USE OF A PDE11 OR PDE2 INHIBITOR FOR 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 PDE2 inhibitor, a PDE11 inhibitor or a combination thereof, optionally together with a GUCY2C agonist.

Claims

1-7. (canceled)

8. A method of increasing phosphorylation of the serine residue at position 40 of human tyrosine hydroxylase (S40 Th) by a dopaminergic cell, the method comprising contacting said cell with a PDE11 inhibitor, a PDE2 inhibitor or a combination thereof

9. A method of increasing dopamine production by a dopaminergic cell, the method comprising contacting said cell with a PDE11 inhibitor, a PDE2 inhibitor or a combination thereof

10. The method of claim 8, further comprising contacting said cell with a GUCY2C agonist.

11. 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 PDE11 inhibitor, a PDE2 inhibitor or a combination thereof to the individual in need thereof.

12. A GUCY2C agonist comprising the sequence NDDCELCVNVACTGCLL; NDCCELCCNVACTGCL; NDDCELCVNVVCTGCL; QEECEL[Abu]INMACTGY; QEECELCINMACTGCL; NTFYCCELCCNPACAGCY; NTFYCCELCCAPACTGCY; or NTFYCCELCCNPaCAGCY.

13. The method of claim 11, wherein the treatment further comprises a guanylate cyclase 2C receptor (GUCY2C) agonist.

14. The method of claim 11, wherein the individual has Parkinson's disease stage 1, 2, 3 or 4.

15. The method of claim 11, wherein the PDE2 inhibitor is EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine); BAY 60-7550 (2-[(3,4-Dimethoxyphenyl)methyl]-7-[(2R,3R)-2-hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one); PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one; or oxindole (2,3-dihydroindol-2-on).

16. The method of claim 11, wherein the PDE11 inhibitor is BC11-15; BC11-19; BC11-28; BC11-38; BC11-38-1; BC11-38-2; BC11-38-3 or BC11-38-4.

17. The method of claim, wherein the GUCY2C agonist is guanylin or a functional derivative thereof.

18. The method of claim 13, wherein the GUCY2C agonist comprises the sequence NDDCELCVNVACTGCLL; NDCCELCCNVACTGCL; NDDCELCVNVVCTGCL; QEECEL[Abu]INMACTGY; QEECELCINMACTGCL; NTFYCCELCCNPACAGCY; NTFYCCELCCAPACTGCY; or NTFYCCELCCNPaCAGCY.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1. Multiple sequence alignment of Th-regulatory domain across different species. The three highlighted serine residues (Ser19, Ser31 and Ser40) are susceptible to phosphorylation and are well conserved throughout evolution. The phosphorylation of Ser31 and Ser40 directly correlates with an increased activity of the enzyme. Note that the amino acids surrounding both residues are virtually unchanged, thereby conserving the consensus sequence and explaining why those serines are phosphorylated in different organisms. ‘*’ represents maximal conservation, followed by ‘:’ and ‘.’.

[0088] FIG. 2. A hypothetical pathway by which GUCY2C activation could lead to an increased Th activity. One alternative is the direct activation of PKG by cGMP, while the other consists in the indirect activation of PKA via the cGMP-mediated inhibition of PDE3, an enzyme involved in degrading cAMP into its non-cyclic form.

[0089] FIG. 3. HEK cells are a suitable model to study GUCY2C-induced Ser40 phosphorylation. (A) RT-qPCR data from intact HEK extracts. Input material was 10 ng of total RNA. Cp corresponds to the cycle number in which the expression starts to be considered positive. (−) represents the water control, which is negative in every case. (B) HEK cultures were transfected with Th and treated with a cGMP analogue 48 hours post-plating. Changes in Ser40 phosphorylation were studied. The graph represents the statistical analysis (n=6; Mean±SEM). Unpaired Student's t-test (p-value: ****<0.0001). Hereon, ‘Fold Change’ corresponds to the ratio P-Ser40/Total Th.

