peptides 28 (2007) 17461752

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Neurotrophic effects of PACAP in the cerebellar cortex

Beatrice Botia a,b,c, Magali Basille a,b,c, Aurelie Allais a,b,c, Emilie Raoult a,b,c,
Anthony Falluel-Morel a,b,c, Ludovic Galas c, Valerie Jolivel a,b,c, Olivier Wurtz a,b,c,
Hitoshi Komuro d, Alain Fournier a,e, Hubert Vaudry a,b,c,*, Delphine Burel a,b,c,
Bruno J. Gonzalez a,c, David Vaudry a,b,c
a

Inserm U413, International Associated Laboratory Samuel de Champlain, 76821 Mont-Saint-Aignan, France
Laboratory of Cellular and Molecular Neuroendocrinology, University of Rouen, 76821 Mont-Saint-Aignan, France
c
European Institute for Peptide Research (IFRMP23), University of Rouen, 76821 Mont-Saint-Aignan, France
d
Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
e
INRSInstitut Armand-Frappier, University of Quebec, Pointe-Claire, H9R 1G6, Canada
b

article info

abstract

Article history:

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are

Received 15 March 2007

present on granule cells during both the development period and in adulthood. Treatment of

Received in revised form

granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and

16 April 2007

induces differentiation. PACAP also protects cerebellar granule cells against the deleterious

Accepted 24 April 2007

effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated

Published on line 1 May 2007

through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but

Keywords:

PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional

Cerebellar granule neurons

PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of

PACAP

expression very similar to that described in rodents, suggesting that PACAP could also exert

cAMP/PKA

neurodevelopmental and neuroprotective functions in the cerebellum of primates including

Apoptosis

human.

Caspase-3

# 2007 Elsevier Inc. All rights reserved.

Neuroprotection

1.

PACAP and its receptors

Pituitary adenylyl cyclase-activating polypeptide (PACAP) has

been originally isolated from the ovine hypothalamus on the
basis of its ability to stimulate cAMP formation in rat anterior
pituitary cells [36]. PACAP belongs to the vasoactive intestinal
polypeptide (VIP)-glucagon-secretin superfamily that also

comprises growth hormone-releasing hormone and helodermin [50]. The sequence of PACAP has been remarkably well
conserved during evolution, suggesting that this peptide is
involved in the regulation of important biological functions
[50]. The actions of PACAP are mediated through three
receptor subtypes i.e. the PACAP-selective receptor PAC1,
and the PACAP/VIP mutual receptors VPAC1 and VPAC2 [25].

* Corresponding author. Tel.: +33 235 14 6624; fax: +33 235 14 6946.
E-mail address: hubert.vaudry@univ-rouen.fr (H. Vaudry).
Abbreviations: Bcl-2, B-cell lymphoma 2; cAMP, cyclic adenosine-mono-phosphate; EGL, external granule cell layer; ERK, extracellular
signal-regulated kinase; IGL, internal granule cell layer; MAPK, mitogen-activated protein kinase; PACAP, pituitary adenylyl cyclaseactivating polypeptide; PAC1, type-1 PACAP receptor; PKA, protein kinase A; Shh, Sonic hedgehog; VIP, vasoactive intestinal polypeptide;
VPAC1, type-1 VIP/PACAP receptor; VPAC2, type-2 VIP/PACAP receptor
0196-9781/$ see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2007.04.013

peptides 28 (2007) 17461752

All three receptors are positively coupled to the adenylyl

cyclase, and the PAC1 receptor also stimulates the phospholipase C and the extracellular regulated kinase (ERK) pathways
[46]. PACAP and its receptors are widely distributed in the
brain and peripheral organs, and the peptide has been found to
exert pleiotropic effects on hormonal release, vasodilation,
regulation of the inflammatory process and neuronal survival
[56]. This report reviews the current knowledge concerning the
neurotrophic activities of PACAP in the cerebellar cortex.

