Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a member of
the deubiquitylating enzymes and is one of the most abundant
proteins in the brain. Whereas other UCH members are
ubiquitously expressed, UCH-L1 is selectively expressed in
neurons and testes/ovaries in the adult (Wilkinson et al.,
1989). UCH-L1 is also known as PGP9.5 and is used as a
neuropathological studies (Dickson et al., 1994; McQuaid et
al., 1995). Recent studies suggest that UCH-L1 is involved in
neurodegeneration. The I93M mutation and the S18Y
polymorphism in UCH-L1 are implicated in Parkinson’s
disease (Leroy et al., 1998; Satoh and Kuroda, 2001). Using
gracile axonal dystrophy (gad) mice, we previously
demonstrated that the dying-back type of axonal degeneration
is caused by a deletion of the Uchl1 gene (Saigoh et al., 1999).
UCH-L1 has an affinity for ubiquitin and ensures its stability
within neurons in vivo (Osaka et al., 2003). Furthermore,
UCH-L1 has ubiquitin ligase activity (Liu et al., 2002). Thus,
in neuroanatomical and
UCH-L1 might have multiple functions and more roles in
biological phenomena than previously expected.
UCH-L1 mRNA is first detected at embryonic day (E) 8.5-
9 in the neural tube and in the neural epithelium (Schofield et
al., 1995). In addition, UCH-L1 immunoreactivity has been
observed in the neural tube at E10.5 (Sekiguchi et al., 2003).
However, its functional role in embryonic neurogenesis is not
well understood. CDK5 and Dab1 are involved in regulating
the migratory behavior of postmitotic neurons. Both p35,
which is a CDK5 kinase, and Dab1 are degraded by the
ubiquitin-proteasome pathway (Arnaud et al., 2003; Bock et
al., 2004; Patrick et al., 1998). Thus, the ubiquitin system might
be important in the migration and differentiation of postmitotic
neurons and for the lamination pattern of the cerebral cortex.
Neural progenitor cells (NPCs) differentiate into neurons,
astrocytes and oligodendrocytes (Qian et al., 1998; Qian et al.,
2000; Shen et al., 1998). In the embryonic brain,
neuroepithelial cells and radial glia are present in the
ventricular zone (VZ); neurogenesis occurs first, followed by
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a
component of the ubiquitin system, which has a
fundamental role in regulating various biological activities.
However, the functional role of the ubiquitin system in
neurogenesis is not known. Here we show that UCH-L1
regulates the morphology of neural progenitor cells (NPCs)
and mediates neurogenesis. UCH-L1 was expressed in
cultured NPCs as well as in embryonic brain. Its expression
pattern in the ventricular zone (VZ) changed between
embryonic day (E) 14 and E16, which corresponds to the
transition from neurogenesis to gliogenesis. At E14, UCH-
L1 was highly expressed in the ventricular zone, where
neurogenesis actively occurs; whereas its expression was
prominent in the cortical plate at E16. UCH-L1 was very
weakly detected in the VZ at E16, which corresponds to the
start of gliogenesis. In cultured proliferating NPCs, UCH-
L1 was co-expressed with nestin, a marker of
undifferentiated cells. In differentiating cells, UCH-L1 was
highly co-expressed with the early neuronal marker TuJ1.
Furthermore, when UCH-L1 was induced in nestin-positive
progenitor cells, the number and length of cellular
processes of the progenitors decreased, suggesting that the
progenitor cells were differentiating. In addition, NPCs
derived from gad (UCH-L1-deficient) mice had longer
processes compared with controls. The ability of UCH-L1
to regulate the morphology of nestin-positive progenitors
was dependent on its binding affinity for ubiquitin but not
on hydrolase activity; this result was also confirmed using
gad-mouse-derived NPCs. These results suggest that UCH-
L1 spatially mediates and enhances neurogenesis in the
embryonic brain by regulating progenitor cell morphology.
