Abstract. Introduction. Gregory Kapatos*

Transcription

1 Critical Review The Neurobiology of Tetrahydrobiopterin Biosynthesis: A Model for Regulation of GTP Cyclohydrolase I Gene Transcription within Nigrostriatal Dopamine Neurons Gregory Kapatos* Center for Molecular Medicine and Genetics and Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA Abstract Within the brain, the reduced pteridine cofactor 6R-L-erythro- 5,6,7,8-tetrahydrobiopterin (BH4) is absolutely required for the synthesis of the monoamine (MA) neurotransmitters dopamine (DA), norepinephrine (NE), epinephrine (E), and serotonin (5-HT), the novel gaseous neurotransmitter nitric oxide (NO) and the production of yet to be identified 1-O-alkylglycerol-derived lipids. GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the BH4 biosynthetic pathway, which is now thought to involve up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. Gene expression analysis across different regions of the human brain shows the abundance of transcripts coding for all eight of these proteins to be highly correlated with each other and to be enriched within MA neurons. The potential for multiple BH4 biosynthetic pathways therefore exists within the human brain. GTPCH expression is particularly heterogeneous across different populations of human and rodent MA-containing neurons, with low expression levels and therefore BH4 being a characteristic of nigrostriatal DA (NSDA) neurons. Basic knowledge of how GCH1 gene transcription is controlled within NSDA neurons may explain the distinctive susceptibility of these neurons to genetic mutations that result in BH4 deficiency. A model for cyclic adenosine monophosphate-dependent GCH1 transcription is described that involves a unique combination of DNA regulatory sequences and transcription factors. This model proposes that low levels of GCH1 transcription by NSDA neuronsaredrivenbydistinctive cell physiology, suggesting that pharmacological manipulation of GCH1 gene transcription can be used to modify BH4 levels and therefore DA synthesis in the basal ganglia. VC 2013 IUBMB Life, 65(4): , 2013 Keywords: GTP cyclohydrolase I (GTPCH); tetrahydrobiopterin (BH4); monoamine (MA); dopamine (DA); norepinephrine (NE); serotonin (5- HT); nigrostriatal dopamine neurons (NSDA); camp response element binding protein (CREB); CCAAT enhancer binding protein beta (C/EBPb); nuclear factor-y (NF-Y); stimulatory protein 3 (Sp3); RNA polymerase II (Pol II) Introduction VC 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 65, Number 4, April 2013, Pages *Address for correspondence to: Dr. Gregory Kapatos, Center for Molecular Medicine and Genetics, Department of Pharmacology, Wayne State University School of Medicine, 3127 Scott Hall, 540 E. Canfield, Detroit, Michigan 48201, USA. gkapato@med.wayne.edu. Received 21 December 2012; accepted 7 January 2013 DOI: /iub.1140 Published online 4 March 2013 in Wiley Online Library (wileyonlinelibrary.com) Tetrahydrobiopterin (BH4) first identified in rat liver (1) is the fully reduced and functional form of biopterin (6-(L-erythro-1 0,2 0 -dihydroxypropyl)-2-amino-4-hydroxypteridine). The established roles for BH4 are in the hydroxylation of the aromatic amino acids by their respective mixed-function monooxygenases, including the detoxification of phenylalanine by phenylalanine hydroxylase (PAH), the synthesis of the neurotransmitters DA, NE, and E from tyrosine by tyrosine hydroxylase (TH) and 5-HT from tryptophan by tryptophan IUBMB Life 323

2 IUBMB LIFE FIG 1 Multiple de novo and salvage pathways for BH4 synthesis. BH4 biosynthesis involves up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. Abbreviations: GTPCH, GTP cyclohydrolase I; GFRP, GTPCH feedback regulatory protein: PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; AKR1C3, aldo-keto-reductase family 1 member C3; AKR1B1, aldo-keto-reductase family 1 member B1; CBR1, carbonyl reductase; DHFR, dihydrofolate reductase. There are six alternate de novo routes to BH4 synthesis identified by the enzymes involved: the preferred SR>SR>SR, and the less preferred AKR1C3>SR>SR, SR>AKR1B1, AKR1C3>AKR1B1, AKR1B1>SR, and CBR1>SR. Note that the only alternate de novo pathway that does not require SR uses AKR1C3 and AKR1B1. There are two alternate salvage pathways, SR>DHFR and CBR1>DHFR. Nonenzymatic reaction (n.e.) (for details, see Ref. 3). hydroxylase (TPH1 and TPH2), the production of NO from arginine by the family of NO synthases (NOS1, NOS2, and NOS3), and the cleavage of ether lipids by alkylglycerol monooxygenase (AGMO) (for review, see refs. 2,3). De Novo and Salvage Pathways for BH4 Biosynthesis The BH4 biosynthetic pathway will be described