2.
Expression of PACAP and its receptors in
the rodent cerebellum
Early studies have shown that, in the brain, the highest
concentrations of PACAP-like immunoreactivity occur in the
hypothalamus and cerebellum [30]. PACAP is expressed in the
rat brain as early as embryonic day 14 (E14) and the
concentration of the peptide gradually increases throughout
pre- and postnatal development [45]. In the cerebellum, the
concentration of PACAP culminates after birth and declines to
reach adult levels within a few weeks [38]. In newborn rat, a
few PACAP-containing cells and PACAP-positive nerve fibers
are localized in the Purkinje cell layer. In the postnatal day 7
(P7) and adult rat cerebellum, PACAP is present in most
Purkinje cells soma and dendrites as well as in nerve fibers
surrounding granule cells [38]. The expression of PACAP by
Purkinje neurons has been confirmed by the detection of
PACAP precursor mRNA in these neurons [40].
Soon after the discovery of the peptide, the presence of
PACAP binding sites has been demonstrated in the rat
hypothalamus, cerebellum and brainstem [32] as well as in
postmortem human brain tissues [42]. The highest densities of
recognition sites are found in the rat hypothalamus and
brainstem but substantial binding also occurs in the cerebellum. Autoradiographic studies revealed that the distribution
pattern of PACAP binding sites markedly differs from that of
VIP recognition sites [34]. In the perinatal rat cerebellum,
PACAP binding sites are transiently expressed in a germinative
matrix, the external granule cell layer (EGL), and in the
medulla, from P8 to P25 [9,10]. PACAP binding sites are also
present in the internal granule cell layer (IGL) in both

1747

developing and in the mature cerebellum [9]. Emulsion-coated

cytoautoradiography, performed on cultured granule cells
from P8 rat cerebellum, demonstrated that high levels of
PACAP binding sites are expressed by granule neurons [10]. In
situ hybridization studies have shown that, in P8 and P12 rats,
both PAC1 and VPAC1 mRNAs, but not VPAC2 mRNAs, are
expressed in the EGL [11]. Treatment of cultured granule cells
with PACAP induces a dose-dependent stimulation of cAMP
production (ED50  0.2 nM), indicating that PACAP binding
sites correspond to functional receptors [10]. Besides adenylyl
cyclase, PACAP also stimulates phospholipase C activity in
granule neurons leading to polyphosphoinositide breakdown
(ED50  20 nM) [8,22].

3.
Effects of PACAP on cell survival,
differentiation and proliferation in vitro
PACAP has been shown to exert neuroprotective activities
both on immature granule cells [14,24] and differentiated
cerebellar neurons [54]. When grown in serum free medium,
most granule cells die within 48 h of culture and those which
survive exhibit very short neurites (Fig. 1). Treatment of the
same cells with subnanomolar concentrations of PACAP
promotes cell survival [14,24] and induces the appearance of
a dense network of long neurites (Fig. 1) [24]. The differentiation of granule cells is associated with an accumulation of
actin at the emergence cone and phosphorylation of the tau
protein [20]. The fact that VIP is at least 1000 times less potent
than PACAP in promoting granule cell survival and differentiation suggests that the neurotrophic effects of PACAP are
mediated through PAC1 receptor. In support of this hypothesis, it has been shown that granule cells primarily express the
short and hop variants of the PAC1 receptor [14,17]. The
expression of these receptors can be regulated as it has been
shown that several neurotrophins including nerve growth
factor or insulin-like growth factor-1 can increase the
expression level of the PAC1 receptor in the rat cerebellum
[28].
The neurotrophic activity of PACAP on granule cells is
mimicked by cAMP stimulators or PACAP analogs [15,55] and
blocked by a dominant negative mutant of cAMP-dependent

Fig. 1 Micrographs illustrating the effect of PACAP on cell survival and neurite outgrowth in rat cerebellar granule cells.
Granule neurons from 8-day-old (P8) rats were cultured for 48 h in control conditions in serum free medium (left) or in the
presence of 10S8 M PACAP (right). Scale bar: 25 mm. Reprinted from Gonzalez et al. [24].

