Specific Increase of Dopamine Synthesis THrough Targeting of the Guanylate Cyclase 2C Receptor in the Treatment of Parkinson's Disease

Abstract

In embodiments the invention relates to means and methods for the treatment of Parkinson's disease. In some embodiments the means and methods involve a GUCY2C agonist. The invention also relates to test systems and cells that are suited to identify new candidate compounds for the treatment of Parkinson's disease.

Claims

1. A GUCY2C agonist for use in the treatment of an individual that has Parkinson's disease.

2. The GUCY2C agonist of claim 1, further comprising administering a PDE inhibitor to the individual.

3. The GUCY2C agonist of claim 1 or claim 2, wherein the individual has Parkinson's disease stage 1, 2, 3 or 4.

4. The GUCY2C agonist of any one of claims 1-3, wherein the agonist is guanylin or a functional derivative thereof.

5. The GUCY2C agonist of any one of claims 1-4, wherein the treatment further comprises a administering a PDE inhibitor to the individual.

6. A method of increasing dopamine production by a dopaminergic cell, the method comprising increasing signaling by Guanylate Cyclase 2C (GUCY2C) in said cell.

7. A method of increasing the level of phosphorylation of Ser40 of tyrosine hydroxylase in a dopaminergic cell, the method comprising increasing signaling by GUCY2C in said cell.

8. The method of claim 6 or claim 7, wherein the signaling by GUCY2C is increased by contacting said cell with a GUCY2C agonist.

9. The method of claim 8, wherein said agonist is guanylin or a functional derivative thereof.

10. The method of any one of claims 6-9, further comprising providing said cell with a PDE inhibitor.

11. The method according to any one of claims 6-10, wherein the cell is a dopaminergic neuron, preferably a midbrain dopaminergic neuron.

12. An isolated or recombinant human cell comprising ectopic expression of human GUCY2C and/or ectopic expression of human tyrosine hydroxylase.

13. The isolated or recombinant human cell of claim 12 comprising ectopic expression of human GUCY2C and ectopic expression of human tyrosine hydroxylase.

14. A method for identifying a candidate compound for modifying dopamine production by a dopaminergic cell, the method comprising culturing a cell according to claim 12 or claim 13; contacting said cell with the candidate compound and determining the activity of tyrosine hydroxylase in said cell.

15. The method of claim 14, wherein the level of phosphorylation of Ser40 of tyrosine hydroxylase is determined.

16. Use of a GUCY2C gene, an RNA or a protein encoded by the gene as a target for identifying a compound that is active in modulating tyrosine hydroxylase activity in a dopaminergic cell.

17. A method of treatment of an individual that has Parkinson's disease, or is at risk of developing the disease, the method comprising administering a GUCY2C agonist to the individual in need thereof.

Description

EXAMPLES

Materials and Methods

[0076] Cell culture

[0077] HEK cells were maintained in 100-mm Petri dishes and grown in DMEM medium supplemented with L-glutamine, penicillin-streptomycin (Pen&Strep) and 10% heat-inactivated fetal bovine serum (HIFBS). Growing conditions were 37 C. and 5% CO2. For every-other-day passages, HEK cultures were split at a 1:3 dilution. From Friday to Monday, the split proportion was doubled for all cell lines. For passaging, cultures were rinsed with PBS and incubated with 1 mL of trypsin during 5 minutes. Cells were resuspended in growth medium and finally split to the proper dilution. The experiments were performed in 12-well plates. For immunocytochemistry analysis, sterile 18-mm coverslips were added prior to the plating. 500 L of cell resuspension was seeded in each well. If experiments were to be performed 48 hours after the plate preparation, 1:5 was the seeding ratio for HEK cells. When the experiments took place 96 hours post-plating, 1:7 was the working dilution for HEK cells. Unless otherwise stated, HEK cultures were processed 96 hours post-plating.

