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The Putative Neuroprotective Role of Neuropeptide Y in the Central Nervous System Ana P. Silva* ,1, Sara Xapelli1 , Eric Grouzmann2 and Cláudia Cavadas3 1Center
for Neuroscience and Cell Biology, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal 2Division
of Clinical Pharmacology and Toxicology, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland 3Center
for Neuroscience and Cell Biology and Faculty of Pharmacy, University of Coimbra, Largo D. Dinis, 3020-123 Coimbra, Portugal Abstract: Neuropeptide Y (NPY) is one of the most abundant and widely distributed neuropeptides in the mammalian central nervous system (CNS). An overview of the distribution of the G-protein coupled NPY receptor family (Y1 , Y2 , Y4 , Y5 receptors) in the brain is described. The coexistence of NPY with other neurotransmitters and its wide distribution in several brain areas predict the high importance of NPY as a neuromodulator. Thus, the effect of NPY on the release of several neurotransmitters such as glutamate, gammaaminobutyric acid (GABA), norepinephrine (NE), dopamine, somastotatin (SOM), serotonin (5-HT), nitric oxide (NO), growth hormone (GH) and corticotropin releasing factor (CRF) is reviewed. A neuroprotective role for NPY under physiological conditions and during hyperactivity such as epileptic-seizures has been suggested. We have shown previously that NPY inhibits glutamate release evoked from hippocampal nerve terminals and has a neuroprotective effect in rat organotypic hippocampal cultures exposed to an excitotoxic insult. Moreover, changes in NPY levels have been observed in different pathological conditions such as brain ischemia and neurodegenerative diseases (Huntington’s, Alzheimer’s and Parkinson’s diseases). Taken together, these studies suggest that NPY and NPY receptors may represent pharmacological targets in different pathophysiological conditions in the CNS.
Keywords: Neuropeptide Y, NPY receptors, glutamate release, excitotoxicity, neuroprotection, epilepsy, neurodegenerative diseases. 1. NEUROPEPTIDE Y (NPY) NPY is a 36-amino acid peptide that possesses an amidated C-terminal residue and a large number of tyrosine residues (which are abbreviated by the letter Y) included in both ends of the molecule. It was first isolated from the pig brain in 1982 [1,2]. The peptide family, which includes NPY, peptide YY (PYY) and pancreatic polypeptide (PP), is named NPY family or “PP-fold” family [3]. NPY is one of the neuropeptides with the highest degree of phylogenetic preservation, while the PP differs considerably between species [4]. The NPY gene is located on human chromosome 7 at the locus 7p15.1 [5]. It is composed of four exons and results in the synthesis of a 97 amino acid Pre-pro NPY [6]. The Prepro-NPY generated after translation is directed into the endoplasmic reticulum where a 28 amino acid peptide is removed and Pro-NPY produced. This NPY precursor, ProNPY, is a 69 amino acid peptide formed by NPY1-39 where the carboxylic group is flanked by a group of 33 amino acids called C-flanking peptide of NPY (CPON). The following processing step is the cleavage of the precursor Pro-NPY at a dibasic site by prohormone convertases, which generates *Address correspondence to the author at the Center for Neuroscience and Cell Biology, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal; Tel: +351239833369; Fax: +351239822776; E-mail:
[email protected]
1568-007X/05 $50.00+.00
NPY1-39 and CPON. Then, a truncation at the C-terminal end by a carboxypeptidase generates NPY1-37 , which is a substrate for the enzyme peptidylglycine alpha-amidating monooxygenase and leads to the biologically active amidated NPY1-36 (NPY) [7]. The mature NPY can be further processed by two enzymes, the dipeptidyl peptidase IV and the aminopeptidase P, resulting in NPY3-36 and NPY2-36 , respectively (Y2/Y5 agonists) [7,8]. 2. BRAIN LOCALIZATION OF NPY NPY is one of the most abundant and widely distributed neuropeptides in the mammalian CNS. This neuropeptide is expressed preferentially in interneurons but it is also present in long projection neurons [9-11]. In order to understand the putative neuroprotective effects of NPY we will describe its brain distribution and co-localization with other neurotransmitters/modulators. NPY-immunoreactivity (NPY-ir) is abundantly found in numerous rat brain areas, including the hypothalamus, nucleus accumbens, septum and locus coerulus. Moreover, moderate levels are also found in the hippocampus, cerebral cortex, basal ganglia and in the thalamus [10-13]. In the rat cerebral cortex, NPY-ir is detectable within 1-2 % of all neurons [14] and all dinucleotide phosphate-diaphorase (NADPH-d) positive neurons are also NPY-ir [15]. Studies on rat, cat and monkey neocortex have shown that nearly all NPY-ir neurons are also immunoreactive for GABA and for © 2005 Bentham Science Publishers Ltd.