1748

peptides 28 (2007) 17461752

protein kinase A (PKA) [29]. Downstream of PKA, PACAP

induces the phosphorylation of ERK through Rap1 and Ras
activation (Fig. 2) [39]. ERK activation is required for the long
lasting inhibition of caspase-3 activity by PACAP [19,49] and

Fig. 2 Schematic representation of the intracellular

mechanisms involved in the neurotrophic activity of
PACAP on cerebellar granule cells. Full lines indicate some
established pathways. Dotted lines represent transduction
pathways potentially involved in the neurotrophic effects
of PACAP.#: Activation; ?, inhibition; AC, adenylyl cyclase;
Apaf-1, apoptotic protease activating factor-1; ATP,
adenosine triphosphate; Bcl-2, B-cell lymphoma 2; cAMP,
cyclic adenosine-monophosphate; caspase, cysteinylaspartate-cleaving protease; CREB, cAMP response
elementbinding protein; DAG, diacylglycerol; Epac,
exchange factor directly activated by cAMP; ERK,
extracellular signal-regulated kinase; IP3, inositol 1,4,5triphosphate; MEK, mitogen-activated protein kinase;
PAC1, type-1 PACAP receptor; PACAP, pituitary adenylyl
cyclase-activating polypeptide; PI3K, phosphatidylinositol
30 -OH kinase; PKA, protein kinase A; PKC, protein kinase
C; PLC, phospholipase C.

contributes to the neuroprotective effect of PACAP [54]. In fact

inhibition of caspase-3 activity is sufficient to mimic the effect
of PACAP on granule cell survival and differentiation [49]. It
should be pointed out that PACAP has also been shown to
rescue cerebellar granule cells from the deleterious actions of
toxic molecules such as 4-hydroxynonenal [27], ethanol [53],
hydrogen peroxide [52] and ceramides [19,46].
It has long been known that PACAP regulates c-fos gene
expression through the PKA pathway (Fig. 2) [47]. More
recently, it has been shown that activation of c-fos transcription is involved in the neuroprotective activity of PACAP [6] by
regulating B-cell lymphoma 2 (Bcl-2) expression [6,19]. Downstream of Bcl-2, PACAP prevents cytochrome C release and
inhibits caspase-9 activation, which in turn regulates caspase3 (Fig. 2). This signaling cascade appears to be essential for the
maintenance of mitochondrial activity [52]. PACAP inhibits
potassium channels, and the potassium channel blocker
triethanolamine (TEA) prevents ethanol-induced cell death
and caspase-3 activation, which indicates that regulation of
caspase-3 activity is at least in part under the control of
potassium currents [35]. Finally, it has been proposed that the
phosphatidylinositol 30 -OH kinase neuroprotective pathway
may synergize with the PKA cascade to promote granule cell
survival after PACAP treatment (Fig. 2) [12]. CREB which is
rapidly phosphorylated by PACAP [12] may participate to this
neurotrophic activity (Fig. 2).
A recent study indicates that PACAP acting through
exchange factor directly activated by cAMP (Epac), stimulates
Rap and p38 MAPK, which mobilize intracellular Ca2+ stores
(Fig. 2) [41]. This new pathway may facilitate the maturation of
granule precursors into excitable neurons. In support of this
notion, PACAP has been shown to enhance the release of
glutamate induced by granule cells depolarization [4]. Activation of calcium influx through L-type voltage-dependent
calcium channels (L-VDCCs) by PACAP also induces VIP
expression [23]. In addition to VIP, PACAP can increase its
own expression thus promoting in an autocrine manner
cerebellar granule cell survival and turning an acute exposure
into a long term action [43,44,51].
In the EGL, granule cells proliferate actively and then stop
to divide to migrate through the different layers of the
cerebellar cortex [3]. Although the different stages of cerebellar development are well described, the molecular mechanisms that transition granule neurons from proliferation to
differentiation remain largely unknown. Sonic hedgehog
(Shh), which is produced by Purkinje neurons, stimulates
granule cell proliferation [18]. The effect of Shh on granule
cells is suppressed by increasing cAMP and activating PKA
[21,26]. It has been shown that PACAP markedly inhibits Shhinduced thymidine incorporation in both rat and mouse
granule neurons [37]. The anti-proliferative action of PACAP
on granule cell is similar to that previously reported on cortical
neuroblasts [33]. Concurrently, the zinc finger transcription
factor Lot1, which acts as a tumor suppressor gene, is regulated
by PACAP in a cAMP-dependent manner through the PKA and
ERK signaling cascade [16]. Whether Lot1 is actually responsible for the growth inhibitory effect of PACAP on granule
neurons remains to be determined.
Besides its activities as a neurotrophic factor, PACAP
induces transient expression of the clock gene Per1 in

peptides 28 (2007) 17461752

cerebellar granule cells through a cAMP/PKA-dependent but

ERK-independent pathway [1]. Although the function of Per1 in
the cerebellum is still unknown, it may contribute to the
effects of PACAP in the control of circadian rhythms.

