(PDF) Does Phosphodiesterase 11A (PDE11A) Hold Promise as a...

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Send Ord ers for Reprints to reprints@benthamscience.net Current Pharmaceutical Design, 2015, 21, 389-416 389 Does Phosphodiesterase 11A (PDE11A) Hold Promise as a Future Therapeutic Target? Michy P. Kelly* University of South Carolina School of Medicine, Department of Pharmacology, Physiology Neuroscience, Columbia, SC 29209 Abstract: Phosphodiesterase 11A (PDE11A) is the most recently discovered 3’, 5’-cyclic nucleotide phosphodiesterase. By breaking down both cAMP and cGMP, PDE11A is a critical regulator of intracellular signaling. To date, PDE11A has been implicated to play a role in tumorigenesis, brain function, and inflammation. Here, we consolidate and, where necessary, reconcile the PDE11A literature to evaluate this enzyme as a potential therapeutic target. We compare the results and methodologies of numerous studies that report conflict-ing tissue expression profiles for PDE11A. We conclude that PDE11A expression is relatively restricted in the body, with reliable ex-pression reported in tissues such as the brain (particularly the hippocampus), the prostate, and the adrenal gland. Each of the four PDE11A splice variants (PDE11A1-4) appears to exhibit a distinct tissue expression profile and has a unique N-terminal regulatory re-gion, suggesting that each isoform could be individually targeted with a small molecule or biologic. Progress has been made in identify-ing a tool PDE11A inhibitor as well as an activator; however, the functional effects of these pharmacological tools remain to be deter-mined. Importantly, PDE11A knockout mice do exist and appear healthy into late age, suggesting a potential safety window for targeting this enzyme. Considering the implication of PDE11A in disease-relevant biology, the potential to selectively target specific PDE11A variants, and the possibility of either activating or inhibiting the enzyme, we believe PDE11A holds promise as a potential future thera-peutic target. Keywords: Phosphodiesterase 11 (PDE11), cAMP, cGMP, psychiatric illness, endocrine, immune, hippocampus, memory, glutamate, in-flammation, lithium. 1. INTRODUCTION Cyclic nucleotides are key regulators of numerous physiologi-cal processes, including gene transcription, glucose and insulin metabolism, endothelial permeability, neuronal plasticity, behavior, cardiac and smooth muscle contractility, secretory processes, cell growth, survival and differentiation, and more (c.f., [1, 2]). Effec-tive cAMP and cGMP signaling requires a tightly choreographed ballet of generating enzymes, effector molecules, and degrading enzymes. Though cyclic nucleotides may appear to be ubiquitous, they are in fact exquisitely confined to subcellular microdomains due to an intricate network of degradative enzymes called phos-phodiesterases (PDEs) [3, 4]. PDEs are the only known enzymes to degrade cyclic nucleotides and, thus, are integral to the regulatio n of intracellular signaling [5, 6]. PDEs themselves have been impli-cated in a wide range of biological functions including—but not limited to—sperm and cardiac physiology, platelet aggregation, smooth muscle contractions, as well as behavior and cognition (c.f., [3, 5, 7, 8]). There are 11 families of PDEs (PDE1-11) that are en-coded by 21 genes [5]. Each of the 21 PDE isoforms shows a unique tissue expression profile and a distinctive subcellular local-ization pattern, making each a unique target capable of modulating separable physiological processes (c.f., [3, 5, 7, 8]). The most recently discovered of the eleven PDE families is PDE11, which hydrolyzes cAMP and cGMP equally well [9-13]. The PDE11 family is comprised of a single gene, PDE11A [9-12, 14]. The catalytic domain is located w ithin the C-terminus of PDE11A, whereas the N-terminus appears to serve a regulatory function [13]. In mice and rats, full-length PDE11A demonstrates ~95% protein sequence homology with full-length human PDE11A4. This high degree of homology increases confidence that results obtained in preclinical rodent models will be applicable to *Address correspondence to this author at the Department of Pharmacology, Physiology Neuroscience, University of South Carolina School of Medicine, 6439 Garners Ferry Road, VA Bldg 1, 3rd Floor, D-12, Columbia, SC 29208; Tel: 803-216-3546; Fax: 803-216-3538; E-mail: Michy.Kelly@uscmed.sc.edu human PDE11A4. Which tissues express PDE11A has been a sub-ject of controversy in the literature, in part due to a number of poor quality commercially-available antibodies, but well-controlled stud-ies consistently show PDE11A is expressed in brain (particularly the hippocampus), the adrenal gland, and the prostate (Table 1). A growing body of evidence suggests that PDE11A signaling plays a role in brain function ([15-19] but see [20, 21]), tumor physiology [22-32] but see [33]), and, possibly, inflammation and immune function [34-37]. We review here what is known about PDE11A to determine whether or not this enzyme holds promise as a future therapeutic target, particularly in the context of neuropsychiatric disease and endocrine dysfunction. 2. GENOMIC AND BIOCHEMICAL FEATURES OF PDE11A 2.1. PDE11A Yields 4 Splice Variants Residing at the chromosomal locus 2q31.2, the PDE11A gene contains 23 exons and gives rise to four known splice variants PDE11A1-4 [9-12, 14] (Fig. 1). Yuasa et a l provided an early analysis of the genomic organization and phylogeny of the Homo sapien PDE11A gene (HSPDE11A)[14]. The coding sequence for PDE11A1, 3, and 4 begins 151, 99, and 317 bases, downstream from their respective transcription start sites [14]. Each PDE11A variant appears to be driven by a unique promoter, and alternative splicing also contributes to the unique N-terminal of each isoform [14]. PDE11A1 and 3 contain consensus TATA motifs in the 5’ promoter region, while the region 5’ to PDE11A4 is GC rich and contains a consensus Sp1 transcription factor binding site [14]. The promoters of PDE11A1, 3, and 4 each contain consensus binding sites for the Sex-determining Region Y (SRY) and the SRY-related high mobility group box (Sox-5) DNA binding proteins, and the promoter of PDE11A1 addition ally contains consensus binding domains for the Myogenic Regulatory Factor MyoD [14]. Although these consensus sequences have been identified, none have been functionally verified to date. Further, it h as yet to be elucidated what signal transduction cascades lead to activation of PDE11A transcription. The fact that each isozyme is driven by a /15 $58.00+.00 © 2015 Bentham Science Publishers 390 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly Table 1. Tissue expression patterns reported for PDE11A are highly contradicto ry. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 391 (Table 1) Contd…. 392 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly (Table 1) Contd…. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 393 (Table 1) Contd…. 