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glutamic acid decarboxylase (GAD) and that they represent a small subpopulation of the cortical GABAergic neurons [1620]. In human brain, high levels of NPY mRNA are found in some areas, such as the dentate gyrus (DG), caudate nucleus and putamen [21]. In cerebral cortex the amount of NPY-ir varies between different cortical areas, with highest concentration in the cingulated and temporal cortices and the lowest in the occipital lobe [13,22,23]. The laminar distribution of NPY positive cell bodies is similar through the cerebral cortex [21,24,25]. NPY-labeled cells are also found in human subcortical white matter while no NPYpositive cells are found in white matter areas away from the cortex [21,25]. In the temporal cortex, all NPY-ir neurons express the glutamate receptor subunit GluR2/4 and 17 and 16 % of neurons that express GluR/2 and GluR5/6/7 subunits, respectively, there is also NPY-ir [25]. In the human neocortex, more than 90 % of NPY-ir neurons are NADPH-d positive [26]. NPY is colocalized with SOM in rat, monkey and human cortical neurons [18,27,28]. The coexistence of GABA/NPY/SOM is also found in neurons of cerebral cortex and subcortical white matter [14]. There is also a wide distribution of NPY-ir and NPY mRNA containing cells in the hippocampus of different species, including humans [9-12,21,29-31]. None, or few, of the NPY-containing neurons are pyramidal; many are likely to be local circuit neurons, but some appear to have extrinsic connections [29]. In the rat hippocampus, NPY-ir cells are found in the hilus of the DG, stratus oriens of CA2 and CA3 subfields and within and around the stratum pyramidale of the CA1 subfield [9-11]. In the human hippocampus, high densities of NPY positive cells are seen in the dentate hilus, the CA1 area, the subicular complex and in the alveolus [31]. NPY-expressing neurons in the rat hippocampus contain few or no AMPA glutamate receptor subunits (GluRl, GluR2/3) [32]. One of the highest densities of brain NPY-ir is found in the hypothalamus [9,12,13,33]. Within the hypothalamus of the adult rodent, NPY neurons are largely restricted to the arcuate [34,35] and some of these neurons co-localized with GABA [36,37]. NPY also shows a widespread distribution in the amygdaloid complex of the rat [9,11,38], cat [39], monkey [40], and human brains [21,41]. Virtually all of the NPY-ir neurons in the amygdala are also SOM-ir [28,40,42]. In the rat medial amygdala, NPY and GABA coexist within the same neurons and NPY producing neurons make close contacts with GABA-containing neurons [43]. The striatum contains high levels of NPY that is found in a subset of medium-sized aspiny interneurons [11-13,4447]. The human striatum shows NPY mRNA levels with a heterogeneous signal [21]. Higher expression is observed in the nucleus accumbens and the ventral region of the caudate [48]. In the rat striatum, nearly all NPY neurons also contain SOM and NADPH-d/nNOS [28,44,49-51]. About 80 % of SOM positive neurons contain NPY and about 90 % of the NPY-ir neurons in the caudate putamen and nucleus accumbens colocalize with NADPH-d [15]. Some studies suggest that GABA is also present in striatal NPY-ir
Silva et al.