4.

Effects of PACAP on cell migration

Another important aspect of the postnatal development of the

cerebellar cortex is the migration of immature granule
neurons from the EGL towards the inner part of the IGL [31].
Time-lapse videomicroscopy recording of cultured granule
cells has shown that PACAP reduces cell motility [13,20]. A
recent study conducted on organotypic tissue slices has
revealed that PACAP inhibits granule cell migration in the
EGL and the molecular layer [13,20]. The average rate of
granule cell migration in the molecular layer decreased from
16.6 mm/h in control conditions to 5.2 mm/h in the presence of
1 mM PACAP. This action of PACAP is mediated through both
the adenylyl cyclase and phospholipase C pathways. Although
the effect of PACAP on cell migration is robust, it only lasts for
about 2 h as the result of a desensitization process that
involves protein kinase C activation. This finding may explain
why a 1 h treatment with PACAP is sufficient to induce a
maximum neuroprotective effect on cultured granule neurons
[47].
It has previously been reported that granule cell migration
slows down at the level of Purkinje neurons where cell soma
remain stationary for approximately 2 h [31] but little is known
regarding the signaling mechanisms underlying this transient
pause. Interestingly, the PACAP antagonist PACAP(638) accelerates granule cell migration in the Purkinje cell layer, but does
not modify the velocity of granule neurons in the EGL, the
molecular layer and the IGL [13]. These data revealed that
PACAP is the first regulatory messenger that has been shown so
far to regulate the migration process of granule cells at the level
of Purkinje neurons. The observation that, in the rat cerebellum,

1749

PACAP is expressed by Purkinje neurons strongly suggests that

endogenous PACAP plays a physiological role in the control of
granule cell migration. The functional significance of this pause
of granule neurons at the level of Purkinje cells during
development remains to be elucidated. PACAP signaling may
be a key process for final neuronal migratory steps in the
absence of radial glial guidance within the IGL

5.
PACAP acts in vivo on rat cerebellar granule
cells during development
Injection of PACAP in the subarachnoid space, at the surface of
the cerebellum of P8 pups, induces a transient increase of the
volume of the cerebellar cortex with a maximum effect at P12,
after 4 days of treatment [48]. The action of PACAP can be
essentially accounted for by an enlargement of the thickness
of the IGL due to a 14% increase of the number of granule cells
(Fig. 3). The neurotrophic effect of PACAP is abolished by the
antagonist PACAP(638) that induces by itself a slight inhibition of the number of granule cells in the IGL, suggesting that
endogenous PACAP may exert a physiological role in the
development of the cerebellum [48]. Consistent with this
notion, a recent study has revealed that PACAP knockout mice
exhibit a significant reduction of the thickness of the EGL at P4
(27%) and IGL at P7 (33%) associated with a decrease of
synaptophysin expression in the molecular layer and IGL and
an increase of caspase-3 activity [2].

6.
Expression of functional PACAP receptors
in the primate cerebellum
In order to investigate whether PACAP could also exert
neurotrophic and/or neuroprotective activities in the brain
of primates, the expression of PACAP and its receptors has
been investigated in the marmoset, macaque and human

Fig. 3 Effects of PACAP on the histogenesis of the cerebellar cortex. Eight-day-old (P8) rats were injected with saline
(control) or 1 mg PACAP at the surface of the cerebellar cortex, and the thickness of the external granule cell layer (EGL),
molecular layer (Mol) and internal granule cell layer (IGL) were visualized at P12. Scale bar: 100 mm. Reprinted from Vaudry
et al. [48].