394 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly (Table 1) Contd…. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 395 (Table 1) Contd…. unique promoter likely accounts for the differential tissue distribu-tion of each of the PDE11A isozymes (Table 1). An analysis of the amino acid sequence of the catalytic domain reveals that PDE11A demonstrates a high degree of sequence simi-larity with the GAF-domain containing PDEs PDE2A, PDE5A, PDE6A-C, and PDE10A, suggesting relatedness between these enzymes [14] (Fig. 2). The phylogenic relatedness of these genes is also supported by similarities in the organization of intron-exon boundaries [14]. Based on these lines of evidence, Yuasa et al. proposed a phylogenic tree in which PDE11A, PDE5A, and PDE6A-C diverged from a common ancestral gene, while the other GAF containing PDEs PDE2A and PDE10A diverged separately [14]. We conducted a phylogenic analysis using the full length amino acid sequences of the Homo sapiens phosphodiesterases: PDE1A (NP_005010.2), PDE1B (NP_000915.1), PDE1C (NP_001177987.1), PDE2A (NP_001137311.1), PDE3A (NP_000912.3), PDE3B (NP_000913.2), PDE4A (NP_001104777.1), PDE4B (NP_001032418.1), PDE4C (NP_000914.2), PDE4D NP_001098101.1), PDE5A (NP_001074.2), PDE6A (NP_000431.2), PDE6B (NP_001138763.1), PDE7A (NP_001229247.1), PDE7B (NP_061818.1), PDE8A (NP_002596.1), PDE8B 396 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly (NP_001025024.1), PDE9A (NP_002597.1), PDE10A (NP_001124162.1), and PDE11A (NP_058649.3). Phylogenetic analysis of the full-length amino acid sequences of the various PDE families yielded similar results to those based on the catalytic do-mains alone (Fig. 2). 2.2. Each PDE11A Variant Exhibits a Unique N-terminal Regu-latory Region Although the C-terminal portion of PDE11A is shared by each PDE11A isoform, the N-terminal region is unique in each of the four variants (Fig. 1). All four PDE11A variants contain exons 8-23, which encode a portion of the regulatory GAF-B domain (cGMP binding PDE, Anabaena ad enylyl cyclase and E. coli FhlA domain) and the catalytic domain [38]. The N-terminal for PDE11A4—often referred to as \"full-length” PDE11A despite lack-ing exons 2 and 7—is encoded by exons 3-6. Exon 3 is found only in PDE11A4 (Fig. 1). The portion of the N-terminal region of PDE11A encoded by exon 3 contains two validated phosphoryla-tion sites (S117 and S162) and the beginning of the GAF-A domain [11] (Fig. 1). As we will rev iew in greater detail below , these phos-phorylation sites and GAF domains may be important for in-tramolecular signaling [13, 39]. Because PDE11A1, PDE11A2 and PDE11A3 lack exon 3, these isoforms do not contain the validated N-terminal phosphorylation sites nor a full GAF-A domain [11, 14]. The PDE11A3 transcript is encoded by exons 1, 2, 4, 5, and 6; however, translation of PDE11A3 does not begin until exon 2 [14] (Fig. 1). This results in PDE11A3 containing a truncated GAF-A domain and a full GAF-B domain upstream of the C-terminal cata-lytic domain. PDE11A2 translation begins at exon 5 and, thus, PDE11A2 is missing even more of the GAF-A domain than is PDE11A3 [9, 38]. PDE11A2 does contain a full GAF-B domain and catalytic region. Translation of the shortest isoform, PDE11A1, does not begin until exon 7; thus, PDE11A1 contains only a portion of the GAF-B domain within its N-terminal region [14] (Fig. 1). The unique nature of the N-terminal regulatory region of each PDE11A isozyme [9, 14, 38] may provide novel inroads for selec-tively targeting a single PDE11A isozyme with high selectivity. The N-terminal region of PDE11A clearly serves an important regulatory role with regard to controlling PDE11A function. Re-moving the regulatory N-terminal region in its entirety does affect the catalytic properties of PDE11A [13]. A comparison of the four PDE11A isoforms found that the affinity of the catalytic domain for cAMP and cGMP inversely correlated with the length of the N-terminus [13]. Whereas the catalytic site of full-length PDE11A4 has the lowest affinity for cAMP and cGMP, the catalytic sites of the shortest isoforms (PDE11A1 and PDE11A2) have the highest affinities for cAMP and cGMP [13]. Similarly, in a chimeric pro-tein consisting of the PDE11A4 N-terminus linked to the catalytic domain of a bacterial adenylyl cyclase, deletion of the first 196 amino acids (i.e., all amino acids upstream of the GAF-A domain) increases basal catalytic activity [39]. Together, these studies sup-port a regulatory role for the PDE11A N-terminal region. 2.2.1. The N-terminus of PDE11A4 has 2 Confirmed PKA/PKG Phosphorylation Sites As mentioned above, PDE11A4 is the only PDE11A variant with confirmed phosphorylation sites within the N-terminal regula-tory region [11]. PDE11A4 exhibits 2 consensus protein kinase A/protein kinase G (PKA/PKG) phosphorylation sequences center-ing on S117 and S162 [11]. Prediction software suggests that S117 and S162 may also be the target of other phosphorylating kinases (Fig. 3). In vitro phosphorylation assays confirmed the ability of both PKA and PKG to phosphorylate PDE11A4, which contains S117 and S162, but not PDE11A3, which does not include these amino acids. In addition, the majority of PKA and PKG-induced phosphorylation of PDE11A4 was ablated when S117 and S162 were mutated to phosphomutant alanines, confirming these specific Fig. (1). Each of the 4 known PDE11A isoforms has a unique N-terminal domain and a shared PDE11A catalytic domain. A bent arrow indicates the transla-tion initiation sites for each isoform. The GAF-A domain is represented by vertical bars, the GAF-B domain by diagonal bars, and the catalytic domain by diamonds. The amino acid position of the beginning and ending of each domain is labeled immediately above each depicted isoform. The two confirmed phos-phorylation sites (P) are labeled, as is the glutamine (Q) within the catalytic site that is essential for substrate binding [49]. PDE11A1 is based on the reference protein sequence NP_001070664.1 (489 amino acids—aa) and the reference mRNA sequence NM_001077196.1 (7786 base pairs—bp). PDE11A2 is based on the reference sequences NP_001070826.1 (575 aa) and NM_001077358.1 (7869 bp). PDE11A3 is based on the reference sequences NP_001070665.1 (683 aa) and NM_001077197.1 (8316 bp). PDE11A4 is based on the reference sequences NP_058649.3 (933 aa) and NM_016953.3 (9285 bp). GAF — cGMP binding PDE, Anabaena adenylyl cyclase and E. coli FhlA domain. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 397 residues being phosphorylated [11]. The remaining PKA and PKG-induced phosphorylation observed in the S117A/S162A PDE11A4 mutant may reflect phosphorylation of S124, which is also pre-dicted to be phosphorylated by PKA and PKG (Fig. 3). Interest-ingly, PKG phosphorylated PDE11A4 to a lesser extent than did PKA, raising the possibility that PKG may be less efficient than PKA in phosphorylating PDE11A4 or that PKG may actually only phosphorylate 1 of the 2 serine residues [11], even though it is pre-dicted to phosphorylate both (Fig. 3). Despite the fact that phos-phorylation of PDE11A4 has been confirmed in vitro, the func-tional consequences of these phosphorylation events are still poorly understood. Gross-Langenhoff et al. used a chimeric protein con-sisting of the PDE11A4 N-terminus linked to the catalytic domain of a bacterial adenylyl cyclase to explore th e functional role S117/S162 phosphorylation [39]. In that chimeric protein, the phosphorylation state of the N-terminal regulatory region affected intramolecular regulation of the GAF-A domain. Specifically, when both S117 and S162 were phosphorylated, or were replaced with amino acids that mimicked the phosphorylated state, the down-stream GAF-A regulatory domain had an increase in affinity for cGMP [39]. Interestingly, deleting the first 176 amino acids simi-larly increased cGMP affinity for the GAF-A domain [39], suggest-ing that phosphorylation of S117/S162 may produce a conforma-tional change that exposes the cyclic nucleotide binding pocket of the GAF-A domain. 2.2.2. The PDE11A GAF-A Domain Binds cGMP Although cyclic nucleotide binding of a PDE GAF domain does affect catalytic activity of some PDE families, this is not always the case. For example, cGMP binding the GAF-B domain of PDE2A increases catalytic activity [40, 41], cGMP binding the GAF-A domain of PDE5A increases affinity for substrates or inhibitors (c.f., [3]), cGMP binding the GAF-A domain of PDE6 enhances protein-protein interactions that inhibit PDE6 catalytic activity [42], and cAMP binding to the GAF-B domain of PDE10A does not appear to affect catalytic activity [43, 44]. In the case of PDE11A, several laboratories have shown that cGMP (but not cAMP) binds the GAF-A domain (but not GAF-B) [39, 43-45]. Further, it appears that cGMP (and select cGMP analogues) may bind the GAF-A domain with a lower Ki versus the catalytic domain [44]. That said, the preponderance of evidence suggests that cGMP binding the GAF-A domain of PDE11A does not directly affect catalytic activity. In the case of the PDE11A-cyclase chimeric protein de-scribed above, cGMP binding to the PDE11A GAF-A domain did increase catalytic activity [39, 45]; however, subsequent studies conducted with full-length recombinant PDE11A failed to replicate this effect [43, 44]. Interestingly, th e ability of cGMP to allosteri-cally stimulate catalytic activity of the PDE11A4-cyclase chimera was dependent on the presence of an aspartate within a conserved NKFDE motif that is present in all m ammlian PDE GAF domains [45]. Mutating D355 to an alanine abolished the intramolecular signaling events triggered by cGMP; however, it is not clear if this mutation simply prevented cGMP from binding the GAF-A domain or if the mutation impacted the ability of bound cGMP to elicit some comformational change [45]. Although it does not appear that cGMP can allosterically regulate catalytic activity of full-length PDE11A, Jager et al did show that a cGMP analog Rp-8-pCPT-PET-cGMPs, which binds the PDE11A GAF domain, increases the catalytic activity of both recombinant and native PDE11A har-vested from mouse hippocampus [43]. Unfortunately, the use of this particular compound as a tool PDE11A activator is severeley limited by off-target activities (namely inhibition of PKG and, likely, many other cGMP-binding molecules). That said, the ability of Rp-8-pCPT-PET-cGMPs to allosterically stimulate PDE11A function does provide an exciting proof of concept for how we might target PDE11A in ord er to increase catalytic activity. In addition to providing a mechanism for cyclic nucleotide feedback, GAF domains also provide critical points of contact for protein-protein binding interactions. Like all PDEs [3, 46], PDE11A spliciforms exist as o ligomers [13]. The C-terminal por-tion of the GAF-B domain appears to be necessary for dimerization, although contacts within the catalytic domain may also contribute to this protein-protein interaction [13]. Whereas isolated PDE11A catalytic domains exist as a monomers or low affinity dimers, PDE11A1-4 all oligomerize [13]. Interestingly, the isoform with the shortest N-terminus, PDE11A1, forms homotetramers whereas PDE11A2-4 form homodimers. This suggests that the GAF-B do-main is the primary point of oligomerization and that a longer N-terminus precludes the ability to form tetramers. It is completely unknown whether or not oligomerization affects full-length Fig. (2). PDE11A is most closely related to the other GAF-domain containing PDEs. Full-length amino acid sequences (reference sequence given to the right of each PDE) were aligned using the European Bioinformatics Institute (EBI) ClustalW multiple sequence alignment tool (http: //www.ebi.ac.uk/Tools/msa/clustalw2). Phylogenic analysis and tree generation were performed using the neighbor-joining algorithm in the EBI ClustalW Phylogeny tool (http: //www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny). These analyses identify PDE5A and PDE6A/B as the PDEs most closely re-lated to PDE11A, with PDE2A and PDE10 the next most closely related. GAF — cGMP binding PDE, Anabaena adenylyl cyclase and E. coli FhlA domain. 398 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly PDE11A function. Weeks et al found that the dimeric PDE11A2 and the tetrameric PDE11A1 have nearly identical substrate affini-ties for cAMP and cGMP, suggesting that quaternary structure does not greatly impact affinity of the cyclic nucleotides for the catalytic site [13]. In the case of the PDE2, PDE4, and PDE5 families, in vitro studies suggest that dimerization is not necessary for catalytic activity but does influence susceptibility to post-translational modi-fications, which indirectly modify activity and subcellular localiza-tion [3, 47]. Much work remains to understand the functional role of PDE11A oligomerization as well as what other proteins PDE11A may potentially bind. Together, these studies suggest that the N-terminal region offers many potential avenues for regulating PDE11A intramolecular signaling in an isozyme-specific nature. Cyclic nucleotide binding of the GAF-A domain may provide feedback or feed-forward sig-nals with regard to substrate levels. Protein-protein binding via the GAF-B domain may impact catalytic activity and subcellular local-ization by dictating quaternary structure as well as interactions with other binding partners. Finally, phosphorylation and other predicted post-translational modifications (Fig. 3) may offer yet another mechanism to regulate catalytic activity and subcellular localization of PDE11A. The fact that the N-terminal regulatory region of each PDE11A isozyme is unique [9, 14, 38] may provide a mechanism to selectively target the function of a single PDE11A isozyme with high selectivity. Indeed, isozyme-specific targeting of PDE11A activity has already been demonstrated as vinpocetine can inhibit PDE11A3 but not PDE11A4 [11]. 