in enough detail to support further discussions. For a comprehensive description, the reader is directed to reviews on this subject (2,3). Figure 1 shows that BH4 biosynthesis can involve up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. The first, common, and generally rate-limiting step in the de novo pathway is catalyzed by the enzyme GTP cyclohydrolase I (GTPCH), which converts GTP to 7,8-dihydroneopterin triphosphate. In some cells, GTPCH forms a complex with GTP cyclohydrolase Feedback Regulatory Protein (GFRP). GFRP has no intrinsic enzymatic activity but confers regulation on GTPCH by BH4, L-phenylalanine, and the chemical inhibitor of BH4 synthesis 2,4-diamino-6-hydroxypryimidine (4,5). In the second common step, 7,8-dihydroneopterin triphosphate is converted to 6-pyruvoyltetrahydropterin by the enzyme 6-pyruvoyltetrahydropterin synthase (PTPS). Although GTPCH activity is considered limiting, BH4 and D-erythro-neopterin (neopterin), a metabolite of 7,8-dihydroneopterin triphosphate, are both decreased in the cerebral spinal fluid (CSF) of patients with Parkinson s disease (6), suggesting that reaction rates for GTPCH and PTPS are closely matched within nigrostriatal dopamine neuron (NSDA) neurons. In what is considered the primary alternate de novo pathway, the intermediate 6-pyruvoyltetrahydropterin is reduced by the enzyme sepiapterin reductase (SR) 324 The Neurobiology of Tetrahydrobiopterin Biosynthesis

3 to form 1 0 -hydroxy-2 0 -oxopropyl-tetrahydropterin, which is reduced to 1 0 -oxo-2 0 -hydroxypropyl-tetrahydropterin and then to BH4 in two additional reactions also catalyzed by SR. In what are thought to be secondary alternate de novo pathways, 6-pyruvoyltetrahydropterin is reduced by aldo-keto-reductase family 1 member C3 (AKR1C3; 3-a-hydroxysteroid dehydrogenase type II) to produce the 1 0 -hydroxy-2 0 -oxopropyl-tetrahydropterin intermediate or by aldo-keto-reductase family 1 member B1 (AKR1B1; aldose reductase) or carbonyl reductase (CBR1) to produce the 1 0 -oxo-2 0 -hydroxypropyl-tetrahydropterin intermediate. The 1 0 -hydroxy-2 0 -oxopropyl-tetrahydropterin intermediate is then reduced directly to BH4 by AKR1B1, whereas the oxo-2 0 -hydroxypropyl-tetrahydropterin intermediate can be reduced to BH4 by SR. Therefore, there are six possible alternate de novo routes to BH4 synthesis. According to the enzymes involved, they are listed as follows: SR>SR>SR, AKR1C3>SR>SR, SR>AKR1B1, AKR1C3>AKR1B1, AKR1B1> SR, and CBR1>SR. The only de novo pathway for BH4 synthesis that does not require SR uses the aldo-keto-reductase family members AKR1C3 and AKR1B1. In cases of SR deficiency, this pathway appears to be active in the liver but not in the brain, resulting in severe CNS deficits in BH4 synthesis and monoamine (MA) and NO neurotransmission (7,8). In the BH4 salvage pathway, a nonenzymatic reaction converts 1 0 -oxo-2 0 -hydroxypropyl-tetrahydropterin to 6-lactoyl- 7,8-dihydropterin (sepiapterin), which is then reduced by either SR or CBR1 to generate 7,8-dihydrobiopterin. 7,8-Dihydrobiopterin is subsequently reduced to BH4 by dihydrofolate reductase (DHFR). The metabolic flow through the salvage pathway has not been established but may be substantial based on the accumulation in the CSF of patients with SR deficiency of sepiapterin derived from 1 0 -oxo-2 0 -hydroxypropyl-tetrahydropterin and 7,8-dihydrobiopterin derived from sepiapterin (7,8). BH4 Localization and Metabolism in the Rodent Brain The first indication that the BH4 biosynthesis in the brain is restricted to MA neurons came from the pioneering work of Levine (9), who showed that BH4 is heterogeneously distributed across rat brain regions in a manner highly correlated with TH and TPH enzyme activities. Further research revealed that BH4 and GTPCH are concentrated within the nerve terminals of rat NSDA neurons, demonstrating that BH4 is not transported down the axon from the cell body but is synthesized in the nerve terminal (10,11). Subsequent studies of BH4 synthesis and degradation in primary cultures of NSDA neurons, hypothalamic DA neurons, and post-ganglionic NE sympathetic neurons showed that the rate of BH4 synthesis actually exceeds that of these neurotransmitters, with 25% of the intracellular pool of BH4 turning over each hour (12,13). Whether this turnover involves catabolism or secretion (14) is unknown. Nevertheless, the rapid synthesis and degradation of the carbon backbone of BH4 is surprising, given that quinoid 6,7[8]-dihydrobiopterin is regenerated to BH4 by quinoid dihydropteridine reductase. The concentration of BH4 within MA nerve terminals remains controversial. Within the nerve terminals of NSDA neurons, BH4 levels