[0090] FIG. 4. GUCY2C activation increases Ser40 phosphorylation. Immunocytochemistry images (A) and western blotting analysis (B) of HEK cultures transfected with Th or Th+GUCY2C. (C) Co-transfected HEK cells were treated with guanylin (G) or uroguanylin (UroG) and changes in Ser40 phosphorylation were studied. The graph represents the statistical analysis (n=3 for Control, n=6 for Guanylin, n=9 for Uroguanylin; Mean±SEM). Unpaired Student's t-test (p-value: **<0.01).

[0091] FIG. 5. The increase in Ser40 phosphorylation is proportional to the produced amount of cGMP upon GUCY2C stimulation. (A) HEK cultures were treated with guanylin, in the presence or absence of IBMX. The graph shows the statistical analysis (n=3 for Control±Guanylin, n=6 for IBMX±Guanylin; Mean±SEM). Unpaired Student's t-test (p-values: ****<0.0001, **<0.01, *<0.05). (B) HEK cells were co-transfected with Th and either the wt GUCY2C or a gain-of-function mutant (R792S). Ser40 phosphorylation was studied following guanylin treatment (G). The graph shows the statistical analysis (n=4 for wt, n=3 for wt+Guanylin, n=4 for R792S±Guanylin; Mean±SEM). Unpaired Student's t-test (p-values: ***<0.001, **<0.01).

[0092] FIG. 6. PDE2a is an important effector regulating Ser40 phosphorylation in MN9D cells. MN9D cultures were treated with IBMX or BAY-60-7550 (50 μM), a PDE2A-selective inhibitor. Cells were analyzed on western-blot for phosphorylation of Ser40 , total Th, and actin. The graph shows the analysis (n=2, Mean+SEM).

[0093] FIG. 7. PDE2a is an important effector regulating Ser40 phosphorylation in MN9D cells. BAY-60-7550 dose-curve and representative examples in MN9D (n=9, red) and HEK cells (n=4, blue) (Mean±SEM). Cells were treated with various concentrations of BAY 60-7750 and analyzed for Th phosphorylation on Ser40 , total Th and actin. Note that the IC50 in the dopaminergic model is 1 μM, a concentration at which HEK cells hardly exhibit any effect.

[0094] FIG. 8. Relative enrichment in RNA expression between human prefrontal cortex neurons and human midbrain dopamine neurons. A negative value (Log2) represents over representation of the transcript in midbrain dopamine neurons. Originating from postmortem RNAseq analysis.

[0095] FIG. 9. PDE1la is enriched in the area containing midbrain dopamine neurons. Graph showing the relative enrichment of PDE11A in the area containing dopaminergic neurons as compared to whole brain. Data was collected from the publically available website Allen Brain Atlas and the database of micro array data of human subjects. The 3 bars indicate 3 different micro-array probes.

[0096] FIG. 10. PDE11 is an important effector regulating Ser40 phosphorylation in MN9D cells. BC 11-38 dose-curve and representative examples in MN9D (n=2) (Mean±SEM). IBMX served as a control and was used at 100 μM. Cell were analyzed for phosphorylation of Th on Ser40 and total Th was used as a loading control to determine phosphorylation specific effects.

[0097] FIG. 11. Structure of some PDE2a inhibitors.

[0098] FIG. 12. IC50 values of some PDE2a inhibitors.

[0099] FIG. 13. Bay 60-7550 and Guanylin synergize to induce Ser40 phosphorylation Analysis of striatal slices treated with indicated compounds. WES data showing the phosphorylation of Th on Serine 40 is shown as well as total Th levels (a). From left to right are sequential striatal slices from rostral to caudal. Every slice is split into left and right hemisphere and either treated with compounds or vehicle. Left a positive and negative control is shown (Neuro2a cells transfected with Th or empty vector). The amount of Th Ser40 is corrected for the amount of total Th and shown in the bar graph (b). Each individual slice is indicated by a different symbol. Signficance is determined using a 2-tailed paired t-test. 2-tailed paired t-test: p=0.006.

[0100] FIG. 14. Bay 60-7550 and Guanylin synergize to induce Ser40 phosphorylation Analysis of striatal slices treated with indicated compounds. WES data showing the phosphorylation of Th on Serine 40 is shown as well as total Th levels (a). From left to right are sequential striatal slices from rostral to caudal. Every slice is split into left and right hemisphere and either treated with compounds or vehicle. Left a positive and negative control is shown (Neuro2a cells transfected with Th or empty vector). The amount of Th Ser40 is corrected for the amount of total Th and shown in the bar graph (b). Each individual slice is indicated by a different symbol. Significance is determined using a 2-tailed paired t-test. 2-tailed paired t-test: p==0.403.