2.3. PDE11A Hydrolyzes both cAMP and cGMP at the C-terminal Catalytic Domain The catalytic site of PDE11A hydrolyzes both cAMP and cGMP under physiological conditions (cf. [38]). Though the cata-lytic properties vary between isoforms, each of the PDE11A vari-ants break down cAMP and cGMP with similar affinities and maximal catalytic rates [9-13]. The shortest isoform PDE11A1 binds cGMP with reported Km values ranging from 0.4 M [13] to 0.52 M [10] and cAMP with reported Km values ranging from 0.45 M [13] to 1.05 M [10]. The longest isoform PDE11A4 Fig. (3). PDE11A contains numerous sites that may serve as potential targets for posttranslational modification. Several tools were used to predict posttransla-tional modifications that are conserved between human PDE11A4 (NP_058649.3) and mouse PDE11A (NP_001074502.1) PDE11A4. The GPS bioinformat-ics phosphorylation prediction tool GPS 2.1.2 (http: //gps.biocuckoo.org, high stringency [190]) was used to predict conserved phosphorylation sites. 105 residues are predicted to be phosphorylated by more than 70 different enzymes. For sake of graphical clarity, we have only featured residues for which 10 individual enzymes are predicted to phosphorylate PDE11A and have binned enzymes by family (e.g., \"PKC” indicates predictions for any PKC isoform, including alpha, delta, eta, etc.). Additional GPS tools (SUMOsp 2.0 and CSS-Palm3.0, both high stringency) were used to predict potential sites for sumoyla-tion, the covalent linkage of a Small Ubiquitin-like Modifier protein, and palmitoylation, an attachment of a fatty acid that usually leads to membrane traffick-ing. Finally, the Ubpred tool (http: //www.ubpred.org, medium confidence) was used to identify potential sites of ubiquitination, a covalent attachment of ubiquitin that often targets a protein for degradation. GSK—Glycogen Synthase Kinase; MAPK—Mitogen-activated protein kinase; CDK—Cyclin-dependent kinase; TKL—Tyrosine Kinase-like; SGK—serum/glucocorticoid-regulated kinase; PIKK—Phosphatidylinositol 3-kinase-related protein kinase; PKB—protein kinase B (also known as AKT, which is not an acronym); AUR—Aurora Kinase; PKA—Protein kinase A, a cAMP-activated kinase; PKG—Protein Kinase G, a cGMP-activated kinase; PKC-Protein Kinase C; DMPK—dystrophia myotonica protein kinase; RSK—Ribosomal protein S6 kinase; GRK—G-protein coupled receptor kinase; CAMK = Ca2+/calmodulin-dependent protein kinases; STE—Homologs of yeast Sterile 7, Sterile 11, Sterile 20 Kinases; Wnk—With No Lysine (K) Kinase; DYRK—Dual Specificity YAK1-Related Kinase; CK1e--casein kinase 1, epsilon; VRK—Vaccinia Related Kinase; Eph—Ephrin Kinase; Met—mesenchymal epithelial transition factor transmembrane tyrosine kinase; Ret—rearranged during transfection tyrosine Kinase; Abl—Ableson leukemia oncogene cellular homolog; CSK—carboxyl-terminal Src kinase; Src—Rous sarcoma oncogene cellular homolog; GFRs—Growth Factor Receptor Tyrosine Kinases; JAK A—Janus-family tyrosine kinase; PEK—Pancreatic eukaryotic initiation factor 2a-subunit kinase; KIS—kinase interacting with stathmin; GAF—cGMP binding PDE, Anabaena adenylyl cyclase and E. coli FhlA domain. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 399 binds cGMP with reported Km values from 0.97 M [48] to 1.4 M [11] and cAMP with reported Km values from 1.6 M [13] to 3.0 M [11]. Although the shorter isoforms have the highest affinities for their cyclic nucleotide substrates [12, 13], the longer isoforms PDE11A3 and PDE11A4 have substantially greater catalytic turn-over rates [12, 13]. A comparison of rat PDE11A found similar results to those obtained using Homo sapien PDE11A [12]. These studies show that each of the four PDE11A isoforms hydrolyze cAMP and cGMP comparably well, although the long and the short isoforms may have distinct catalytic properties. PDE5A inhibitors have already been shown to bind to the PDE11A catalytic site and decrease PDE11A enzymatic activity [49]. As noted above, the catalytic domain of PDE11A is most similar to that of PDE5A, so it is not surprising that PDE5A inhibi-tors would have this cross reactivity [10]. Identifying important structural differen ces between the catalytic domains of PDE11A and PDE5A will improve the ability to effectively design therapeu-tics that selectively target PDE11A versus PDE5A. Unfortunately, the crystal structure for PDE11A (in its entirety or an individual domain) has yet to be published. Weeks et al. conducted site-directed mutagenesis of the PDE11A catalytic site and systemati-cally altered cGMP analogs to identify trends that affected intermo-lecular bond formation [49]. These studies determined that one important contributor to PDE11A4-substrate binding is a hydrogen bond involving Q869 [49]. It is likely that this Q869 residue drives the preferential binding of the PDE5A inhibitor sildenafil to PDE5A versus PDE11A [49]. Clearly, additional work is needed to more fully understand the 3-dimensional structure of PDE11A and its catalytic domain. 3. TISSUE EXPRESSION PROFILE FOR PDE11A A prime consideration in evaluating a drug target is knowing where it is expressed in the body. Unfortunately, exactly where PDE11A is expressed in the body is somewhat controversial, due to a number of conflicting reports in the literature (Table 1). Some of these discrepancies, particularly those conflicting as to whether or not PDE11A is detected in a given tissue (as opposed to conflicting reports about which specific isoform is present), may be related to species differences or the fact that PDE11A is expressed only in a select sub-compartment of certain organs. For example, initial stud-ies failed to detect PDE11A expression in brain [10, 11]; however, subsequent studies have convincingly shown that PDE11A4 is in fact expressed within certain regions of the brain, particularly the hippocampus in mouse, rat, and human [19, 43, 50]. Expression of PDE11A4 in brain was first verified by comparing several molecu-lar probes and antibodies in tissue taken from PDE11A wild-type versus KO mice [19]. These validated tools were then also used to identify PDE11A4 expression in tissue taken from rat brain and human hippocampus [19]. Indeed, it is these very studies that high-light the danger in interpreting PDE11A western blot results in absence of negative controls, because all three PDE11A antibodies (PD11A-112AP, PD11A-101AP, and Abcam 14624) not only relia- Fig. (4). PDE11A mRNA and protein expression in brain are enriched in the hippocampus. A) The left panel shows images of sagittal sections of mouse brain labeled with either a thionin tissue stain (top) or an autoradiographic probe for PDE11A mRNA (middle) (conducted as previously described [19]). The merged images (lower left and right panels) show how PDE11A mRNA aligns with the dorsal hippocampal formation (DHIPP) and ventral hippocampal for-mation (VHIPP). The pseudo-colored magnification of the HIPP (right panel; tissue stain in purple) illustrates how PDE11A mRNA (shown in green) is re-stricted to CA1 and subiculum (Sub) and is 2-3 fold higher in VHIPP vs. DHIPP. An identical pattern of PDE11A mRNA expression is observed whether using a probe that recognizes PDE11A4 only or all PDE11A isoforms [19]. B) The same pattern of PDE11A mRNA expression is observed in rat brain. West-ern blots using Fabgennix PD11A-112AP (as previously described [19]) to probe brain tissue taken from C) PDE11A wild-type (WT) and knockout (KO) littermates and D) Sprague Dawley rats confirms that PDE11A4 protein is e nriched in the VHIPP versus DHIPP. Low levels of PDE11 A4 expression also seen in prefrontal cortex (PFC) and negligible expression is observed in the striatum (STR). Absolutely no PDE11A protein is seen in the cerebellum (CBLM). The fact that reliable PDE11A4 protein expression is observed in the PFC in absence of measurable mRNA expression suggests that the PDE11A4 protein found in dissected PFC may be localized to axonal projections originating from the ventral hippocampus. The color version of the figure is available in electronic of the article. 