are reported to be subsaturating for TH (15 17), whereas BH4 is reported to be either saturating (18) or subsaturating (19,20) for TPH within the nerve terminals of midbrain raphe 5-HT neurons. Differences in BH4 concentrations between NSDA and 5-HT neurons are likely driven by differences in the expression of the genes coding for GTPCH and GFRP (21 24). The Neuroscience of Human BH4 Deficiencies For a comprehensive description of the various BH4 deficiencies, the reader is directed to reviews on this subject (7,25). Neuroscientists first became interested in BH4 in the mid- 1970s when clinicians began to publish case reports of hyperphenylalaninemia (HPA) associated with mental retardation, developmental delay, convulsions, and hypotonia of the trunk and hypertonia of the limbs despite dietary control of blood phenylalanine levels and normal PAH protein. Complicating the situation are more recent reports of mutations in BH4 biosynthetic enzymes that impair BH4 and neurotransmitter synthesis in the brain but do not produce HPA and therefore go undetected by neonatal screening. Patients with BH4 deficiency are unable to synthesize DA, NE, and 5-HT in quantities sufficient to maintain functions that depend on these neurotransmitters. BH4 deficiency also severely reduces NO production in the brain (26) and would be expected to reduce AGMO activity as well. A number of studies have shown that BH4 deficiency can affect some brain functions but not others. For example, patients can present with postural abnormalities, indicative of dysfunction of NSDA neurons yet have normal hypothalamic DA neuron control of prolactin secretion (27). Select functional deficits in central and peripheral MA systems resulting from BH4 deficiency were originally attributed to differences in MA turnover rates. Although differences in MA turnover, no doubt, contribute to the diversity of the BH4 deficiency phenotype, an alternative hypothesis is that different populations of MA neurons normally have different capacities to synthesize BH4. MA neurons that maintain the lowest levels of BH4 would therefore be most vulnerable to deficiencies in BH4 synthesis or regeneration (28). The latter hypothesis received support in 1994 with the report by Ichinose that patients heterozygous for mutations in GCH1 exhibit a mild dystonia referred to as hereditary progressive dystonia (HPD) also known as DA-responsive dystonia (29). HPD is not detected by neonatal screening for HPA, is progressive in nature because motor symptoms worsen as the day progresses, and is effectively treated with low doses of the DA precursor L-dopa. HPD is characterized by low concentrations of BH4, neopterin, and DA in the CSF and postmortem basal ganglia, along with the virtual absence of TH protein Kapatos 325

4 IUBMB LIFE within what otherwise appear to be normal NSDA nerve terminals (30 32). The loss of MA is specific to NSDA neurons; levels of 5-HT and NE in the CSF of these patients are generally within the normal range (33). Selective deficits in NSDA neuron function have since been reproduced in transgenic mouse models of BH4 deficiency (34,35). These studies indicate that the unique susceptibility of NSDA neurons to BH4 deficiency is conserved across evolution and therefore based on the intrinsic properties of these neurons. This animal research ultimately resulted in the discovery that BH4 protects TH from protein degradation (36), which may explain the severe loss of TH protein in the basal ganglia of HPD patients. HPD is an autosomal dominant disease and, while the inheritance pattern is not sex linked, the disorder primarily affects females. Genetic studies of maternal versus paternal transmission and gender-related penetrance show no evidence for genetic imprinting of the GCH1 locus (37). Studies in rodents do indicate that GCH1 gene expression is sexually dimorphic in 5-HT, NSDA, and NE neurons and in the liver, with lower levels in females than in males but that this difference is not dependent on adult sex hormones (23,38). Moreover, gender-related differences in GCH1 mrna expression are not found in the postmortem human cerebellum (39) or substantia nigra (unpublished observations) of adult males and females. The 4:1 ratio of female to male HPD patients must therefore be related to some more subtle aspect of GCH1 gene control within NSDA neurons. One such possibility is that estrogen acting through either the a or b estrogen receptor antagonizes cyclic adenosine monophosphate (camp)-dependent GCH1 transcription (see below) (40). The HPD phenotype raises a number of intriguing questions about GCH1 gene expression, not the least of which is how a heterozygous mutation in GCH1 