[0101] FIG. 15. Bay 60-7550 and Guanylin synergize to induce Ser40 phosphorylation Analysis of striatal slices treated with indicated compounds. WES data showing the phosphorylation of Th on Serine 40 is shown as well as total Th levels (a). From left to right are sequential striatal slices from rostral to caudal. Every slice is split into left and right hemisphere and either treated with compounds or vehicle. Left a positive and negative control is shown (Neuro2a cells transfected with Th or empty vector). The amount of Th Ser40 is corrected for the amount of total Th and shown in the bar graph (b). Each individual slice is indicated by a different symbol. Significance is determined using a 2-tailed paired t-test. 2-tailed paired t-test: p=0.018.

[0102] FIG. 16. Bay 60-7550 and Guanylin synergize to induce Ser40 phosphorylation Analysis of striatal slices treated with indicated compounds. WES data showing the phosphorylation of Th on Serine 40 is shown as well as total Th levels (a). From left to right are sequential striatal slices from rostral to caudal. Every slice is split into left and right hemisphere and either treated with compounds or vehicle. Left a positive and negative control is shown (Neuro2a cells transfected with Th or empty vector). The amount of Th Ser40 is corrected for the amount of total Th and shown in the bar graph (b). Each individual slice is indicated by a different symbol. Significance is determined using a 2-tailed paired t-test. 2-tailed paired t-test: p=0.073.

[0103] FIG. 17. Bay 60-7550 and Guanylin synergize to induce Ser40 phosphorylation Analysis of striatal slices treated with indicated compounds. WES data showing the phosphorylation of Th on Serine 40 is shown as well as total Th levels (a). From left to right are sequential striatal slices from rostral to caudal. Every slice is split into left and right hemisphere and either treated with compounds or vehicle. Left a positive and negative control is shown (Neuro2a cells transfected with Th or empty vector). The amount of Th Ser40 is corrected for the amount of total Th and shown in the bar graph (b). Each individual slice is indicated by a different symbol. Significance is determined using a 2-tailed paired t-test. 2-tailed paired t-test: p=0.024.

[0104] FIG. 18. GUCY2C ligand analysis.

[0105] Potential GUCY2C ligands were tested in HEK-293 cells transfected with GUCY2C and Th and subsequently treated and analyzed by automated western (WES). Th Ser40 was corrected for total Th. Controls were set to 1. Significance is determined by two-tailed t-test on n=3. * p<0.05, **p<0.01, ***p<0.005, “”p<0.001. A) Guanylin control 10 μM. B) Peptides 1 to 5 in an amount of 10 μM. C) Peptides 6 to 11 in an amount of 10 μM.

EXAMPLES

Example 1

Materials and Methods

[0106] IBMX or BAY-60-7550 were obtained from chemcruz biochemicals. HEK-cells were obtained from ATCC.

Cell Culture

[0107] 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 !μL 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.

Cell Transfection

[0108] 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 μL of growth medium before transfection. For each pair of wells, plasmids of interest did not exceed 2.5 μg and were filled up to 5μg with the empty construct pBlueScript. The plasmid mixture was taken into a final concentration of 250 mM CaCl2 in a total volume of 110 μL. A complementary tube was filled with 110 μL of HEPES-buffered saline 2X (HEBS 2X: 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, 554 of the final mixture was added to each well. The medium was replaced within the next 24 hours.

Chemical Treatment

[0109] 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.

Western Blotting

[0110] Cells were harvested in 150 μL 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).

[0111] Up to 35 μL 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 μm 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.

Immunocytochemistry

[0112] 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 60×20-mm slides. Final preparations were allowed to harden O/N at 4° C. before performing the analysis under the fluorescence microscope (Leica).