400 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly bly labeled PDE11A4 (i.e., they detected a band at the correct mo-lecular weight in wild-type tissue but not knockout (KO) tissue), they also labeled many non-specific bands at or near the predicted molecular weights of the 4 PDE11A isoforms (i.e., bands that ap-peared in tissue from both wild-type and KO mice) [43] (also, see Figs. 5 and 6). Several studies, including our own conducted using tissue from PDE11A wild-type and KO mice (Figs. 5 and 6), have also failed to detect PDE11A expression in the adrenal gland as a whole [10, 11, 43]. Other studies, however, have been able to detect PDE11A expression in human adrenal gland [30, 51] and suggest PDE11A expression is restricted to the adrenal cortex and adrenal nodules (i.e., it is not expressed in the adrenal medulla) [30, 52]. A study by Horvath and colleagues, strongly suggests that it is PDE11A4 that is specifically expressed in human adrenal tissue. PDE11A4 signals were detected in human adrenal tissue by PCR and western blot, and the western blot signals were diminished in patients with a truncating deletion in PDE11A (a human equivalent to rodent studies using knockout mice as a negative control) [30]. The fact that we are unable to detect PDE11A protein in mouse adrenal gland may reflect a species difference, or the fact that PDE11A expression may only be driven in the adrenal gland under conditions of stress and/or disease. The imperfection of antibodies and lack of adequate negative controls for human samples (outside of the PDE11A truncating mutation found in select adrenal tumors) may partially explain why reports are very conflicted with regard to PDE11A tissue expression profiles. With regard to establishing PDE11A expression in the prostate and skeletal muscle, a handful of studies have made attempts to increase the stringency of their expression studies. Unfortunately, this has done little to improve clarity in the field. For example, Fawcett and colleagues took the approach of using 3 different anti-bodies (although only 1 was shown) coupled with loading recombi-nant hPDE11A1 as a positive control on their western blots [10]. With this approach they identified PDE11A1 as the predominant isoform in prostate and PDE11A3 as the predominant isoform in skeletal muscle [10]. Loughney and colleagues took a similar ap-proach, using 2 different antibodies and loading recombinant PDE11A1, 2, 3 4 as positive controls, yet they identified PDE11A4 as the predominant isoform in prostate and no isoform in skeletal muscle [53]. The discrepant findings may be related to different sources of tissue (Table 1) or the antibodies themselves (rabbit polyclonal antibodies were used in the first study and mouse monoclonal antibodies in the second). That said, each of these two studies included an antibody that was raised to the same region of PDE11A (corresponding to amino acids 454-467/468 of PDE11A1), suggesting that the conflicting reports are not due to differences in isoform-specificity of the antibodies used (each of which were designed to recognize all PDE11A isoforms). A synthe-sis of expression studies to date equally support expression of PDE11A4 [11, 30, 53, 54] and PDE11A1 in prostate ([10, 30, 55]; Fig. 5) particu larly in the glandular epithelium [53, 56]. Reports of PDE11A expression in skeletal muscle are so variable that no con-sistent theme emerges (Table 1). We did not observe any PDE11A isoform in any of the sk eletal mu scles examined, which spanned fast-twitch and slow-twitch fiber types; however, we did detect several non-specific bands that could easily have been misinter-preted as PDE11A in absence of tissue from a KO mouse (Figs. 5 and 6). It remains to be determined whether discrepancies with regard to PDE11A expression in prostate and skeletal muscle may reflect biological differences in the tissues examined, for example with regards to disease state, or simply inadequate technical tools. In summary, a synthesis of tissue expression studies to date suggests that PDE11A expression is relatively restricted throughout the body (Table 1). PDE11A1 and/or PDE11A2 may be expressed in kidney (moderate levels), islet cells of the pancreas (moderate level), and prostate (moderate level). PDE11A3 is likely expressed in testes and seminal vesicles (moderate levels within restricted compartments). PDE11A4 is most likely expressed in brain (high levels particularly in the ventral hippocampus) and the human adre-nal gland (moderate level). Some evidence suggests PDE11A4 may also be expressed in heart (low level), liver (low level), pituitary (moderate level), and prostate (moderate level), but our own studies employing tissue from PDE11A KO mice as negative controls fail to detect PDE11A4 outside of brain (Figs. 5 and 6). Finally, PDE11A (isoform unknown) may be expressed at low levels in mammary and salivary glands. This limited tissue expression pro-file suggests that a PDE11A-targeted therapeutic would stand to impact very selective systems of interest without eliciting wide-ranging toxicological side effects. 4. PDE11A AS A TUMOR SUPPRESSOR An increased risk for tumor development, particularly of endo-crine tissue, has been associated with mutations in a number of proteins along the cAMP signal transduction cascade (c.f., [26]). For example, inactivating mutations in the PKA regulatory subunit PRKAR1A, which result in unregulated PKA catalytic activity, are inherited in an autosomal dominant fashion and cause Carney Complex [26, 57-60]. Carney Complex is a d isease characterized by a constellation of symptoms that includes tumors of the skin, con-nective tissue, peripheral myelin, liver, and endocrine tissues [26, 57-60]. Endogenous Cushing s syndrome can result from adrenal hyperplasia, a condition linked to excessive cAMP signaling down-stream of ACTH or other hormone receptors [61]. Cushing’s Syn-drome is caused by pathologically high cortisol levels and typically presents with obesity, diabetes mellitus, dyslipidemia, hypertension, muscle weakness, and depression [62]. Activating mutations in Gs and the ectopic expression of Gs coupled receptors, which are caused by either sporadic or dominantly inherited mutations, can lead to ACTH-independent macronodular adrenal hyperplasia (AI-MAH) [61, 63]. Activating mutations of Gs are also known to cause McCune-Albright Syndrome (MAS), another disease associ-ated with adrenal and other tumors [63]. Given that uncontrolled increases in cAMP signaling have been implicated in tumorigene-sis, it is not surprising that a growing body of evidence suggests that PDE11A may act as a tumor suppressor. 