can have such a selective and devastating effect on NSDA neurons. We have proposed that HPD is a cell type-specific haploinsufficiency. Specifically, we suggest that low basal levels of GCH1 gene expression and therefore BH4 synthesis are an intrinsic or regulated property that makes NSDA neurons more susceptible than other MA cell types to BH4 deficiency (41). Knowing how GCH1 gene transcription is regulated within NSDA neurons would therefore seem essential if we are to understand the HPD phenotype and the eightfold decline in GCH1 gene expression that occurs within NSDA neurons across the human life span and may be responsible for age-related declines in motor performance (42). This conclusion is perhaps best illustrated by recent literature, describing mutations in the human GCH1 proximal promoter that result in HPD (43 45). Expression in the Human Brain of Genes Coding for BH4 Biosynthetic and BH-dependent Enzymes With the exception of a very preliminary study performed on a single human brain which showed that GCH1 mrna is enriched in MA cell types and more abundant in 5-HT than in DA neurons, virtually nothing is known about the expression of genes coding for BH4 biosynthetic enzymes in the human brain (46). SR deficiency reveals, however, that BH4 synthesis catalyzed by the AKR1C3 and AKR1B1 alternate de novo pathway (Fig. 1) cannot support MA or even NO neurotransmission in the human brain (7,26). One possible explanation for this is that genes coding for AKR1C3 and AKR1B1 are not expressed within the human central nervous system. The Allen Brain Institute offers a comprehensive public database of microarray gene expression profiles across human brain regions that have been dissected under strict anatomical control ( Brain tissue for this analysis was obtained postmortem from 18- to 68-year-old males and females with no known neuropsychiatric or neuropathological history. Gene expression levels for GCH1, GCHFR, PTS, SPR, CBR1, AKR1B1, AKR1C3, and DHFR of the de novo and salvage pathways as well as three representative BH4-dependent enzymes, TH, NOS1, and AGMO, were mined from this database. Figure 2A is a global heat map showing gene expression levels for these 11 transcripts normalized by Z-scores and organized in rows with brain regions arranged in columns. In this plot, increasing red saturation represents increasing levels of gene expression, black average gene expression, and increasing green saturation decreasing levels of gene expression. As evidenced by concentrated patches of green (cerebral cortex) and red (hypothalamus and basal ganglia) across different brain regions, gene expression levels for all of the known and predicted enzymes of the de novo and salvage pathways are highly correlated with each other and with the expression of genes coding for BH4-dependent enzymes. Although correlative, the coexpression of genes long established to be involved in BH4 synthesis, namely GCH1 and PTS, with genes coding for enzymes of the alternative de novo pathways and salvage pathways, SPR, CBR1, AKR1B, and AKR1C3, provides strong evidence supporting the existence of alternative pathways for BH4 synthesis in the human brain. Figure 2B expands select regions of the global heat map to show expression levels for these 11 genes in 5-HT, NSDA, and NE neurons as well as in the corpus callosum, a brain region chosen as a negative control because it contains no intrinsic neurons. This expanded heat map shows that 5-HT neurons of the midbrain raphe express very high levels of GCH1 and GCHFR mrnas. Identical results were observed for all of the 5-HT cell groups represented on the global heat map (data not shown). GCH1 mrna expression in NSDA neurons of the substantia nigra is also high relative to other regions but, as indicated by the lower level of red saturation, not as high as in 5-HT neurons. In contrast to 5-HT neurons, the abundance of GCHFR mrna is very low in NSDA neurons. NE neurons of the locus coeruleus express GCH1 mrna at levels similar to those observed in NSDA neurons and, like NSDA neurons, are relatively devoid of GCHFR mrna. This analysis supports previous studies in the rodent and human brain, which showed that GCH1 mrna is more abundant in 5-HT than in NSDA neurons or NE neurons of the 326 The Neurobiology of Tetrahydrobiopterin Biosynthesis