RNA Isolation and RT-qPCR

[0113] 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 μL 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 μg of glycogen and 500 μL of isopropanol were added to the preparations. After a 10-minute incubation, samples were centrifuged at 4° C. during 10 minutes at 12,000 ref. 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 ref. Supernatants were discarded and, once the pellets were moderately dry, resuspension took place in 30 μL of RNAse-free water. Regarding the RT-qPCR analysis, purified RNA samples were diluted to a final concentration of 2.6 ng/A. For each reaction well, volumes of the different reagents were the following: 5 μL SYBR Green buffer 2X, 0.14 reverse transcriptase, 0.1 μL RNAse inhibitor, 0.5 μL forward primer 10 μM, 0.5 μL reverse primer 10 μM, 3.8 μL purified RNA 2.6 ng/μL. 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.

Results

HEK Cells Can Be Employed to Study the Induction of Ser40 Phosphorylation Upon GUCY2C Activation

[0114] 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).

[0115] 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).

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

[0116] 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).

[0117] 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 (Muller 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.

[0118] 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).

[0119] IBMX is a non-selective PDE inhibitor, therefore we investigated the effect of inhibiting a specific PDE selectively on Ser40 phosphorylation. To study the effect of PDE2A inhibition on Ser40 phosphorylation we treated the dopaminergic cell-line MN9D with Bay BAY-60-7550 and analyzed the effects. BAY-60-7750 resulted in a more than 4-fold increase in the phosphorylation of Ser40 and performed better than IBMX although at half the concentration (FIG. 6). Since BAY 60-7750 performed very well in the dopaminergic cell line MN9D we compared the effect of BAY 60-7750 on MN9D cells to non-dopaminergic HEK-293 cells transfected with Th. A dose-response curve of cells treated with BAY 60-7550 clearly indicates that PDE2A inhibition has more profound effects in MN9D cells. Low concentrations of BAY 60-7750 result in a clear fold change increase in Ser40 phosphorylation, whereas there is almost no effect observed in HEK-293 cells (FIG. 7) suggesting specificity for dopaminergic cells.

[0120] Next we investigated the effect of a PDE11 inhibitor on Th phosphorylation on Ser40 as an additional selective PDE inhibitor. Expression data collected from allen brain atlas (https://www.brain-map.org/) and laser captured dopamine neurons shows that PDE11a is expressed in the human dopaminergic midbrain area and is also enriched in dopamine neurons (FIG. 8, 9).

[0121] Treatment of MN9D cells with the specific PDE11 inhibitor BC 11-38 resulted in an increase in the phosphorylation of Th on Ser40 as compared to total Th (FIG. 10). BC 11-38 at the highest concentration used performed less well than the positive control IBMX, also used at the same concentrations, which may be due to lower levels of PDE11A as compared to PDE2A.

[0122] The present invention shows methods to increase Th activity specifically in the nigrostriatal pathway. We focused on the phosphorylation of its regulatory domain. Ser40 phosphorylation has been reported to promote such an effect (Dunkley P R et al., 2004).

[0123] The present invention shows that a GUCY2C ligand, a PDE inhibitor and/or a combination thereof serves to increase dopamine production by dopaminergic cells. This finding is used as a therapy for PD.

[0124] 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 component provided by the wide expression of PDEs. An 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). Specific targeting of PDEs expressed in the dopaminergic midbrain, such as PDE2A or PDE11A can be used as a method to increase specificity and/or effectivity of the treatment.

Example 2

Slice Preparation and Treatment

[0125] Adult mice with an average of 5 weeks were sacrificed and the brain was quickly removed and sliced into coronal sections of 250 μm using a vibratome. Sections containing striatum were micro-dissected to remove non-striatal structures. Hemispheres were separated and one hemisphere was treated with compounds whereas the counter-hemisphere was treated with vehicle. Treatment of slices occurred in carbonized artificial cerebrospinal fluid (aCSF). After treatments, slices were lyzed in SDS containing sample buffer, sonicated, and heated at 95° C. Samples were analyzed on a WES automated western system (Protein Simple) according to manufacturer's instructions.