4.1. PDE11A Truncating Mutations Mutations, expression changes, and functional alterations of PDE11A have been reported in patients with endocrine dysfunction. In 2006, the Stratakis group published the first association between PDE11A mutations and adrenal tumors, as identified in a small cohort of patients with Cushing Syndrome presenting with primary pigmented nodular adrenocortical disease (PPNAD) or micronodu-lar adrenal hyperplasia [30]. This particular cohort of patients was of interest because they lacked mutations in PRKAR1A [30]. In this original study, 3 PDE11A mutations were identified that resulted in early stop codons prior to the start of the catalytic domain; hence, these mutations were termed \"PDE11A inactiv ating mutations” [30] (Table 2). Two of these truncating mutations were shown to be associated with a decrease in PDE11A4 protein expression in adre-nal tissue, and all three mutations were shown to associate with an increase in cAMP and cGMP in adrenal tissue [30]. Although this original report did not identify these truncating mutations in any controls examined, their follow-up report did [29]. Three out of the 17 patients (12%) examined exhibited PDE11A inactivating muta-tions, as did 12 out of 745 controls (1.6%; 2 = 14.62, P 0.0001) [29]. The fact that these PDE11A inactivating mutations do exist in controls but are found significantly more frequently in patients suggests these mutations are not sufficient to cause adrenal tumors but may increase the risk for developing disease [29]. 4.2. PDE11A Missense Mutations The suggestion that abnormal PDE11A signaling is not suffi-cient to cause adrenal dysfunction, but may increase risk for dis-ease, is further supported by findings examining PDE11A missense Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 401 mutations. In addition to the PDE11A truncating mutations noted above, the Stratakis group also identified 2 PDE11A missense mu-tations in their initial report, R804H and R867G [29, 30]. The authors initially reported decreased PDE11A4 protein expression and increased levels of cAMP and cGMP in adrenal tumor tissue taken from a patient with the R804H missense mutation relative to healthy adrenal tissue, suggesting a direct effect of the PDE11A missense mutation [29]. Subsequent studies, however, clarified that decreased PDE11A protein expression [52] and increased cAMP and cGMP levels can be found in adrenal tumors whether or not PDE11A missense mutations are present [31, 33]. Further, the R804H and R867G mutations, in particular, appear to occur at equal rates in healthy controls versus patients with either adrenocor-tical, testicular germ cell tumors (TGCT), prostate, or pitu itary tumors [23, 28-31, 64]. Although several synonymous, missense, and nonsense PDE11A mutations have been identified that occur with equivalent frequency in both healthy controls and patients, there are a number of rare PDE11A mutations that have only been observed in patients with tumors (adrenal, testicular or prostatic) to date (Table 2) [31]. Individual PDE11A missense mutations are rarely reported to occur significantly more frequently in patients with adrenal, testicu-lar or prostate tumors (potentially due to low n’s); however, several reports have associated an increased frequency of PDE11A muta-tions in general with each of these tumor types. Two studies found that the overall frequency of PDE11A missense/nonsense mutations was significantly greater in patients with AIMAH versus controls (24% versus 9%, P = 0.05 [31]; 28% versus 7.2%, P = 5 x 10-5 [27]), although this was not the case for patients with secreting adrenocortical adenomas or adrenocortical cancer [31]. The overall Fig. (5). PDE11A protein appears to be expressed most robustly in the hippocampus (PDE11A4), seminal vesicle (PDE11A3) and prostate (PDE11A1) of male mice. Tissue from male PDE11A wild-type (WT) and male knockout (KO) mice was homogenized in lysis buffer consisting of ice cold 20 mM Tris-HCl, 2 mM MgCl2, protease inhibitor, and phosphatase inhibitor cocktail. Protein was quantified using the BioRad DC assay and 66 g of protein from the total homogenate of each sample was run out on a denaturing 4-12% Bis-Tris SDS-PAGE gel, using the SeeBlue 2 Ladder as a molecular weight Marker (as previ-ously described [19]). Membranes were probed with Fabgennix PD11A-112AP antibody, which recognizes an epitope common to all PDE11A isoforms, and a horseradish-peroxidase conjugated secondary antibody. Bands were visualized using a chemiluminescent substrate and apposing membranes to film. PDE11A WT hippocampus (HIPP) and purified human PDE11A4 (hPDE11A4) were loaded on each gel as a positive control and tissue from PDE11A KO mice was loaded for each organ as a negative control. In general, many non-specific bands are noted in most tissues examined. Though many of these bands correspond to the predicted weights of the various PDE11A isoforms, they do not disappear in tissue from the PDE11A KO, confirming they are non-specific in nature. As observed in Figure 4, PDE11A4 is specifically expressed in hippocampus of male PDE11A WT mice but not male KO mice—no other tissues examined ex-pressed PDE11A4 in male mice. Although we did not detect any reliable PDE11A expression in testes, we did appear to detect PDE11A3 in the seminal vesi-cles of mice as tissue from the male WT mouse showed a doublet ~80 kDa, but tissue from the male KO showed only a single band at that molecular weight. Finally, a protein band corresponding to the molecular weight of PDE11A1 is observed in prostate of male PDE11A WT mice but not male KO mice. To-gether, these results suggest that PDE11A expression is quite restricted in the male body. EPIDIDYM—epididymis; L. INTEST—large intestine; S. IN-TEST—small intestine; M. ADIPOSE—mesentery adipose; GASTROC—gastrocnemius; PLANT—plantaris; SEM VESS—seminal vesicles; E. ADIPOSE—epididymal adipose. 