[0078] Cell Transfection

[0079] Plasmids encoding mouse Th and human GUCY2C had a pcDNA3.1 backbone. HEK cells were transfected using the calcium-phosphate method. 12-well plates were refreshed with 500 L of growth medium before transfection. For each pair of wells, plasmids of interest did not exceed 2.5 g and were filled up to 5 g with the empty construct pBlueScript. The plasmid mixture was taken into a final concentration of 250 mM CaCl2 in a total volume of 110 L. A complementary tube was filled with 110 L of HEPES-buffered saline 2 (HEBS 2: 1.5 mM Na2HPO4, 50 mM HEPES pH 7.05, 280 mM NaCl). The tube with CaCl2 was pipetted drop-wise into the HEBS and mixed gently. After 60 seconds of incubation, 110 L of the final mixture was added to each well. The medium was replaced within the next 24 hours.

[0080] Chemical Treatment

[0081] 24 hours prior to the administration of the different compounds, cells were serum-starved with DMEM medium supplemented with L-glutamine and Pen&Strep. Unless otherwise stated, the concentration of the different reagents is reported in Table 1 and the duration of the treatment was 1 hour.

[0082] Western Blotting

[0083] Cells were harvested in 150 L of Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris-Cl pH 6.8, 0.01% bromophenol blue, 50 mM freshly added DTT). Wells were duplicated in pairs and pooled into the same tubes to minimize variability. Samples were sonicated at maximum potency during 3 minutes, heated at 95 C. for 5 minutes and spun down briefly before loading. Running gels were 10% polyacrylamide except for the GUCY2C detection, which was optimal in 7% gels (375 mM Tris-Cl pH 8.8, 0.1% APS, 0.1% SDS, 0.04% TEMED). Stacking gels were 5% polyacrylamide (125 mM Tris-Cl pH 6.8, 0.1% APS, 0.1% SDS, 0.04% TEMED).

[0084] Up to 35 L of sample was loaded in each slot and run in the presence of tris-glycine buffer and 0.1% SDS. Running conditions were 100 V during the first 20 minutes, followed by 160 V until the migration front reached the bottom of the gel. The transference was performed at 100 V onto 0.2 m nitrocellulose membranes, in the presence of tris-glycine buffer and 20% methanol. The blotting duration was 140 minutes except for the detection of GUCY2C, which was transferred during 240 minutes in the presence of 0.1% SDS. Membranes were then submerged in Ponceau S solution to check blotting efficiency (0.1% Ponceau S, 5% acetic acid). After several washings with DEMI water, blots were incubated during 1 hour in the presence of 5% milk powder and TBS-T (154 mM NaCl, 49.5 mM Tris-Cl pH 7.4, 0.1% Tween-20). The incubation with the primary antibodies was performed O/N at 4 C. in TBS-T (consult Table 2 for dilution and species). Membranes were rinsed in TBS-T during 1 hour to remove the excess antibody. The incubation with the secondary antibodies took place during 60 minutes at room temperature (1:10,000 dilution in TBS-T, with the exception of the goat secondary which was diluted 1:20,000). Secondary antibodies were fused to the horseradish peroxidase and raised against the host species of the primary antibody. After additional washings during 1 hour, blots were exposed to an enhanced chemiluminescence solution and the signal was detected using an Odyssey imager (LI-COR). Band densitometry was performed with LI-COR Image Studio Lite. For graph quantifications, Fold Change represents the ratio P-Ser40/Total Th. Statistical comparisons between pair of groups correspond to unpaired Student's t-tests, as calculated with GraphPad Prism. Asterisks denote the following p-values: *<0.05, **<0.01, ***<0.001, ****<0.0001.