PDE2 Inhibition and GUCY2C Activation Result in Tyrosine Hydroxylase Activation in the Mouse Striatum

[0126] To investigate the effect of PDE2 inhibition in the mouse brain, an ex-vivo approach using brain slices was utilized. Coronal slices of mouse striatum were prepared and treated with BAY-60 7550 diluted in aCSF and subsequently analyzed by a WES automated western system. 10μM BAY 60-7550 resulted in an increase in Ser40 phosphorylation as shown in FIG. 13a. As BAY 60-7750 targets PDE2 and we observed that inhibition influences Th phosphorylation this indicates that both proteins are expressed in the same cell (compartment). PDE2 is thus expressed in dopaminergic neurons and PDE2 protein and Th protein are present in the synaptic terminals of the neurons which project from the midbrain dopamine system to the striatum. A concentration of 1 μM BAY 60-7550 did not result in a significant increase in Ser40 phosphorylation and shown in FIGS. 14a and 14b. To investigate the effects of GUCY2C ligands on Th activation, coronal slices of the mouse striatum were prepared, treated with guanylin and analyzed for Th Ser40 phosphorylation as was done for BAY-60-7550. Application of 10μM guanylin resulted in an increase in Ser40 phosphorylation as shown in FIG. 15a. Since guanylin resulted in an increase in Th activity in striatal slices the GUCY2C receptor is expressed in the synaptic terminals of the midbrain dopamine neurons which project to the striatum. The machinery for producing dopamine is thus present in the synaptic terminals where dopamine is released. 1 μM of guanylin did not result in a significant increase in Ser40 phosphorylation (FIG. 16a). Since both BAY 60-7550 and guanylin increased Th phosphorylation at 10μM, but not at 1 μM, we examined the effect of combining BAY 60-7750 and guanylin at 1μM. A combination of guanylin and BAY 60-7550 resulted in a significant increase in Th phosphorylation indicating that a combination of both compounds is more effective than either compound alone (FIG. 17a). It also indicated that the GUCY2C receptor, the PDE protein and the Th protein are all present in the same cellular environment and that upstream products/signals are able to influence downstream components of the pathway such as Th phosphorylation and dopamine production.

Novel Peptides That Activate GUCY2C

[0127] We designed novel peptides with different activities with respect to endogenous GUCY2C ligands (Table 3). The peptides were analyzed in Th and GUCY2C transfected HEK-293 cells and subsequently tested ex-vivo in mice striatal slices. Commercially available rat/mouse guanylin (Chemcruz) resulted in a modest, but significant increase, in Ser40 phosphorylation in HEK-293 cells as can be seen in FIG. 18 indicating that the WES system is comparable to the results obtained by traditional western blotting. We synthezised uroguanylin and heat stable toxin as controls for the synthezised peptides and they thus are from a different source than the commercially available uroguanylin and heat stable toxin. Surprisingly peptide 1 did not show any significant effects when tested in HEK-293 cells or mouse striatal slices, whereas peptide 2, 4, 6, 8, 9, 10 and 11 showed significant effects in HEK-293 cells and peptide 5, 9, 10 in striatal slices. Although peptide 2 was not significant in striatal slices it did show the largest fold change. Possibly the effects of the peptides can differ depending on the area in the striatum which is stimulated.

Cited Art

[0128] Ahlskog J E, Tyce G M, Kelly P J, van Heerden J A, Stoddard S L, Carmichael SW (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.

[0129] Allen Brain Atlas (http://mouse.brain-map.org/).

[0130] 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.

[0131] Bender A T, Beavo J A (2006). Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006 September;58(3):488-520.

[0132] 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.

[0133] 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.

[0134] Carta M, Carlsson T, Kirik D, Björklund 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.

[0135] Clarke C E (2004). Neuroprotection and pharmacotherapy for motor symptoms in Parkinson's disease. Lancet Neurol. 2004 August;3(8):466-74.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] Fiskerstrand T, Arshad N, Haukanes B I, Tronstad R R, Pham K D, Johansson S, Håvik B, Tønder 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.

[0140] 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.

[0141] 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 Sep. 16;333(6049):1642-6. doi: 10.1126/science.1207675. Epub 2011 Aug. 11.

[0142] Ikemoto S, Yang C, Tan A (2015). Basal ganglia circuit loops, dopamine and motivation: A review and enquiry. Behav Brain Res. 2015 Sep. 1;290:17-31. doi: 10.1016/j.bbr.2015.04.018. Epub 2015 Apr. 20.

[0143] 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.

