distribution of
doublecortin have been divergent. Using primary
neurons in culture, Francis and colleagues [65]
observed an enrichment of doublecortin in the
distal part of the neurites. On the other hand,
Gleeson and colleagues [66] reported the detection
of doublecortin in migrating Purkinje cells and
cultured neurons, predominantly at the periphery of
the cell soma with little immunoreactivity in the
neurites. Using cultures of human embryonic
cortical neural stem cells, we could detect high
levels of doublecortin along the entire length of
processes in newly differentiated neurons. There is
nevertheless agreement on the co-localization of
doublecortin and the microtubule network, without
apparent association to the actin or neurofilament
networks [65, 66]. In vitro experiments revealed that
doublecortin could interact directly with purified
tubulin subunits and thereby reduce the critical
concentration required for microtubule assembly
[65, 67]. Doublecortin facilitation of microtubule in
vitro polymerization results in an increase of total
number of microtubules, without an apparent effect
on the average length of the polymers [66]. This
activity is believed to originate from the
microtubule-bundling capacity of doublecortin.
This capacity can readily be observed in COS-7
cells overexpressing doublecortin (Fig. (3)). Upon
doublecortin expression, the microtubule network
reorganizes into large bundles. These microtubule
bundles become more resistant to depolymerizing
agents, such as colchicine [66], as well as to cold-
induced depolymerization [66, 68]. In parallel,
684 Current Molecular Medicine, 2001, Vol. 1, No. 6
over-expression of doublecortin also increases the
percentage of tubulin protein recovered in the
insoluble fraction. Whereas approximately half of
the tubulin dimers are normally recovered in the
soluble fraction when cells are homogenized in
mild conditions, the overexpression of doublecortin
increases this percentage to more than 80% [67].
The amino-terminus half of the protein appears to
encode the microtubule binding-domain. Although
no canonic microtubule-binding domain has been
detected by sequence analysis, a polypeptide
containing the first 198 amino acid can efficiently
bind to microtubules, whereas a polypeptide
representing the last 110 amino acids does
apparently not interact [67, 69, 70]. Expression of
several doublecortin mutants in non-neuronal cells
yielded variable phenotypes. While some mutants
induced similar microtubule bundling as observed
following overexpression of the wild type allele,
other mutants did not alter the microtubule
distribution, whereas a third group of doublecortin
mutants provoked protein aggregates [69, 70].
Therefore, the mechanism by which doublecortin
mutations lead to lissencephaly remains to be
elucidated.
Interestingly, a direct interaction between the
LIS1 and doublecortin proteins has been reported
[71]. It has been proposed that LIS1 can form
heterodimers with doublecortin, thereby competing
with doublecortin homodimerization. The impact of
doublecortin mutations was also scrutinized in
respect to the doublecortin-LIS1 interaction, but no
difference was observed when compared to the
behavior of doublecortin wild type allele [71].
Reelin and Lissencephaly
Some cases of autosomal recessive
lissencephaly with cerebellar hypoplasia from two
different pedigrees have recently been linked to
mutations in the reelin gene [72]. Deletion of this
protein was already reported to cause migration
disorders in an animal model, the reeler mouse [46].
The pathological similarities between some
individuals affected by recessive lissencephaly with
cerebellar hypoplasia and reeler mice prompted the
investigators to look for reelin mutations in the
selected families. The "reeler phenotype" consists
of a hypoplasia of the cerebellum and migration
disorders affecting the neocortex, the cerebellum,
the hippocampal formation and the brain stem.
One hallmark of this phenotype is the "inversion" of
the cortical layers, where the earlier born neurons
occupy a more superficial position in respect to the
later born neurons. At the origin of this aberrant
lamination is the failure of the first cortical plate
neurons to bisect the pre-plate into its two
components: the marginal zone and the subplate.
Each subsequent cohort of migrating neurons
simply accumulate beneath the preceding ones,
Couillard-Despres et al.
leading to an outside-in addition of new neurons
[73].