5 FIG 2 Expression of genes coding for proteins known and hypothesized to be involved in BH4 synthesis in the human brain. A. Gene expression levels in human brain regions for GCH1, GCHFR, PTS, SPR, CBR1, AKR1B1, AKR1C3, and DHFR of the de novo and salvage pathways and the BH4-dependent enzymes TH, NOS1, and AGMO were mined using the Allen Brain Institute public database of microarray gene expression profiles. Transcript levels are normalized by Z-scores with increasing red saturation representing increased levels of gene expression, black average gene expression, and increasing green saturation decreased levels of gene expression. Concentrated patches of red and green, some of which have been underlined, show that across human brain regions gene expression for all of the enzymes of the de novo and salvage pathways are highly correlated with each other and with the expression of genes coding for the three BH4-dependent enzymes. B. Regions of the global heat map are expanded to show expression levels of these eleven genes in 5-HT, NSDA, and NE neurons as well as in the corpus callosum, a brain region that contains no intrinsic neurons. Transcripts coding for BH4 synthetic enzymes are enriched and coexpressed within MA neurons, supporting the conclusion that CBR1, AKR1B1, and AKR1C3 are components of the BH4 biosynthetic pathway. Although TH and NOS1 transcripts are distributed in a predictable manner across brain regions, AGMO mrna is enriched within the non-neuronal cells of the corpus callosum. AMGO might therefore play an important role in the functioning of oligodendrocytes. locus coeruleus (21,23,46). Moreover, it supports an earlier study in rat brain, showing that GCHFR mrna is highly expressed by 5-HT neurons but is virtually absent from NSDA and NE neurons of the locus coeruleus (24). Figure 2B also shows that mrnas coding for PTS and SPR are expressed at average to high levels in 5-HT, NSDA, and NE neurons. Transcript abundance for other enzymes involved in alternate de novo pathways shows more heterogeneity, however, with CBR1 at low levels in 5-HT neurons but at average to high levels in NSDA and NE neurons and AKR1B1 and AKR1C3 at high to very high levels in 5-HT and NE neurons but average to high levels in NSDA neurons. Expression of the Kapatos mrna coding for DHFR, the second enzyme in the salvage pathway, is very low in 5-HT and NE neurons but average in NSDA neurons. Transcripts of genes coding for established and proposed BH4 synthetic enzymes are therefore coenriched and coexpressed within MA neurons. Why then is the pathway for BH4 synthesis catalyzed by AKR1C3 and AKR1B1 unable to produce sufficient BH4 for MA and NO synthesis in the brains of patients with SR deficiency (7,8)? The physiological relevance of differences in GCH1 and GCHFR gene expression to the synthesis of 5-HT, DA and NE in central MA neurons remains open to conjecture. The fact that these differences are conserved across evolution, 327

6 IUBMB LIFE however, suggests their importance. In the rat brain, higher levels of GCH1 mrna are translated into higher levels of GTPCH protein (22). 5-HT neurons therefore have a greater capacity to synthesize BH4 than do NSDA or NE neurons. Similarly, the expression of GFRP by 5-HT neurons confers responsiveness to BH4 and L-phenylalanine on GTPCH activity and BH4 synthesis that is not found in NSDA and NE neurons (24). It is interesting to note that GFRP is also absent from MA cell types located outside of the central nervous system, including the pineal gland and the adrenal medulla (24). Data from microarray experiments often lead to unexpected findings, such as the high levels of AGMO mrna associated with the non-neuronal cells of the corpus callosum. The natural substrates and products of AMGO are currently being determined. Based on AMGO gene expression in the corpus callosum, however, this novel BH4-dependent transmembrane enzyme might eventually be found to play an important role in the myelination of axons by oligodendrocytes. It is interesting to note that expression of CBR1 and DHFR transcripts coding for an alternate salvage pathway are also enriched in this tissue. Regulation of BH4 Synthesis within MA Cell types After extended periods of increased neurotransmitter release, BH4 biosynthetic capacity is increased as part of the co-ordinated induction of MA biosynthetic enzymes. For example, BH4 content and GTPCH enzyme activity (47,48), GCH1 and PTS mrna (49,50), and GCH1 gene transcription (51) are all elevated in the rat adrenal gland after extended periods of increased NE turnover. First messengers that control GCH1 gene expression in various MA cell types include the neurotrophin nerve growth factor (52 54), epidermal growth factor (52), the neuropoietic cytokines leukemia inhibitory factor and ciliary neurotrophic factor (55), glial cell-derived neurotrophic factor (56), glucocorticoids (51), estrogen (57), vasoactive intestinal peptide (58), lithium (59), and membrane depolarization (14). Taken together, these studies show that GCH1 gene