[0085] Immunocytochemistry

[0086] The growth medium was removed from the 12-well plates containing 18-mm coverslips. Following a washing step with ice-cold PBS, cells were fixed in 4% paraformaldehyde during 20 minutes and subsequently washed 3 times with PBS (137 mM NaCl, 2 mM KH2PO4, 100 mM Na2HPO4, 2.7 mM KCl ). The blocking was performed during 1 hour in the presence of 4% fetal donkey serum and 0.2% Triton X-100. Coverslips were then incubated O/N at 4 C. with the primary antibodies, diluted in PBS and 0.2% Triton X-100 (see Table 2 for antibody information). After rinsing the cells 3 times with PBS, the incubation with the secondary antibody took place during 2 hours at room temperature (1:1,000 dilution in PBS). An additional washing step with PBS preceded the 5-minute incubation with DAPI, diluted 1:3,000 in PBS. After a final rinse with PBS, coverslips were embedded with Fluorosave onto 6020 -mm slides. Final preparations were allowed to harden O/N at 4 C. before performing the analysis under the fluorescence microscope (Leica).

[0087] RNA Isolation and RT-qPCR

[0088] The growth medium was discarded from the culture dish and 1 mL of Trizol was added to each well. Cells were directly lysed in the plate and harvested into Eppendorf tubes. 200 L of chloroform was added and the mixture was incubated during 3 minutes after intense shaking. Samples were centrifuged at 4 C. during 15 minutes at 12,000 rcf. The upper aqueous phase was then collected into a new tube and the lower phases were disposed of. 10 g of glycogen and 500 L of isopropanol were added to the preparations. After a 10-minute incubation, samples were centrifuged at 4 C. during 10 minutes at 12,000 rcf. The supernatant was cautiously removed from the tubes and the pellet was subsequently rinsed with 1 mL of 75% ethanol. A brief vortex step was followed by a centrifugation at 4 C. during 5 minutes at 12,000 rcf. Supernatants were discarded and, once the pellets were moderately dry, resuspension took place in 30 L of RNAse-free water. Regarding the RT-qPCR analysis, purified RNA samples were diluted to a final concentration of 2.6 ng/L. For each reaction well, volumes of the different reagents were the following: 5 L SYBR Green buffer 2, 0.1 L reverse transcriptase, 0.1 L RNAse inhibitor, 0.5 L forward primer 10 M, 0.5 L reverse primer 10 M, 3.8 L purified RNA 2.6 ng/L. Reactions were carried out in a LightCycler 480 (Roche) according to the QuantiTect SYBR Green RT-PCR handbook (Quiagen). The ribosomal RNA 18S was used as loading control. Primers were designed using Primer-BLAST and their specificity was tested in a 1.5% agarose gel once the RT-qPCR was terminated.

[0089] Results

[0090] HEK Cells Can be Employed to Study the Induction of Ser40 Phosphorylation Upon GUCY2C Activation

[0091] We studied the regulation of Th via GUCY2C activation in vitro. Among the various cell lines which are available, we firstly employed HEK cells since they are easily transfectable and had been previously used to study the role of GUCY2C in cGMP production (Fiskerstrand T et al., 2012; Muller T et al., 2015). We checked the presence of the mRNAs encoding the kinases. RT-qPCR data showed a positive expression of the different isoforms of PKG (PRKG1 and PRKG2) and the catalytic centers of PKA (PRKACA and PRKACB), suggesting that HEK cells are a suitable model for our purposes (FIG. 3A). However, neither TH nor GUCY2C are endogenously expressed in this cell line. With the aim of checking if the cGMP-derived signaling could promote Ser40 phosphorylation, we transfected HEK cells with a Th-encoding plasmid and treated them with an artificial cGMP analogue (i.e. 8-Bromo-cGMP). As expected, we detected a small but significant increase in P-Ser40 levels upon the compound administration (FIG. 3B).