In humans, the reelin gene is localized on
chromosome 7q22 [46]. The 12kb reelin transcript is
encoded by 65 exons translated into a pro-protein
containing a cleavable signal peptide. At the
amino-terminus of the 388 kDa mature protein, a
domain sharing homology with F-sponding is
described, followed by 8 repeats containing EGF-
like motifs [46]. Reelin possesses a secretion signal
located at its carboxy-terminus, allowing for the
accumulation of the protein into the extracellular
matrix [74]. There are two potential reelin mRNA
polyadenylation signals reported, as well as two
alternative splice isoforms, only distinguished by the
presence or absence of 6 nucleotides from exon 64
[74, 75]. The presence of exon 64 adds two amino
acids, valine and serine, and therefore offers an
extra phosphorylation site. More substantial
changes are predicted for the use of the most 5'
polyadenylation signal, since the resulting mRNA
would encode a protein devoid of a secretion
signal. The significance of these various reelin
mRNA isoforms is still unknown. In vivo, a N-terminal
directed antibody detects three forms of reelin,
approx. 400 kDa, 300 kDa and 180 kDa. The two
smaller bands detected are likely to be cleavage
products of the full length reelin by a protease
located either in the post-endoplasmic reticulum
secretory pathway or in the extracellular space [76].
The Cajal-Retzius cells residing in the marginal
zone, i.e. layer 1 of the cortex, are among the first
post-mitotic neurons. They also represent one of the
main sources of reelin in the developing embryo.
Yet, the next generations of migrating neurons
building the cortical plate do not, or at very low
levels, express this protein [46]. Therefore, in
contrast to molecules presented in the two previous
sections, mutations in reelin cause migration
disorders in a non-cell autonomous fashion.
Recent studies shed more light on the possible
mechanisms of action of reelin on neuronal
migration. Dulabon and colleagues [77] reported
that reelin interacts with the 31 integrins present
on the migrating neurons of the cortical plate. In
response to reelin, the rate of neuronal migration
decreases and neurons detach from the radial glia.
Hence the vicinity of Cajal-Retzius cells producing
a reelin-filled extracellular milieu is interpreted by
neurons as a stop signal during their outward
migration on the radial glia scaffold.
Immunoprecipitation experiments established that
31 integrins can specifically bind reelin [77]. In
absence of this integrin type, the radial glia-guided
migration of embryonic neurons becomes
unresponsive to the presence of reelin. In neurons,
the 31integrin colocalizes with a cytoplasmic
molecule named mammalian Disabled (mDab1)
[77]. The spontaneous null mutation of this
molecule described in the scrambler mouse
Neuronal Migration Disorders, Quo Vadis
provokes a phenotype equivalent to the reeler
phenotype, suggesting that the molecules are
components of the same signaling pathway [78,
79]. Two other observations substantiate this
hypothesis. First, in response to the presence of
reelin, mDab1 becomes tyrosine-phosphorylated
[80]. In contrast, in the absence of reelin, such as in
the reeler mouse, mDab1 accumulated at levels
several times higher than those observed in normal
mice [81]. Although mDab1 is a cytoplasmic
protein, it possesses a protein interaction /
phosphotyrosine-binding domain, allowing for
interaction with the cytoplasmic moiety of
transmembrane receptors [82]. In response to reelin,
the phosphorylated mDab1 can interact with non-
receptor tyrosine kinases such as Abl, Fyn and Src
[82]. Surprisingly, in the absence of 31 integrin
as a consequence of 3 integrin targeted
disruption, a reduction of approximately 50% in
mDab1 protein content is described [77]. This
contrasts with the reeler mice, in which an
accumulation of mDab1 is reported [81]. Therefore,
the integrins and mDab1 are likely to be part of a
common reelin-signaling pathway, where 31 and
reelin directly or indirectly stabilize mDab1. Yet,
application of reelin on cortical embryonic neurons
Figure (4). Schematic representation of molecules involved in the regulation of neuronal precursor cell migration.