expression in MA cells is dynamic and controlled by a diverse and growing collection of extracellular signaling molecules and signal transduction pathways that ultimately converge on GCH1 gene transcription (for review, see ref. 60). The GCH1 Proximal Promoter Confers basal and camp-dependent Transcription The biological functions of many first messengers are mediated by G-protein-coupled receptors (GPCRs) that are positively or negatively coupled to adenylyl cyclase (AC) and the production of the second messenger 3 0,5 0 -cyclic adenosine monophosphate (camp). Application of camp analogues or antagonists to cells can therefore mimic GPCR activation. BH4 synthesis is increased by camp in adrenal medullary cells (47), NSDA and hypothalamic DA neurons (14), PC12 cells (58) and the human neuroblastoma SKN- BE(2)M17 (61) but not in the pineal gland (62), the astrocytoma C6, or the fibroblast Rat2 cell lines (63 65). This implies that the transcriptional response of the GCH1 gene to camp involves a novel combination of cis-elements and trans-acting factors. Our studies have focused on GCH1 transcription in the PC12 cell line because it is impossible to obtain pure preparations of NSDA neurons and because like NSDA neurons, PC12 cells are a MA cell type that responds to camp with an increase in GCH1 transcription. Based on deletion analysis of GCH1 promoter-luciferase reporter constructs performed in cell lines that do or do not respond to camp with an increase in GCH1 mrna, it was concluded that the first 140 bp upstream from the rat and human GCH1 transcription start sites contains all of the DNA regulatory elements necessary and sufficient for cell type-specific campdependent transcription (61,63 65). Detailed analysis of the rat and human proximal GCH1 promoters has identified a number of DNA sequences that bind nuclear protein extracts prepared from rat and human cell lines that respond to camp with an increase in GCH1 transcription. These regulatory elements include ( ) a repeating GC-box, a noncanonical camp response element (CRE), another GC-box found in the human but not the rat sequence, a canonical CCAAT-box and a canonical E-box found in the rat but not the human sequence (Fig. 3A). Inasmuch as both the rat and human GCH1 proximal promoters drive camp-dependent transcription, cis-elements common to both must be candidates for conferring camp responsiveness, including the 5 0 GC-box, the CRE and the CCAAT-box. A broad array of techniques was used to determine which proteins are recruited to the common cis-elements of the GCH1 proximal promoter (Fig. 3B). These studies reveal that the 5 0 repeating GC-box is actually a triad of GC-rich elements that bind specificity proteins Sp1 and Sp3 (65). Sp3 occupies two sites within the GC-box triad, whereas Sp1 can occupy only one. Cyclic-AMP-response element-binding protein (CREB), activating transcription factors (ATFs) 2 and 4 and CCAAT/enhancer binding protein beta (C/EBPb) are the cognate proteins bound by the CRE (61,63,64). Also, recruited to the CRE are basic region leucine zipper transcription factors including C-jun, C/EBPa, and C/EBPd (66,67). The heterotrimeric transcription factor nuclear factor-y (NF-Y) is the only protein recruited by the CCAAT-box (61,63,68). Although RNF4 may act as a coactivator for NF-Y binding to the GCH1 CCAAT-box (68), in the absence of RNF4 recombinant NF-Y protein binds the CCAAT-box with high affinity (61). Acting alone, Sp3 and to a lesser degree Sp1, NF-Y, and C/EBPb but not CREB are each able to trans-activate the GCH1 promoter and to synergistically activate transcription when bound in combinations of any two or all three proteins (60,65). Both the CRE and the CCAAT-box can act independently to confer camp-dependent transcription on the CGH1 promoter although enhancement from the CRE predominates (63,64). In contrast to the CRE and the CCAAT-box, the GC-box is not involved in camp-dependent transcription but is a major 328 The Neurobiology of Tetrahydrobiopterin Biosynthesis