1750

peptides 28 (2007) 17461752

cerebellum. Immunohistochemical labelling has shown that,

in monkeys, PACAP is located in Purkinje cells and in radial
glial fibers [5]. In the cerebellum of primates, the mRNAs
encoding PAC1 and VPAC1 receptors are actively expressed in
granule cells and VPAC1 mRNA is also expressed by Purkinje
cells. Similarly, in humans, PAC1 and VPAC1 receptors are
expressed in the EGL and IGL of the cerebellum from 15-weekold fetuses to adults [7]. The distribution patterns of these
receptors are very similar to those described in rodents [11]
and monkeys [5]. Pharmacological characterization of PACAP
binding sites in the human cerebellum has shown that, in the
EGL and IGL, these receptors exhibit the typical profile of PAC1
whereas the cerebellar medulla of adults possesses a
heterogeneous population of PAC1 and VPAC receptors [7]
indicating that the peptide may exert different stage-related
functions. Nevertheless, the VPAC2 receptor is only
expressed at a low level in the adult human cerebellum.
Functional studies have demonstrated that PACAP is a potent
activator of adenylyl cyclase in tissue slices of macaque
cerebellum [5] suggesting that the neuroprotective effects of
PACAP documented in rodents may have parallels in primates
including humans.

7.

Conclusion

The expression of PACAP in Purkinje cells, the ocurrence of

PAC1 and VPAC1 receptors in granule neurons, and the effects
of PACAP on granule cell survival, migration and differentiation indicate that PACAP is a major regulator of the
histogenesis of the cerebellar cortex in rodents. Studies
conducted in monkeys and human suggest that the neurotrophic and neuroprotective effects of PACAP could be
extrapolated to the cerebellum of primates. As part of the
mechanisms involved in the control of neuronal cell death
occurring during proper brain development are similar to
those observed in some neurodegenerative diseases, the
present observations support the view that selective and
stable PACAP agonists may have a therapeutic potential for
the treatment of such pathologies.

references

[1] Akiyama M, Minami Y, Nakajima T, Moriya T, Shibata S.

Calcium and pituitary adenylate cyclase-activating
polypeptide induced expression of circadian clock gene
mPer1 in the mouse cerebellar granule cell culture. J
Neurochem 2001;78:499508.
[2] Allais A, Burel D, Isaac ER, Gray SL, Basille M, Ravni A et al.
Altered cerebellar development in mice lacking pituitary
adenylate cyclase-activating polypeptide. Eur J Neurosci, in
press.
[3] Altman J. Postnatal development of the cerebellar cortex in
the rat 3. Maturation of the components of the granular
layer. J Comp Neurol 1972;145:465513.
[4] Aoyagi K, Takahashi M. Pituitary adenylate cyclaseactivating polypeptide enhances Ca(2+)-dependent
neurotransmitter release from PC12 cells and cultured
cerebellar granule cells without affecting intracellular
Ca(2+) mobilization. Biochem Biophys Res Commun
2001;286:64651.