402 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly Table 2. Genetic studies have linked PDE11A to brain function, tumorigenesis, and inflammation. PDE11A Mutation Associated Disease/Pharmacology Effect of Mutation Refs SNPs rs11684634 allergic asthma in PRAM [34] rs11684634 asthma in B58C adult cohort [34] (not child cohort) rs1405716 asthma in childhood asthma MP [34] rs1997209 asthma in childhood asthma MP [34] rs11687573 asthma in FHS [34] rs4893980 extrapulmonary tuberculosis [35] rs7585543 Li+ responsivity in Bipolar Disorder [77] rs3770018 rs4893975 Major depressive disorder (MDD) MDD [191] [80] rs3770018 MDD remission with fluoxetine [191] rs3770018 not associated with duloxetine [21] antidepressant response rs1880916 MDD remission with fluoxetine/desipramine [191] rs1880916 not associated with citalopram [80] antidepressant response rs1880916 not associated with duloxetine [21] antidepressant response rs959157 Cushing Syndrome hemizygous loss [30] Nonsense/Missense fs41X AT/TT/PC/PT cases controls decreased PDE11A protein [28-30, 32, 64] fs15X AT cases controls predicted truncation [28-30] R52T TT case diminished ability to hydrolyze cAMP in HEK293 and MLTC-1 cells [27, 28] R202C PC case diminished PDE11A function in HEK293 and PC3M cells [192] F205L AT case [31] L218F AT case [32] F258Y TT case diminished ability to hydrolyze cAMP in HEK293 and MLTC-1 cells [28] S275X AT case [32] G291R TT case diminished ability to hydrolyze cAMP in HEK293 and MLTC-1 cells [28] R307X AT/TT cases controls decreased PDE11A mRNA and protein [29, 30, 32] A349T AT/PC cases = controls diminished ability to hydrolyze cAMP in HEK293 and MLTC-1 cells [27, 31, 32, 192] E382X AT case predicted truncation [32] Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 403 (Table 2) Contd…. PDE11A Mutation Associated Disease/Pharmacology Effect of Mutation Refs D609N AT case control diminished ability to decrease global cAMP signal-ing in H295R, MLTC-1, and HEK293 cells [31, 192] Y658C PC case loss of PDE11A function in HEK293 and PC3M cells [27, 192] H664G AT case [31] Y727C AT/PC/TT/PT cases controls diminish ed ability to hydrolyze cAMP in MLTC-1 cells [27, 28, 31, 32, 64, 192] R804H AT/PC/TT/PT cases = controls diminished or lost ability to hydrolyze cAMP in HEK293, HeLA, and MLTC-1 cells; loss of ability to hydrolyze cGMP in HeLA but not HEK293 cells [27-32, 64, 192] V820M TT case = control diminished ability to hydrolyze cAMP in HEK293 and MLTC-1 cells [28] E840K PC case loss of PDE11A function in PC3M cells, decreased PDE11A function in HEK293 cells [192] R867G AT/PC/TT/PT cases = controls diminished ability to hydrolyze cAMP in MLTC-1 cells and complete loss of ability to hydrolyze cGMP in HEK293 cells [27-32, 64, 192] Nonsense/Missense I873T AT case [32] M878V AT/PC/TT/PT cases controls diminished ability to decrease global cAMP signal-ing in H295R, MLTC-1, and HEK293 cells [27, 28, 31, 32, 64, 192] M878V (a.k.a. rs74357545) Utah pedigree suicides non-pedigree suicides non-suicide controls [81] Synonomo us L49L AT case and control [31] Q118Q AT cases [31] L160L ATcase [31] C230C AT cases and controls [31] E421E AT/PT cases and controls [31, 64] 2758_2760ins AT/PT cases and controls [31, 64] TCC/S920ins Intronic 1072-3C T cases and controls [31] (intron 4) 1644+26ins cases and controls [31, 64] GTTTATA (intron 11) MP—management program; FHS—Framingham Heart Study; B58C—British 1958 Birth Cohort; CS—Cushing Syndrome; AT—adrenal tumor; TT—testicular tumors; PC—prostate cancer; PT—pituitary tumor frequency of PDE11A missense/nonsense mutations also appears to be significantly greater in patients with Carney Complex relative to healthy controls (25.3% versus 6.8%, P 0.0001), an effect largely driven by those patients with Carney Complex that present with PPNAD versus those that do not (30.8% versus 13%, P = 0.025) [32]. PDE11A mutations may increase risk not just for adrenal tu-mors, but also prostate and testicular tumors. An increase in the frequency of PDE11A mutations (many of the same mutations re- 404 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly ported in the adrenal studies) was observed in patients with prostate cancer relative to healthy controls (16% versus 5%, P 0.001) [23]. Further, the frequency of exon 3 missense mutations (F258Y, G291R, and R52T), but not the total frequency of PDE11A muta-tions, was consistently greater in patients with testicular germ cell tumors (TGCT) versus 3 groups of healthy controls [28]. The fact that exon 3 is specific to PDE11A4, and the preponderance of evi-dence suggests that it is PDE11A3 that is expressed in testes, sug-gests that any potential impact of PDE11A dysfunction on testicular tumor risk may be related to an impact on hormonal signaling else-where in the body (e.g., the adrenal gland) as opposed to altered activity of PDE11A within testicular cells them selves. Indeed, PDE11A missense/nonsense mutations appear to be particularly over represented in male patients with Carney Complex that have both PPNAD and large-cell calcifying Sertoli cell tumors, a type of testicular tumor (81% with PDE11A mutations) [32]. Taken to-gether, these studies show PDE11A missense mutations are not sufficient to cause disease but generally increase the risk for a num-ber of tumors (adrenal, testicular, prostate). 4.3. Functional Effects of PDE11A Missense Mutations In vitro studies suggest that PDE11A missense mutations gen-erally decrease PDE11A function; however, the effects of several PDE11A missense mutations appear to be cell-type specific. Initial studies conducted in HEK293 and HeLA cells suggested the R804H mutation was capable of increasing basal cAMP levels but not basal cGMP levels [29]. A subsequent report by the same group, how-ever, showed that R804H did not change basal cAMP levels in HEK293 cells (i.e., appeared catalytically dead). This subsequent report also showed the R804H was able to decrease cAMP levels below baseline in MLTC-1 cells (a testicular tumor cell line), albeit to a lesser extent than did wild-type PDE11A [28]. Of note, com-plete knockdown of PDE11A was not able to change basal levels of cAMP or cGMP in HEK293 or HeLa cells [29]. Thus, if R804H were able to increase basal cAMP levels in HeLa cells, it would not be explained by a simple loss of PDE11A catalytic activity. Rather, this pattern of results would imply that the R804H mutation acts as a dominant negative—perhaps displacing endogenous PDEs in a way wild-type PDE11A does not, thereby preventing hydrolysis of cAMP by endogenous PDEs. Thus, the effect of the R804H muta-tion on cAMP levels would differ depending on the tissue in ques-tion, given that PDEs are expressed differentially across various tissues (c.f., [3]). Indeed, a tissue-specific effect of the R804H mu-tation is supported by the fact that the R804H construct affected cAMP levels differentially across HEK293, HeLa, and MLTC-1 cells [28, 30]. Further, R804H appeared to function normally in HEK293 cells with regard to cGMP hydrolytic activity (i.e., R804H transfection decreased cGMP levels from baseline), but appeared catalytically dead in HeLa cells (i.e., R804H transfection did not change cGMP levels from baseline). Taken together, these results suggest that the R804H mutation is most likely to diminish PDE11A activity but the specific impact