[0092] Next, in order to study the potential effects of GUCY2C activation on Th phosphorylation, we performed co-transfections with the plasmids encoding both proteins. Western blotting analysis revealed HEK cultures expressing Th and GUCY2C. Sole transfection with Th served as control for GUCY2C expression and recognition (FIG. 4B). Immunocytochemistry analysis allowed us to identify individual cells co-expressing both proteins, essential requirement to study the potential link between the receptor and Ser40 phosphorylation (FIG. 4A). Most importantly, within this experimental paradigm in which the kinases are endogenously expressed while Th and GUCY2C are co-transfected, we were able to increase P-Ser40 levels upon separate administration of different receptor ligands (i.e. guanylin and uroguanylin) (FIG. 4C).

[0093] The Induction of P-Ser40 Upon GUCY2C Activation is Proportional to the Levels of cGMP

[0094] Regarding the two ways by which the receptor activation might induce Ser40 phosphorylation, both depend on the initial production of cGMP (Fiskerstrand T et al., 2012). To test this assumption, we combined guanylin with the administration of IBMX, a general inhibitor of phosphodiesterases (PDEs). This family of enzymes breaks down cyclic nucleotides to their non-cyclic forms (Bender A T and Beavo J A, 2006). Therefore, as the cyclic variants can activate PKA and PKG, we hypothesized that inhibiting PDEs would potentiate the induction of P-Ser40 upon GUCY2C stimulation. Surprisingly, treatment with IBMX potentiated the increase in Ser40 phosphorylation as compared to guanylin administration (FIG. 5A). These data show that the basal pool of cyclic nucleotides in HEK cells is large and continuously subjected to degradation by PDEs as a negative-feedback mechanism. The combined treatment of guanylin with IBMX further increased Ser40 phosphorylation in relation to IBMX alone, suggesting that the basal and the GUCY2C-derived pools of cyclic nucleotides display additive effects (FIG. 5A).

[0095] However, since IBMX is a general PDE inhibitor, its effects on Ser40 phosphorylation result from the increase in the levels of both cAMP and cGMP. To correlate the extent of Ser40 phosphorylation exclusively with the amount of cGMP produced by the receptor, we devised an alternative approach. A previous paper described the existence of human naturally-occurring mutations in the GUCY2C-encoding gene, rendering a set of gain-of-function receptors (Mller T et al., 2015). These variants produce more cGMP than the wt form in the presence of guanylin. The mutant with the highest activity contains an arginine to serine substitution in the position 792 (R792S), located at the starting point of the catalytic domain.

[0096] HEK cultures were co-transfected with the plasmids encoding Th and either the wt or the R792S variant of GUCY2C. When compared to the wt receptor, the boost in Ser40 phosphorylation upon guanylin treatment was 1.5-fold higher when the R792S mutant was co-transfected (FIG. 5B).

[0097] The present invention shows methods to increase Th activity specifically in the nigrostriatal pathway. We focused on the phosphorylation of its regulatory domain. Both Ser31 and Ser40 phosphorylation have been reported to promote such an effect (Dunkley P R et al., 2004).

[0098] In conclusion, our experiments indicate that the signaling pathway underlying GUCY2C activation is specific for each model system. HEK cells show an induction of Ser40 phosphorylation which is proportional to GUCY2C activity levels.

[0099] The present invention shows that the combination of GUCY2C ligands with PDE inhibitors serve to increase dopamine production by dopaminergic cells. This finding is used as a therapy for PD.

[0100] Current levodopa treatments are often supplemented with AADC inhibitors which cannot reach the brain but prevent dopamine synthesis in peripheral tissues (Ahlskog J E et al., 1989). The therapy the invention provides provides a specific component, given the selective brain expression of GUCY2C in the SNpc, and a more or less non-selective component provided by the wide expression of PDE. An unspecific inhibition of PDEs is not a major complication for clinical treatments. In fact, some widely-used drugs are inhibitors of PDEs which are expressed in various off-target tissues. A representative example is sildenafil (Viagra), a PDES-selective inhibitor. This phosphodiesterase is expressed in the penis, where it is intended to exert its effects, but also in the heart, pancreas, kidney or cerebellum (Lin C S, 2004).