neurons [20,52]. In the rat striatum, some aspartate-positive neurons also show NPY-ir [53]. Striatal NPY-ir neurons receive direct glutamatergic inputs from the cerebral cortex, dopaminergic inputs from the substantia nigra and also have major GABAergic innervation [53-56]. Several lines of evidence suggest that the expression of NPY and NPY receptors may not be restricted to neurons but could also extend to glial cells [57-62]. However, the functional role of the NPY in glial cells in vivo remains to be established. 3. NPY RECEPTORS AND THEIR DISTRIBUTION IN THE BRAIN The G-protein coupled NPY receptor family includes the Y1, Y 2, Y 3, Y 4, Y 5 and y6 subtypes [3]. The Y1, Y2 and Y5 receptor subtypes preferentially bind NPY and PYY, whereas Y4 is preferentially activated by PP. The Y 3 receptor has not yet been cloned and its presence in the brain was pharmacologically characterized only in the solitary tract nucleus [63,64]. The y6 receptor is functional only in the mouse and rabbit and is absent in the rat [4,65-69]. In the next part of this review, we will describe the pharmacology and brain distribution of the Y1, Y2, Y4 and Y5 receptors. The knowledge of the localization of each NPY receptor in different brain regions is important to understand the role of each receptor in physiological and pathological conditions. NPY Y1 Receptor The Y1 receptor was the first PP-fold peptide binding receptor to be cloned. Peptides modified on the C-terminal end (i.e. [Pro34]NPY and [Leu31,Pro 34]NPY) retain full activity on the Y1 receptor, but lose their affinity for the Y2 receptor. The Y1 receptor can be internalized after agonist stimulation [70-74].The agonists and antagonists for the Y1 receptor are listed in Table 1. NPY Y1 receptors are abundantly expressed in the CNS and have been implicated in food intake [75-80], anxiety [81-84] and ethanol intake [85-87] (reviewed in [88]). The highest levels of Y1 mRNA in the brain of several mammalian species are consistently seen in forebrain regions, including the cerebral cortex, the hippocampal formation, and several amygdaloid, thalamic, and hypothalamic nuclei [89-91]. Within the rat hippocampus, high to moderate levels of Y 1 receptor mRNA is restricted to the CA3, CA2 and CA1 pyramidal cell layers. The levels of Y1-mRNA expression on DG are discrepant in the literature. Parker and Herzog [90] found relatively low levels of Y1 mRNA expression but others have shown high levels of expression in the DG granule layer [91,92]. The rat thalamus and amygdala contain widespread Y1-receptor mRNA expression [90,91]. The rat basal ganglia contain very low levels of Y1 receptor mRNA [90,91]. Interestingly, although devoid of Y1 receptor mRNA, the rat nucleus accumbens contains Y1-ir [93,94]. This suggests that these Y1containing terminals are projections from more distant sites such as the ventral tegmental area, which contains Y1 mRNA [90,93,94]. A widespread distribution of Y 1 receptor in hypothalamic nuclei in the rat brain is supported by
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several techniques with a good agreement between ligand binding, in situ hybridization and immunohistochemistry, one exception being the magnocellular neurons [33,90,91,95-99]. The Purkinje cell layer of the rat cerebellum expresses Y1 receptor mRNA and Y1-ir is also detected in these tissues [90,91,97-99].
is relatively resistant to the N-terminal deletion and retains a high binding affinity for the C-terminal fragments of NPY (i.e. NPY2-36 , NPY3-36 , NPY13-36 ) [3]. The Y2 receptor does not appear to be internalized after prolonged agonist stimulation, or does so very slowly [71]. The agonists and antagonists for the Y 2 receptor subtype are listed in Table 1.
In human brain significant Y1 receptor mRNA levels are also observed [21,100]. The Y1 mRNA signal is widely distributed in all layers of most limbic and neocortical regions [100]. Moreover, uniquely Y1 mRNA-positive labelled cells are found in the subcortical white matter [100]. The striatum, the caudate, putamen and nucleus accumbens show moderate levels of Y1 receptor mRNA expression, which is non-homogeneous in appearance [21,100]. In the human hippocampus, very high levels of Y1 mRNA are found in the granular cell layer of the DG [21,100]. A positive Y1 mRNA hybridization signal is also detected in the amygdaloid complex and through the hypothalamus [21,100]. The lack of co-localization of Y1 mRNA with NPY mRNA observed in the human brain suggests that the Y1 receptor has a postsynaptic localization (terminal, dendritic, and/or somatic location) in the mammalian brain [21]. However, immunohistochemical studies identified the presence of Y1 receptors in NPY-ir terminals of the rat nucleus accumbens and rat hippocampal neurons in culture, which suggests that the Y1 receptor may also be a presynaptic receptor and may act as an autoreceptor to regulate NPY release [93,101,102].
The presence of the Y2 receptor in the CNS has attracted particular interest because of its possible implication in the control of food intake [104-106], bone formation [107,108], gastrointestinal motility [109,110], circadian rhythms [111114], anxiety [115-117], cardiovascular regulation [118], neuronal excitability and epilepsy [119-124], alcohol dependence [125,126] and learning and memory [127,128]. Y2 receptor mRNA is discretely localized in the rat brain, including the hippocampus, hypothalamus, thalamus, amygdala and brainstem, and is generally consistent with the distribution of Y2-like receptor protein demonstrated by radioligand-binding methods [90,129-131]. A similar distribution occurs in the mouse brain [132]. In general, Y2 receptor mRNA expression is lower when compared to Y1 receptor mRNA [90].