[5] Aubert N, Basille M, Falluel-Morel A, Vaudry D, Bucharles C,

Jolivel V et al. Molecular, cellular and functional
characterizations of PACAP and its receptors in the
cerebellum of new and old world monkeys. J Comp Neurol,
in press.
[6] Aubert N, Falluel-Morel A, Vaudry D, Xifro X, RodriguezAlvarez J, Fisch C, et al. PACAP and C2-ceramide generate
different AP-1 complexes through a MAP-kinase-dependent
pathway: involvement of c-Fos in PACAP-induced Bcl-2
expression. J Neurochem 2006;99:123750.
[7] Basille M, Falluel-Morel A, Vaudry D, Aubert N, Fournier A,
Freger P, et al. Ontogeny of PACAP receptors in the human
cerebellum: perspectives of therapeutic applications. Regul
Pept 2006;137:2733.
[8] Basille M, Gonzalez BJ, Desrues L, Demas M, Fournier A,
Vaudry H. Pituitary adenylate cyclase-activating
polypeptide (PACAP) stimulates adenylyl cyclase and
phospholipase C activity in rat cerebellar neuroblasts. J
Neurochem 1995;65:131824.
[9] Basille M, Gonzalez BJ, Fournier A, Vaudry H. Ontogeny of
pituitary adenylate cyclase-activating polypeptide (PACAP)
receptors in the rat cerebellum: a quantitative
autoradiographic study. Dev Brain Res 1994;82:
819.
[10] Basille M, Gonzalez BJ, Leroux P, Jeandel L, Fournier A,
Vaudry H. Localization and characterization of PACAP
receptors in the rat cerebellum during development:
evidence for a stimulatory effect of PACAP on immature
cerebellar granule cells. Neuroscience 1993;57:32938.
[11] Basille M, Vaudry D, Coulouarn Y, Jegou S, Lihrmann I,
Fournier H, et al. Distribution of PACAP receptor mRNAs
and PACAP binding sites in the rat brain during
development. Ann N Y Acad Sci 2000;921:3047.
[12] Bhave SV, Hoffman PL. Phosphatidylinositol 3-OH kinase
and protein kinase A pathways mediate the anti-apoptotic
effect of pituitary adenylyl cyclase-activating polypeptide
in cultured cerebellar granule neurons: modulation by
ethanol. J Neurochem 2004;88:35969.
[13] Cameron DB, Galas L, Jiang Y, Raoult E, Vaudry D, Komuro
H. Cortical layer-specific control of neuronal migration by
PACAP. Neuroscience 2007;146:697712.
[14] Cavallaro S, Copani A, DAgata V, Musco S, Petralia S,
Ventra C, et al. Pituitary adenylate cyclase activating
polypeptide prevents apoptosis in cultured cerebellar
granule neurons. Mol Pharmacol 1996;50:606.
[15] Chang JY, Korolev VV, Wang JZ. Cyclic AMP and pituitary
adenylate cyclase-activating polypeptide (PACAP) prevent
programmed cell death of cultured rat cerebellar granule
cells. Neurosci Lett 1996;206:1814.
[16] Contestabile A, Fila T, Bartesaghi R, Ciani E. Cyclic AMPmediated regulation of transcription factor Lot1 expression
in cerebellar granule cells. J Biol Chem 2005;280:335415.
[17] DAgata V, Cavallaro S, Stivala F, Canonico PL. Tissuespecific and developmental expression of pituitary
adenylate cyclase-activating polypeptide (PACAP) receptors
in rat brain. Eur J Neurosci 1996;8:3108.
[18] Dahmane N, Ruiz i Altaba A. Sonic hedgehog regulates the
growth and patterning of the cerebellum. Development
1999;126:3089100.
[19] Falluel-Morel A, Aubert N, Vaudry D, Basille M, Fontaine M,
Fournier A, et al. Opposite regulation of the mitochondrial
apoptotic pathway by C2-ceramide and PACAP through a
MAP-kinase-dependent mechanism in cerebellar granule
cells. J Neurochem 2004;91:123143.
[20] Falluel-Morel A, Vaudry D, Aubert N, Galas L, Benard M,
Basille M, et al. Pituitary adenylate cyclase-activating
polypeptide prevents the effects of ceramides on migration,
neurite outgrowth, and cytoskeleton remodeling. Proc Natl
Acad Sci USA 2005;102:263742.