CITED ART

[0101] Ahlskog J E, Tyce G M, Kelly P J, van Heerden J A, Stoddard S L, Carmichael S W (1989). Cerebrospinal fluid indices of blood-brain barrier permeability following adrenal-brain transplantation in patients with Parkinson's disease. Exp Neurol. 1989 August; 105(2): 152-61.

[0102] Allen Brain Atlas (http://mouse.brain-map.org/).

[0103] Baladron J, Hamker F H (2015). A spiking neural network based on the basal ganglia functional anatomy. Neural Netw. 2015 July; 67:1-13. doi: 10.1016/j.neunet.2015.03.002. Epub 2015 Mar. 24.

[0104] Bender A T, Beavo J A (2006). Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006 September; 58(3):488-520.

[0105] Boess F G, Hendrix M, van der Staay F J, Erb C, Schreiber R, van Staveren W, de Vente J, Prickaerts J, Blokland A, Koenig G (2004). Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance. Neuropharmacology. 2004 December; 47(7):1081-92.

[0106] Campbell D G, Hardie D G, Vulliet P R (1986). Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase. J Biol Chem. 1986 Aug. 15; 261(23):10489-92.

[0107] Carta M, Carlsson T, Kirik D, Bjrklund A (2007). Dopamine released from 5-HT terminals is the cause of L-DOPA-induced dyskinesia in parkinsonian rats. Brain. 2007 July; 130(Pt 7):1819-33. Epub 2007 Apr. 23.

[0108] Clarke C E (2004). Neuroprotection and pharmacotherapy for motor symptoms in Parkinson's disease. Lancet Neurol. 2004 August; 3(8):466-74.

[0109] Dale Purves, George J Augustine, David Fitzpatrick, Lawrence C Katz, Anthony-Samuel LaMantia, James O McNamara, and S Mark Williams (2001). Neuroscience, 2nd edition. ISBN-10: 0-87893-742-0.

[0110] Defer N, Best-Belpomme M, Hanoune J (2000). Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol Renal Physiol. 2000 September; 279(3):F400-16.

[0111] Dunkley P R, Bobrovskaya L, Graham M E, von Nagy-Felsobuki E I, Dickson P W (2004). Tyrosine hydroxylase phosphorylation: regulation and consequences. J Neurochem. 2004 December; 91(5):1025-43.

[0112] Fiskerstrand T, Arshad N, Haukanes B I, Tronstad R R, Pham K D, Johansson S, Hvik B, Tnder S L, Levy S E, Brackman D, Boman H, Biswas K H, Apold J, Hovdenak N, Visweswariah S S, Knappskog P M (2012). Familial diarrhea syndrome caused by an activating GUCY2C mutation. N Engl J Med. 2012 Apr. 26; 366(17):1586-95. doi: 10.1056/NEJMoa1110132. Epub 2012 Mar. 21.

[0113] Gershanik O S (2015). Improving L-dopa therapy: the development of enzyme inhibitors. Mov Disord. 2015 January; 30(1):103-13. doi: 10.1002/mds.26050. Epub 2014 Oct. 21.

[0114] Gong R, Ding C, Hu J, Lu Y, Liu F, Mann E, Xu F, Cohen M B, Luo M (2011). Role for the membrane receptor guanylyl cyclase-C in attention deficiency and hyperactive behavior. Science. 2011 Sept. 16; 333(6049):1642-6. doi: 10.1126/science.1207675. Epub 2011 Aug. 11.

[0115] Ikemoto S, Yang C, Tan A (2015). Basal ganglia circuit loops, dopamine and motivation: A review and enquiry. Behav Brain Res. 2015 Sept. 1; 290:17-31. doi: 10.1016/j.bbr.2015.04.018. Epub 2015 Apr. 20.

