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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
present on granule cells during both the development period and in adulthood. Treatment of
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
effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated
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
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
Apoptosis
human.
Caspase-3
Neuroprotection
1.
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
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
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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].
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4.
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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].
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7.
Conclusion
references
[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
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