peptides 28 (2007) 17461752

[21] Fan CM, Porter JA, Chiang C, Chang DT, Beachy PA, TessierLavigne M. Long-range sclerotome induction by sonic
hedgehog: direct role of the amino-terminal cleavage
product and modulation by the cyclic AMP signaling
pathway. Cell 1995;81:45765.
[22] Favit A, Scapagnini U, Canonico PL. Pituitary adenylate
cyclase-activating polypeptide activates different signal
transducing mechanisms in cultured cerebellar granule
cells. Neuroendocrinology 1995;61:37782.
[23] Fukuchi M, Sakuragawa S, Tabuchi A, Tsuda M. Calcium
signal-mediated expression of the vasoactive intestinal
polypeptide gene and its small contribution to activitydependent survival of mouse cerebellar granule cells. J
Neurosci Res 2004;77:2634.
[24] Gonzalez BJ, Basille M, Vaudry D, Fournier A, Vaudry H.
Pituitary adenylate cyclase-activating polypeptide
promotes cell survival and neurite outgrowth in rat
cerebellar neuroblasts. Neuroscience 1997;78:41930.
[25] Gonzalez BJ, Basille M, Vaudry D, Fournier A, Vaudry H.
Pituitary adenylate cyclase-activating polypeptide. Ann
Endocrinol (Paris) 1998;59:364405.
[26] Hammerschmidt M, Bitgood MJ, McMahon AP. Protein
kinase A is a common negative regulator of Hedgehog
signaling in the vertebrate embryo. Genes Dev 1996;10:
64758.
[27] Ito Y, Arakawa M, Ishige K, Fukuda H. Comparative study of
survival signal withdrawal- and 4-hydroxynonenalinduced cell death in cerebellar granule cells. Neurosci Res
1999;35:3217.
[28] Jamen F, Bouschet T, Laden JC, Bockaert J, Brabet P. Upregulation of the PACAP type-1 receptor (PAC1) promoter by
neurotrophins in rat PC12 cells and mouse cerebellar
granule cells via the Ras/mitogen-activated protein kinase
cascade. J Neurochem 2002;82:1199207.
[29] Kienlen Campard P, Crochemore C, Rene F, Monnier D,
Koch B, Loeffler JP. PACAP type I receptor activation
promotes cerebellar neuron survival through the cAMP/
PKA signaling pathway. DNA Cell Biol 1997;16:32333.
[30] Kivipelto L, Absood A, Arimura A, Sundler F, Hakanson R,
Panula P. The distribution of pituitary adenylate cyclaseactivating polypeptide-like immunoreactivity is distinct
from helodermin- and helospectin-like immunoreactivities
in the rat brain. J Chem Neuroanat 1992;5:8594.
[31] Komuro H, Rakic P. Distinct modes of neuronal migration in
different domains of developing cerebellar cortex. J
Neurosci 1998;18:147890.
[32] Lam HC, Takahashi K, Ghatei MA, Kanse SM, Polak JM,
Bloom SR. Binding sites of a novel neuropeptide pituitaryadenylate-cyclase-activating polypeptide in the rat brain
and lung. Eur J Biochem 1990;193:7259.
[33] Lu N, DiCicco-Bloom E. Pituitary adenylate cyclaseactivating polypeptide is an autocrine inhibitor of mitosis
in cultured cortical precursor cells. Proc Natl Acad Sci USA
1997;94:335762.
[34] Masuo Y, Ohtaki T, Masuda Y, Tsuda M, Fujino M. Binding
sites for pituitary adenylate cyclase activating polypeptide
(PACAP): comparison with vasoactive intestinal
polypeptide (VIP) binding site localization in rat brain
sections. Brain Res 1992;575:11323.
[35] Mei YA, Vaudry D, Basille M, Castel H, Fournier A, Vaudry
H, et al. PACAP inhibits delayed rectifier potassium current
via a cAMP/PKA transduction pathway: evidence for the
involvement of Ik in the anti-apoptotic action of PACAP.
Eur J Neurosci 2004;19:144658.
[36] Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang
L, et al. Isolation of a novel 38 residue-hypothalamic
polypeptide which stimulates adenylate cyclase in
pituitary cells. Biochem Biophys Res Commun
1989;164:56774.

1751

[37] Nicot A, Lelievre V, Tam J, Waschek JA, DiCicco-Bloom E.