[0116] Jacobs F M, Smits S M, Noorlander C W, von Oerthel L, van der Linden A J, Burbach J P, Smidt M P (2007). Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency. Development. 2007 July; 134(14):2673-84.

[0117] Joh T H, Park D H, Reis D J (1978). Direct phosphorylation of brain tyrosine hydroxylase by cyclic AMP-dependent protein kinase: mechanism of enzyme activation. Proc Natl Acad Sci USA. 1978 October; 75(10):4744-8.

[0118] Kalia L V, Lang A E (2015). Parkinson's disease. Lancet. 2015 Aug. 29; 386(9996):896-912. doi: 10.1016/S0140-6736(14)61393-3. Epub 2015 Apr. 19.

[0119] Lin C S (2004). Tissue expression, distribution, and regulation of PDES. Int J Impot Res. 2004 June; 16 Suppl 1:S8-S10.

[0120] Lochner A, Moolman J A (2006). The many faces of H89: a review. Cardiovasc Drug Rev. 2006 Fall-Winter; 24(3-4):261-74.

[0121] MacFarland R T, Zelus B D, Beavo J A (1991). High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells. J Biol Chem. 1991 Jan. 5; 266(1):136-42.

[0122] Mahlknecht P, Seppi K, Poewe W (2015). The Concept of Prodromal Parkinson's Disease. J Parkinsons Dis. 2015; 5(4):681-97. doi: 10.3233/JPD-150685.

[0123] Morgenroth V H 3rd, Hegstrand L R, Roth R H, Greengard P (1975). Evidence for involvement of protein kinase in the activation by adenosine 3:5-monophosphate of brain tyrosine 3-monooxygenase. J Biol Chem. 1975 Mar. 10; 250(5):1946-8.

[0124] Mller T, Rasool I, Heinz-Erian P, Mildenberger E, Hlstrunk C, Mller A, Michaud L, Koot B G, Ballauff A, Vodopiutz J, Rosipal S, Petersen B S, Franke A, Fuchs I, Witt H, Zoller H, Janecke A R, Visweswariah S S (2015). Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C. Gut. 2016 August; 65(8):1306-13. doi: 10.1136/gutjnl-2015-309441. Epub 2015 May 20.

[0125] Navailles S, Milan L, Khalki H, Di Giovanni G, Lagire M, De Deurwaerdre P (2014). Noradrenergic terminals regulate L-DOPA-derived dopamine extracellular levels in a region-dependent manner in Parkinsonian rats. CNS Neurosci Ther. 2014 July; 20(7):671-8. doi: 10.1111/cns.12275. Epub 2014 Apr. 28.

[0126] Nikolaev V O, Gambaryan S, Engelhardt S, Walter U, Lohse M J (2005). Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis. J Biol Chem. 2005 Jan. 21; 280(3):1716-9. Epub 2004 Nov. 22.

[0127] Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).

[0128] Postuma R B, Aarsland D, Barone P, Burn D J, Hawkes C H, Oertel W, Ziemssen T (2012). Identifying prodromal Parkinson's disease: pre-motor disorders in Parkinson's disease. Mov Disord. 2012 Apr. 15; 27(5):617-26. doi: 10.1002/mds.24996.

[0129] Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

[0130] Romi H, Cohen I, Landau D, Alkrinawi S, Yerushalmi B, Hershkovitz R, Newman-Heiman N, Cutting G R, Ofir R, Sivan S, Birk O S (2012). Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C. Am J Hum Genet. 2012 May 4; 90(5):893-9. doi: 10.1016/j.ajhg.2012.03.022. Epub 2012 Apr. 19.

[0131] Roskoski R Jr, Vulliet P R, Glass D B (1987). Phosphorylation of tyrosine hydroxylase by cyclic GMP-dependent protein kinase. J Neurochem. 1987 March; 48(3):840-5.

[0132] Scansite3 (http://scansite3.mit.edu/#home).