[0144] 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 U S A. 1978 October;75(10):4744-8.

[0145] Kalia L V, Lang A E (2015). Parkinson's disease. Lancet. 2015 August 29;386(9996):896-912. doi: 10.1016/S0140-6736(14)61393-3. Epub 2015 Apr. 19.

[0146] Lin C S (2004). Tissue expression, distribution, and regulation of PDE5. Int J Impot Res. 2004 June;16 Suppl 1:S8-S10.

[0147] Lochner A, Moolman J A (2006). The many faces of H89: a review. Cardiovasc Drug Rev. 2006 Fall-Winter;24(3-4):261-74.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Müller T, Rasool I, Heinz-Erian P, Mildenberger E, Hüllstrunk C, Müller 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.

[0152] Navailles S, Milan L, Khalki H, Di Giovanni G, Lagière M, De Deurwaerdère 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.

[0153] 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.

[0154] Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).

[0155] 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.

[0156] Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

[0157] 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.

[0158] 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.

[0159] Scansite3 (http://scansite3.mit.edu/#home).

[0160] 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.

[0161] Smidt M P, Burbach J P (2007). How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci. 2007 Jan;8(1):21-32. Erratum in: Nat Rev Neurosci. 2007 February;8(2):160.

[0162] 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 Sep. 2.

[0163] 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 Sep. 19.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 μM cGMP analogue and PKG activator Guanylin 10 μM GUCY2C ligand Uroguanylin 1 μM GUCY2C ligand IBMX 100 μM 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 α Goat 488 Total Th Rabbit 1:1,000 Goat α Rabbit 488

TABLE-US-00003 TABLE 3 Peptides designed and tested for GUCY2C ligand activity. Peptides contain disulfide bridges between C residues. In case peptide contains two C residues, disulfide bridge between N-terminal C en C-terminal C. In case peptide contain 4 C resi- dues, disulfide bridges between first N-terminal C and third C residues and between second and fourth C residue. In case peptide contains six C residues disulfide bridge is between first N-terminal C and fourth C residue. Between second N-terminal C residues and fifth C residue and between third C residue and sixth C residue. The structural arrangement of the disulfide bridges can possibly lead to different isomers. peptide 1 H-NDDCELCVNVACTGCL-OH peptide 2 H-NDDCELCVNVACTGCLL-OH peptide 3 H-NDDCELCVNVACTGC-OH peptide 4 H-NDCCELCCNVACTGCL-OH peptide 5 H-NDDCELCVNVVCTGCL-OH peptide 6 H-QEECEL[Abu]INMACTGY-OH peptide 8 H-QEECELCINMACTGCL-OH peptide 9 H-NTFYCCELCCNPACAGCY-OH peptide 10 H-NTFYCCELCCAPACTGCY-OH peptide 11 H-NTFYCCELCCNPaCAGCY-OH
In peptide 11 the a is the amino acid D-Ala)

TABLE-US-00004 TABLE 4 Striatal slices were treated with indicated peptides and incubated for 1 h before determining Th Ser40 levels and total Th levels using WES automated western system. Concentration Fold change Peptide (μM) Effect p value (average) Peptide 1 10 P-S40 TH/TTH p = 0.473 1.11 Peptide 2 10 P-S40 TH/TTH p = 0.139 2.23 Peptide 3 10 P-S40 TH/TTH p = 0.294 1.20 Peptide 4 10 P-S40 TH/TTH p = 0.872 1.00 Peptide 5 10 P-S40 TH/TTH p = 0.046 1.22 Peptide 6 10 P-S40 TH/TTH p = 0.144 0.88 Peptide 8 10 P-S40 TH/TTH p = 0.255 0.89 Peptide 9 10 P-S40 TH/TTH p = 0.035 1.37 Peptide 10 10 P-S40 TH/TTH p = 0.018 1.44 Peptide 11 10 P-S40 TH/TTH p = 0.154 1.20 Fold change is indicated as well as the p-value for each individual peptide (10-12 slices per mouse were analysed, P-value is calculated on the 10-12 slices, and each slice was split in a control and a treated part). Significance is determined by a two-tailed t-test. Th levels that were lower than 50% of the average were excluded from the analysis.