Pituitary adenylate cyclase-activating polypeptide and
sonic hedgehog interact to control cerebellar granule
precursor cell proliferation. J Neurosci 2002;22:924454.
[38] Nielsen HS, Hannibal J, Fahrenkrug J. Expression of
pituitary adenylate cyclase activating polypeptide (PACAP)
in the postnatal and adult rat cerebellar cortex.
Neuroreport 1998;9:263942.
[39] Obara Y, Horgan AM, Stork PJ. The requirement of Ras and
Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in
cerebellar granule cells. J Neurochem 2007;101:82470.
[40] Skoglosa Y, Patrone C, Lindholm D. Pituitary adenylate
cyclase activating polypepetide is expressed by developing
rat Purkinje cells and decreases the number of cerebellar
gamma-amino butyric acid positive neurons in culture.
Neurosci Lett 1999;265:20710.
[41] Ster J, De Bock F, Guerineau NC, Janossy A, Barrere-Lemaire
S, Bos JL, et al. Exchange protein activated by cAMP (Epac)
mediates cAMP activation of p38 MAPK and modulation of
Ca2+-dependent K+ channels in cerebellar neurons. Proc
Natl Acad Sci USA 2007;104:251924.
[42] Suda K, Smith DM, Ghatei MA, Murphy JK, Bloom SR.
Investigation and characterization of receptors for pituitary
adenylate cyclase-activating polypeptide in human brain
by radioligand binding and chemical cross-linking. J Clin
Endocrinol Metab 1991;72:95864.
[43] Tabuchi A, Koizumi M, Nakatsubo J, Yaguchi T, Tsuda M.
Involvement of endogenous PACAP expression in the
activity-dependent survival of mouse cerebellar granule
cells. Neurosci Res 2001;39:8593.
[44] Tabuchi A, Koizumi M, Tsuda M. Novel splice variants of
PACAP gene in mouse cerebellar granule cells. Neuroreport
2001;12:11816.
[45] Tatsuno I, Somogyvari-Vigh A, Arimura A. Developmental
changes of pituitary adenylate cyclase activating
polypeptide (PACAP) and its receptor in the rat brain.
Peptides 1994;15:5560.
[46] Vaudry D, Falluel-Morel A, Leuillet S, Vaudry H, Gonzalez
BJ. Regulators of cerebellar granule cell development act
through specific signaling pathways. Science
2003;300:15324.
[47] Vaudry D, Gonzalez BJ, Basille M, Anouar Y, Fournier A,
Vaudry H. Pituitary adenylate cyclase-activating
polypeptide stimulates both c-fos gene expression and cell
survival in rat cerebellar granule neurons through
activation of the protein kinase A pathway. Neuroscience
1998;84:80112.
[48] Vaudry D, Gonzalez BJ, Basille M, Fournier A, Vaudry H.
Neurotrophic activity of pituitary adenylate cyclaseactivating polypeptide on rat cerebellar cortex during
development. Proc Natl Acad Sci USA 1999;96:941520.
[49] Vaudry D, Gonzalez BJ, Basille M, Pamantung TF, Fontaine
M, Fournier A, et al. The neuroprotective effect of pituitary
adenylate cyclase-activating polypeptide on cerebellar
granule cells is mediated through inhibition of the CED3related cysteine protease caspase-3/CPP32. Proc Natl Acad
Sci USA 2000;97:133905.
[50] Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry
H. Pituitary adenylate cyclase-activating polypeptide and
its receptors: from structure to functions. Pharmacol Rev
2000;52:269324.
[51] Vaudry D, Hamelink C, Damadzic R, Eskay RL, Gonzalez B,
Eiden LE, et al. PACAP acts as a stress response peptide to
protect cerebellar neurons from ethanol or oxidative insult.
Peptides 2005;26:251824.
[52] Vaudry D, Pamantung TF, Basille M, Rousselle C, Fournier
A, Vaudry H, et al. PACAP protects cerebellar granule
neurons against oxidative stress-induced apoptosis. Eur J
Neurosci 2002;15:145160.

1752

peptides 28 (2007) 17461752

[53] Vaudry D, Rousselle C, Basille M, Falluel-Morel A,

Pamantung TF, Fontaine M, et al. Pituitary adenylate
cyclase-activating polypeptide protects rat cerebellar
granule neurons against ethanol-induced apoptotic cell
death. Proc Natl Acad Sci USA 2002;99:6398403.
[54] Villalba M, Bockaert J, Journot L. Pituitary adenylate
cyclase-activating polypeptide (PACAP-38) protects
cerebellar granule neurons from apoptosis by activating

the mitogen-activated protein kinase (MAP kinase)

pathway. J Neurosci 1997;17:8390.
[55] Zerr P, Feltz A. Forskolin blocks the transient K current of rat
cerebellar granule neurons. Neurosci Lett 1994;181:1537.
[56] Zhou CJ, Shioda S, Yada T, Inagaki N, Pleasure SJ, Kikuyama
S. PACAP and its receptors exert pleiotropic effects in the
nervous system by activating multiple signaling pathways.
Curr Protein Pept Sci 2002;3:42339.