The localization of Y1 receptor protein was studied by autoradiography and immunohistochemistry. [125I][Leu31, Pro34]PYY/BIBP3226 sensitive binding sites (Y1 sites) in rat brain are located in all cortical areas, olfactory nuclei, most thalamic nuclei, dentate gyrus and cerebellum [103]. In the rat brain, Y1-ir is observed within cerebral cortex, hippocampal formation, some hypothalamic nuclei and amygdalar complex [94,99]. NPY Y2 Receptor The Y2 receptor is pharmacologically characterized by high affinity for NPY and PYY but, unlike the Y1 receptor, Table 1.
In the human brain, Y2 receptor mRNA is present in different cerebral cortical areas and labeling is found predominantly over small- and medium-sized nerve cell bodies [21,133]. Moderately labeled nerve cells are also found in the subcortical white matter [133]. In the hippocampal formation, the granular layer of the DG shows the highest overall signal of Y2 receptor mRNA [21,133]. Very high levels of the Y 2 receptor mRNA are also found in the CA2 and CA3 regions [21,133]. A weak Y2 mRNA levels is observed in the caudate nucleus and nucleus accumbens while a moderate signal is detected throughout most of the amygdaloid complex [21,133]. The co-localization of Y2 receptor mRNA with NPY mRNA in some neuronal cells observed in several areas of human brain supports pharmacological studies that indicate a presynaptic localization where it acts as an autoreceptor, inhibiting the release of NPY and other neurotransmitters [21,134-138].
Agonists and Antagonists of NPY Receptors Y1 receptor
Y2 receptor
Y4 receptor
Y5 receptor
Selective agonists
[Phe 7 ,Pro34 ]NPY; [Leu 31 ,Pro34 ]NPY; [Pro 34 ]NPY; [Leu 31 ,Pro34 ]PYY; [Pro 34 ]PYY
NPY3–36 , NPY13-36 ; Ac-[Lys28 ,Glu32 ]NPY 25-36 ; TASP-V
PP; GW1229 (or GR231118 or 1229U91)
[Ala 31 ,Aib32 ]NPY; [hPP1–17 , Ala 31 ,Aib32 ]NPY; [cPP1–7 , NPY19–23 , Ala 31 ,Aib32 ,Gln34 ]hPP
Antagonists
N-terminally truncated analogues; GW1229 (or GR231118 or 1229U91); BIBP3226; SR120819A; BIBO3304; LY357897; J-115814; H394/84c; J-104870; GI264879A; H409/22
BIIE0246; T4 -[NPY 33– 36 ] 4 ; JNJ-5207787
--
CGP71683A; JCF109; NPY5RA-972; 3-[2-[6-(2-tertbutoxyethoxy) pyridin-3-yl]-1Himidazol-4-yl]benzonitrile hydrochloride salt; GW438014A; L-152,804; pyrrolo [3,2-d] pyrimidine derivatives, 2substituted 4-amino-quinazolin derivatives; alpha-substituted N(sulfonamino)alkyl-beta aminotetralins
References
[3,80,87,192-196]
[3,137,141,148,197-202]
[3,68,143,144,146]
[3,68,164,165,167,172,173,203205,206,207-211]
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The brain distribution of Y2 receptor protein was only investigated by autoradiography. The radiolabeled PYY3-36 peptide was initially proposed as an Y2-selective ligand [139]. However, competition binding experiments suggested that [125I]-PYY3-36 could bind to a heterogeneous population of sites [139] and it was later shown that this compound also binds to the Y5 receptor subtype [68]. High density of [125I]-PYY3-36 binding sites are found in several areas of the rodent and primate brain such as the hippocampal formation, substantia nigra, nucleus tractus solitarius and area postrema [131,140]. Ninety percent of these binding sites represent Y2 receptors [131,140,141]. A good Y2 receptor antibody and a selective radiolabeled Y2 receptor antagonist will be useful to confirm the localization of the protein and to evaluate changes in Y2 receptor density under physiological and pathological situations. NPY Y4 Receptor The Y 4 receptor was originally named as PP1 receptor, because of its preference for PP as a ligand. In mammals, the affinity of PP for this receptor is much greater than the affinities of NPY and PYY [67,142,143]. The principal feature of the Y4 receptor is its very high affinity (