Doublecortin, LIS1 and FLN1 are intracellular molecules and mutations in their genes will cause cell-autonomous migration
impairment. In contrast, Reelin is a protein secreted in the extracellular matrix and arrests the migration of the incoming
neuroblasts.
Current Molecular Medicine, 2001, Vol. 1, No. 6 685
lacking 31 integrin nevertheless led to extensive
phosphorylation of mDab1. This indicates that in
addition to 31 integrin, other molecules are
involved in the signal transduction pathway of
reelin across the cytoplasmic membrane.
The very low-density lipoprotein receptor
(VLDLR) and the apolipoprotein E receptor 2
(ApoER2), two members of the low-density
lipoprotein receptor family, have also been
presented as receptors for reelin [83]. These two
receptors are closely related, suggesting possible
functional overlap. Pull-down experiments
demonstrated the mDab1 could directly bind to the
cytoplasmic tail of the VLDLR and the ApoER2
[84]. Although the disruption of either receptor
leads to a mild alteration of the brain
cytoarchitecture, the simultaneous gene
inactivation of the VLDLR and ApoER2 lead to an
aberrant neuronal development analogous to the
reeler/scrambler phenotype, comprising of a
hypoplasia of the cerebellum and inversion of the
cortical layers [84]. Moreover, similarly to the reeler
mice, the double-knockout animals for VLDLR and
ApoE2R show a significant increase in the
accumulated levels of mDab1 proteins.
686 Current Molecular Medicine, 2001, Vol. 1, No. 6
Unfortunately, the level of phosphorylation of
mDab1 was not investigated in these various
knockout animals [84]. However, in a parallel study,
indirect evidence was presented to demonstrate
that competition for the binding of reelin on VLDLR
and ApoER2 reduces the levels of mDab1 tyrosine
phosphorylation [85]. Hiesberger and colleagues
also report in the same study that in absence of
reelin, as well as in the double VLDLR/ApoER2
knockout, the level of phosphorylation of Tau
protein in the brain was greatly increased when
compared to the normal mouse. It is hypothesized
that an increase of Tau phosphorylation would lead
to a decrease in microtubule stability and therefore
affect the axonal cytoskeleton. However, the
histological analysis of mice in which the Tau gene
is selectively targeted did not reveal neuronal
migration impairment [86].
Figure (4) constitutes an attempt to link the
various molecules described in this review into a
schematic representation. Animals bearing
disruption of the p35/cdk5 complex present a
phenotype similar to the reeler mouse [45, 87]. To
date, however, no mutations in these genes were
reported to be linked with migration disorders in
human. Still, animal models bearing genetic
defects that cause neuronal migration disorders are
extremely valuable tools. For instance, some
spontaneous mutations causing migration disorders
may originate from mutations in pathways also
affected in human migrations disorders, albeit at a
different level. Caution must be taken, however,
because defective pathways could affect human
corticogenesis in a different way due to intrinsic
molecular and cytoarchitectural differences. For
example, cortex development in mice bearing a
hemi-depletion of LIS1 is only delayed, whereas in
human, cortical development is completely
disturbed.
ACKNOWLEDGEMENT
We would like to thank Christiana Cooper-Kuhn
helping in preparing the manuscript. We thank Dr.
Ulrich Bogdahn as the head of the Dept. for
Neurology, University of Regensburg, for support.
The work was further supported by the Volkswagen-
Foundation, Hannover, Germany and by the Fritz-
Thyssen Foundation, Kln, Germany.
LIST OF ABBREVIATIONS
AREs = AU-rich elements
ApoER2 = Apolipoprotein E receptor 2
BrdU = Bromo-deoxyuridine
MAP = Microtubule-associated protein
Couillard-Despres et al.
MRI = Magnetic resonance imaging
MTOC = Microtubule-organizing center
mDab1 = Mammalian Disabled
PET = Positron emission tomography
SCLH = Subcortical laminar heterotopia
PNH = Periventricular nodular heterotopia
VLDLR = Very low-density lipoprotein receptor
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