Key words: PGP9.5, UCH-L1, Nestin, Ubiquitin, Cell morphology,
Ubiquitin C-terminal hydrolase L1 regulates the
morphology of neural progenitor cells and modulates
Mikako Sakurai1,2, Koichi Ayukawa1, Rieko Setsuie1,2, Kaori Nishikawa1, Yoko Hara1, Hiroki Ohashi1,3,
Mika Nishimoto1,4, Toshiaki Abe3, Yoshihisa Kudo4, Masayuki Sekiguchi1, Yae Sato1,2, Shunsuke Aoki1,
Mami Noda2and Keiji Wada1,*
1Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira,
Tokyo, 187-8502, Japan
2Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan
3Department of Neurosurgery, Graduate School of Medicine, Jikei University School of Medicine, Minato-ku, Tokyo, 105-8461, Japan
4Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392, Japan
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 27 September 2005
Journal of Cell Science 119, 162-171 Published by The Company of Biologists 2006
Journal of Cell Science
UCH-L1 spatially mediates and enhances neurogenesis in the embryonic brain
gliogenesis. Committed progenitor cells move from the VZ to
the cortical plate (CP) (Noctor et al., 2004). The differentiating
cells migrate by means of radial migration, during which the
migrating cells change their morphology (Kawauchi et al.,
2003; Noctor et al., 2002; Tabata and Nakajima, 2003). Here,
we analyzed the functional role of UCH-L1 using mouse
embryonic NPCs. Our results indicate that UCH-L1 is
expressed in nestin-positive NPCs and might regulate
neurogenesis. The expression pattern of UCH-L1 changed in
parallel with the transition from neuronal generation to glial
generation. Furthermore, UCH-L1 modulated the length of
nestin-positive processes in NPCs. Our results constitute the
first evidence that UCH-L1 is important in neurogenesis and
thus provide the basis for further investigation into the role of
the ubiquitin system in neurogenesis.
UCH-L1 expression in embryonic mouse brain
We first determined the specificity of the UCH-L1 antibody
using immunoblotting (data not shown) and immunostaining.
Because gad mice do not express endogenous UCH-L1
(Saigoh et al., 1999), we used these mice as a negative control.
Heterozygous littermates had UCH-L1 immunostaining,
whereas UCH-L1 immunoreactivity was not detected in the
brains of gad mice (Fig. 1). These results confirmed the
specificity of the antibody against UCH-L1. Using this
antibody, we further compared the distribution and expression
of UCH-L1 with the neural progenitor marker nestin and the
early neuronal marker TuJ1. Nestin was expressed in the VZ
of brains from both gad and heterozygous mice at E13 (Fig.
1). Nestin expression was observed throughout the region,
whereas TuJ1 immunoreactivity was detected at the marginal
zone (MZ). In heterozygous mice, UCH-L1 and nestin
immunostaining overlapped in almost all cells in the VZ,
suggesting that UCH-L1 is expressed in NPCs (Fig. 1A). TuJ1
expression colocalized with that of UCH-L1 in MZ cells,
indicating that UCH-L1 is expressed in embryonic neurons as
well (Fig. 1B). In E13 gad mouse brain, nestin staining differed
compared with that in heterozygous littermates. Nestin staining
was observed in many long radial fibers in the mutant, which
we believed were radial glia; by contrast, staining in the
heterozygotes occurred in radial glia as well as in neuronal
cells at various stages of development (Fig. 1A; arrow and
We then looked for developmental changes in UCH-L1
expression. In the embryonic cerebral cortex, asymmetric cell
division generates one neuron and one neural progenitor
(Roegiers and Jan, 2004; Zhong et al., 1996; Zhong et al.,
1997). These asymmetric cell divisions begin at E11, peak
around E14, and subside after E16. At E14, astrocytes and
oligodendrocytes are not yet present. However, at E16, glial
cell production begins. The regional expression level for both
nestin and TuJ1 did not change between E14 and E16 (Fig.
2A,B). At E14 and E16, nestin immunoreactivity was stronger
in the VZ (Fig. 2A) and was faintly detected only along radial
glial fibers in the CP (Fig. 2A,C; arrowhead) (Malatesta et al.,
2003; Malatesta et al., 2000). TuJ1 immunoreactivity was
predominantly detected in the MZ, CP, intermediate zone and
subventricular zone at E14 and E16 (Fig. 2B,D). In the VZ,
TuJ1 immunoreactivity was detected only in migrating neurons
(Fig. 2D; arrowhead).
By contrast, the pattern of UCH-L1 expression changed
between E14 and E16 (Fig. 2A,B). At both stages of
development, UCH-L1 was expressed in neuronal cells as well
as in progenitor cells. UCH-L1 immunoreactivity was stronger
in the VZ than in the CP at E14; however, the immunoreactivity
Fig. 1. Antibody specificity and expression of UCH-L1 in the
ventricular zone at E13. UCH-L1 expression was detected using
immunohistochemistry with anti-PGP9.5. UCH-L1 is not detected
in the brain of gad mice at E13 (A,B) but is strongly expressed in
heterozygous littermates (A,B). Confocal microscopic images of
coronal sections of gad mice and heterozygous littermates were
double stained with antibodies for the progenitor marker nestin and
UCH-L1 (PGP9.5) (A) or for the early neuronal marker tubulin ?
III (TuJ1) and UCH-L1 (B). Long radial fibers are indicated by
arrowheads, and various phases of progenitor cells are indicated by
arrows (A). TuJ1-positive, migrating neuronal cells are indicated by
arrowheads (B). MZ, marginal zone; VZ, ventricular zone. Bars,
Journal of Cell Science
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