[0133] Scholten A, Aye T T, Heck A J (2008). A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: in vivo characterization and structure/function relationships. Mass Spectrom Rev. 2008 July-August; 27(4):331-53. doi: 10.1002/mas.20166.

[0134] Smidt M P, Burbach J P (2007). How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci. 2007 January; 8(1):21-32. Erratum in: Nat Rev Neurosci. 2007 February; 8(2): 160.

[0135] Steegborn C (2014). Structure, mechanism, and regulation of soluble adenylyl cyclases-similarities and differences to transmembrane adenylyl cyclases. Biochim Biophys Acta. 2014 December; 1842(12 Pt B):2535-47. doi: 10.1016/j.bbadis.2014.08.012. Epub 2014 Sept. 2.

[0136] Stephenson D T, Coskran T M, Kelly M P, Kleiman R J, Morton D, O'Neill S M, Schmidt C J, Weinberg R J, Menniti F S (2012). The distribution of phosphodiesterase 2A in the rat brain. Neuroscience. 2012 Dec. 13; 226:145-55. doi: 10.1016/j.neuroscience.2012.09.011. Epub 2012 Sept. 19.

[0137] Stephenson D T, Coskran T M, Wilhelms M B, Adamowicz W O, O'Donnell M M, Muravnick K B, Menniti F S, Kleiman R J, Morton D (2009). Immunohistochemical Localization of Phosphodiesterase 2A in Multiple Mammalian Species. J Histochem Cytochem. 2009 October; 57(10):933-49. doi: 10.1369/jhc.2009.953471. Epub 2009 Jun. 8.

[0138] Tekin I, Roskoski R Jr, Carkaci-Salli N, Vrana K E (2014). Complex molecular regulation of tyrosine hydroxylase. J Neural Transm (Vienna). 2014 December; 121(12):1451-81. doi: 10.1007/s00702-014-1238-7. Epub 2014 May 28.

[0139] Upadhya M A, Shelkar G P, Subhedar N K, Kokare D M (2016). CART modulates the effects of levodopa in rat model of Parkinson's disease. Behav Brain Res. 2016 Mar. 15; 301:262-72. doi: 10.1016/j.bbr.2015.12.031. Epub 2016 Jan. 8.

[0140] Valentino M A, Lin J E, Snook A E, Li P, Kim G W, Marszalowicz G, Magee M S, Hyslop T, Schulz S, Waldman S A (2011). A uroguanylin-GUCY2C endocrine axis regulates feeding in mice. J Clin Invest. 2011 September; 121(9):3578-88. doi: 10.1172/JCI57925. Epub 2011 Aug. 25.

[0141] Zaccolo M, Movsesian M A (2007). cAMP and cGMP signaling cross-talk: role of phosphodiesterases and implications for cardiac pathophysiology. Circ Res. 2007 Jun. 8; 100(11): 1569-78.

TABLE-US-00001 TABLE 1 Example of a concentration, or concentration range and a function of different compounds used in the present invention Compound Conc. Function 8-Bromo-cGMP 500 M cGMP analogue and PKG activator Guanylin 10 M GUCY2C ligand Uroguanylin 1 M GUCY2C ligand IBMX 100 M General phosphodiesterase inhibitor

TABLE-US-00002 TABLE 2 Antibodies employed in western blotting (blue) and immunocytochemistry (green). The mouse and human GUCY2C were detected with the goat and mouse primary antibodies, respectively. Antibody Dilution Species GUCY2C 1:170 Mouse GUCY2C 1:170 Goat Total Th 1:1,000 Sheep Total Th 1:1,000 Rabbit P-Ser40 1:1,000 Rabbit Actin 1:3,000 Mouse Primary Antibody Dilution Secondary Antibody GUCY2C Goat 1:100 Donkey Goat 488 Total Th Rabbit 1:1,000 Goat Rabbit 488