7 FIG 3 Common cis-elements and trans-acting protein factors in the human and rat GCH1 proximal promoters. A. The 140-bp human and rat GCH1 proximal promoters confers basal and camp-dependent gene transcription and share common cis-elements, including the 5 0 GC-box, CRE, CCAAT-box, and TATAA-box. Interesting differences between the human and the rat promoters include an additional GC-box located between the conserved CRE and the CCAAT-boxes of the human promoter and the E-box in the rat but not the human sequence. The transcription start sites (þ1) for the human and rat promoters are also different with the start site in the human gene located within the nonconserved rat E-box, whereas the start site for the rat gene is located 17 bp downstream of the E-box. B. The conserved GC-box is a triad of cis-elements that bind specificity proteins Sp1 and Sp3. CREB, ATF 2 and 4, and CCAAT/enhancer binding protein beta (C/EBPb) bind to the conserved CRE as do C-jun, C/EBPa, and C/EBPd. The protein(s) recruited to the nonconserved GC-box located between the CRE and the CCAAT-box in the human promoter has not been characterized. The transcription factor nuclear factor-y (NF-Y) is the only protein recruited by the conserved CCAAT-box. RNF4 may serve as a coactivator for NF-Y binding to the human CCAAT-box. The protein(s) that binds to the nonconserved E-box in the rat sequence has not been characterized. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] determinant of basal transcription (65,74). Because of the dual nature of the GCH1 camp-response element (the CRE and CCAAT-box cassette) and recent studies showing protein kinase A (PKA)-independent signaling by camp (69,70), it was hypothesized that camp might act via multiple signaling pathways to regulate GCH1 transcription from either the CRE or the CCAAT-box. Experiments conclusively demonstrate, however, that PKA is necessary and sufficient for camp-dependent control of GCH1 transcription from both promoter elements (64). It was concluded from these experiments that the proteins recruited by the CRE and the CCAAT-box and/or the coactivators recruited by these proteins must therefore be substrates for PKA. This is not to say, however, that camp-dependent signaling is the sole mechanism for activating GCH1 transcription in PC12 cells. For example, nerve growth factor induction of transcription from the human GCH1 proximal promoter operating in PC12 cells is mediated by the Ras/mitogen-activated protein kinase (MEK) pathway (71). Cyclic AMP-dependent GCH1 transcription is, however, unique in that it does not involve increased acetylation of H3 and H4 histones located in proximal promoter-associated nucleosomes nor is the GCH1 CpG island differentially methylated in cells that do or do not respond to camp with an increase in transcription (64,65). A Model of camp-dependent GCH1 Transcription Chromatin immunoprecipitation (ChIP) assays have revealed that proteins that bind in vitro to the conserved GC-box, CRE, and CCAAT-box also bind in vivo to the endogenous GCH1 Kapatos 329

8 IUBMB LIFE FIG 4 A model for camp-dependent GCH1 gene transcription. Under basal conditions, the proximal promoter has CREB bound to the CRE and Sp3 bound to the GC-box. This combination maintains a low level of transcription by recruiting low levels of the general transcriptional machinery, including Pol II. 0.5 h after the application of camp PKA enhances transcription by phosphorylating CREB bound to the CRE. Subunits of NF-Y located in the cytoplasm are also phosphorylated by PKA, which stimulates NF-Y heterotrimer formation in the nucleus and binding to the CCAAT-box. Phosphorylation of CREB and binding of NF-Y to the CCAAT-box results in recruitment of the coactivator CBP and Pol II to the promoter, resulting in an increase in GCH1 transcription. C/EBPb in the cytoplasm and nucleus is also phosphorylated by PKA and nuclear C/EBPb displaces CREB from the CRE. Although NF-Y is displaced from the promoter, C/EBPb recruits additional CBP and Pol II. The stable complex of Sp3, C/ EBPb, and CBP then maintains elevated levels of GCH1 transcription. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] proximal promoter actively transcribing GCH1 within living PC12 cells, namely Sp1, Sp3, CREB, C/EBPb, and NF-Y (64,65). A model for camp-dependent GCH1 transcription that is based on these ChIP experiments is shown in Fig. 4. Under basal conditions, the proximal promoter has CREB bound to the CRE, one molecule of Sp1 or two molecules of Sp3 bound to the GC-box as well as low levels of the general transcriptional machinery, including Pol II. Cyclic-AMP acting through PKA enhances GCH1 transcription by first phosphorylating CREB already bound to the promoter. Subunits of the NF-Y heterotrimer located in the cytoplasm are also substrates for PKA, and their phosphorylation by PKA stimulates NF-Y assembly, transport to the nucleus, and binding to the CCAAT-box. Phosphorylation of CREB and the binding of NF-Y to the promoter recruit the coactivator CREB binding protein (CBP) and Pol II which results in a burst of GCH1 transcription. Activation of PKA by camp also promotes phosphorylation of C/EBPb in the cytoplasm and in the nucleus. C/EBPb in the cytoplasm is translocated to the nucleus where it and nuclear C/EBPb displace CREB from the CRE and eventually NF-Y from the CCAAT-box. The stable complex of Sp3, C/EBPb, and CBP then serves to maintain a stable but elevated level of Pol II activity and GCH1 transcription. A Model for Regulation of GCH1 Gene Transcription within NSDA Neurons Are the low levels of GCH1 transcription maintained by NSDA neurons an intrinsic property of GCH1 gene function, such as DNA hypermethylation, or a regulated property determined by cell physiology? It is known that NSDA neurons maintained in culture have all of the cellular machinery necessary to respond to camp with an increase in GCH1 transcription (14). In addition, GCH1 transcription by NSDA neurons in the intact rodent brain is dramatically increased by reserpine, a drug that produces massive DA depletion in NSDA cell bodies and nerve terminals (49). As GCH1 transcription in NSDA neurons is not enhanced by membrane depolarization (14), which is also a consequence of reserpine treatment, we have proposed that reserpine s effect on GCH1 transcription results not from increased nerve impulse flow but rather from the depletion of intracellular stores of DA (72). An updated version of this model of GCH1 transcriptional regulation is shown in Fig. 5 and is based on the novel physiology of NSDA neurons, which release DA from their dendrites to stimulate a GPCR of the D2 type located on the cell body. D2 GPCRs are negatively coupled to AC through inhibitory G- 330 The Neurobiology of Tetrahydrobiopterin Biosynthesis

9 FIG 5 Regulation of GCH1 gene transcription within NSDA neurons. DA released from the dendrites of NSDA neurons stimulates a GPCR of the D2 type located on the NSDA neuron cell body, which is negatively coupled through inhibitory G-proteins (Gi) to AC. Autostimulation of D2 receptors by DA inhibits the synthesis of camp by AC, thereby decreasing activation of PKA and decreasing GCH1 transcription. Afferent nerve terminals on NSDA neurons release neurotransmitters that stimulate GPCRs that interact with AC through stimulatory G-proteins (Gs), increasing production of camp, and increasing GCH1 transcription. A balance between stimulation and inhibition of camp production is therefore proposed to control GCH1 transcription and under normal conditions this balance is tipped in favor of inhibition. Alterations in GCH1 transcription within the nucleus are translated into changes in the amount of GTPCH protein transported down the axon to DA nerve terminals located in the basal ganglia and drive similar changes in BH4 synthesis. As BH4 is subsaturating for TH in NSDA, nerve terminal changes in BH4 would alter TH catalytic activity and possibly the amount of TH protein, resulting in changes in DA synthesis and release that would ultimately modify the delicate balance of neurotransmission within the basal ganglia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] proteins (Gi) (73). Autostimulation of NSDA neuron somatic D2 receptors by DA inhibits the synthesis of camp thereby decreasing activation of PKA and decreasing GCH1 transcription. Afferent nerve terminals that release neurotransmitters other than DA also stimulate GPCRs located on NSDA neurons but these GPCRs interact with AC through stimulatory G-proteins (Gs), increasing camp production, and GCH1 transcription through the process described in Fig. 4. In this model, GCH1 transcription is dependent upon the balance between neurotransmitters that stimulate camp production and neurotransmitters that inhibit camp production. Under normal conditions, this balance is tipped in favor of inhibition of camp production by the release of dendritic DA by the NSDA neurons themselves. Unique cellular physiology rather than genomic repression would therefore be the principle determinant of GCH1 transcription within NSDA neurons. To be functional, alterations in GCH1 transcription within the nucleus must be translated into changes in the amount of GTPCH protein synthesized within the cell body and transported down the axon to DA nerve terminals located in the basal ganglia (10). Alterations in GTPCH activity in the nerve terminal would drive similar changes in BH4 synthesis and, because BH4 is subsaturating for TH in NSDA nerve terminals (16,17,74), would alter TH catalytic activity and quite possibly the amount of TH protein (36). The resulting changes in DA synthesis and release would ultimately alter the balance of neurotransmission within the basal ganglia. Acknowledgements This study was supported by grants from the National Institute of Neurological Diseases and Stroke. References [1] Kaufman, S. (1963) The structure of the phenylalanine-hydroxylation cofactor.proc. Nat. Acad. Sci. USA 50, Kapatos 331

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