on cAMP versus cGMP hydrolytic activity will vary across tissues. Effects of the R867G mutation have also been inconsistent. An initial report suggested that R867G did not change the ability of PDE11A to hydrolyze cAMP in HEK293 (i.e., R867G decreased cAMP levels from baseline to a similar extent as did wild-type PDE11A) [29]. A subsequent report from the same group, however, showed diminished cAMP hydrolyzing activity of R867G in HEK293 and MLTC-1 cells (i.e., R867G decreased cAMP levels but not to the same extent as wild-type PDE11A) [28]. Only one report examined the effect of R867G on cGMP hydrolytic activity, with the mutation appearing to completely inhibit the cGMP hydro-lytic activity of PDE11A (i.e., R867G transfection did not alter basal cGMP levels) [29]. Clearly, it will be of interest to future studies to test the effects of the R804H and R867G mutations spe-cifically within an adrenocortical cell line (e.g. H295R cells) to determine how the mutations impact signaling specifically within that cellular context. The M878V and D609N PDE11A missense mutations, identi-fied in patients with adrenal tumors and testicular tumors, have been tested in HEK293, H295R, and MLTC-1 cells (a testicular tumor cell line), with somewhat separable results [27]. In H295R cells, transfection with the M878V and D609V mutants decreased cAMP-response element (CRE) transcription, but not to the same extent as did transfection with wild-type PDE11A [27]. Similarly, M878V and D609V decreased cAMP levels in MLTC-1 cells, but not to the same extent as did wild-type PDE11A [28]. This suggests that the M878V and D609V mutants exhibit diminished cAMP hydrolytic activity in H295R and MLTC-1 cells, but their activity in HEK293 cells is less clear. Similar to effects observed in MLTC-1 cells, M878V and D609N mutants both decreased global cAMP levels in HEK293 cells (both basal and forskolin-stimulated), albeit not to the same extent as did wild-type PDE11A [27, 28]. Seem-ingly contrary, transfection of HEK293 cells with the M878V mu-tant increased CRE transcription above baseline, suggesting a domi-nant negative effect, and transfection with the D609N mutant elicited no effect on CRE transcription, suggesting absence of cAMP hydrolytic activity in this mutant [27]. The fact that the M878V mutant retained some ability to decrease global cAMP levels and yet increased CRE transcription in HEK293 cells sug-gests the mutation may have a differential effect within specific HEK293 subcellular compartments. For example, it is possible the M878V mutation changes the ability of PDE11A to interact with certain compartment-specific binding partners, which could result in the displacement of another PDE that would otherwise much more efficiently degrade cAMP. A host of other PDE11A missense mutations, some of which have only been identified in patients with TGCT (Table 2), were tested in HEK293 and MLTC-1 cells with consistent results. In both cell types, R52T, F258Y, G291R, A349T, and V820M were able to decrease cAMP levels somewhat, but not to the extent of wild-type PDE11A [28]. Y727C showed a similar diminished capacity to hydrolyze cAMP in MLTC-1 cells [28]. This suggests an ability of these mutations to decrease PDE11A function. Interestingly, rela-tive to healthy controls, increased methylation of select CpG islands in the PDE11A promoter was identified in leukocytes from the same patients with TGCT that were used in the analyses of PDE11A missense mutations [65]. Increased CpG methylation generally correlates with decreased mRNA expression. Thus, these results together strongly argue for diminished PDE11A function as a risk factor for developing TGCT. The effects of the R202C, Y658C, and E840K mutations identi-fied in patients with prostate can cer have been tested in HEK293 cells and PC3M prostate cancer cells. In these studies, total PDE activity was measured [23]. R202C-transfected HEK293 and PC3M cells showed increased PDE activity relative to mock-transfected cells but decreased PDE activity relative to wild-type PDE11A-transfected cells [23]. In contrast, there was no difference in PDE activity between Y658C-transfected HEK293 and PC3M cells and mock-transfected cells [23]. The effect of the E840K mutation was cell-type specific, with E840K eliciting no effect on PDE activity in PC3M cells but increasing PDE activity quite substantially in HEK293 cells, albeit to a somewhat lesser extent that wild-type PDE11A [23]. Together, these studies point to a consistent trend toward PDE11A missense mutations diminishing PDE11A func-tion. It will be very important for future studies to understand exactly how these various PDE11A missense mutations lead to decreased PDE11A function. Several of the missense mutations fall within the catalytic domain and, thus, may elicit their effect directly by de-creasing substrate affinity or enzymatic activity. Other mutations in the GAF-B domain might decrease PDE11A function by interfering with homodimerization or other protein-protein binding interac- Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 405 tions, thereby displacing PDE11A from its cognate pool(s) of cyclic nucleotides. Still oth er missen se mutations might imp act PDE11A function by interfering with cyclic nucleotide feedback at the GAF-A domain or by impacting post-translational modifications. Under-standing how these various missense mutations impact PDE11A will be key to developing therapeutics that effectively restore PDE11A function in these individuals. 5. PDE11A AND BRAIN FUNCTION PDE11A expression in brain is almost exclusively localized to CA1 and subiculum of the ventral hippocampal formation (a.k.a., the anterior hippocampal formation in primates) [19] (Fig. 4). This makes PDE11A one of the most restrictedly expressed PDE fami-lies in brain (c.f., [7, 8]). Our hypotheses regarding how PDE11A impacts brain function and behavior are directly tied to our under-standing of the functional output of the ventral hippocampal forma-tion. The ventral hippocampal formation in rodents is analogous to the anterior hippocampal formation in primates [66]. As such, we will use the term \"v entral-an terior” hippocampal formation (VHIPP) and \"dorsal-posterior” hippocampal formation (DHIPP) throughout the remainder of this manuscript for consistent referenc-ing of these anatomically and functionally distinct subregions across species. As we will see below, the VH IPP is a small brain region that modulates affective and social behaviors [67]. 5.1. Separable Functions of VHIPP Versus DHIPP Separable functions have been attributed to the VHIPP versus DHIPP (e.g., [68-72]). While the VHIPP appears important for modulation of behaviors related to emotion/affect, sociality, moti-vation, stress, behavioral flexibility, and sensorimotor gating, the DHIPP appears key to learning, memory, contextual representation, and spatial navigation (c.f., [66, 67, 71-73] also see [68, 74, 75]). The separable fun ctions of the VHIPP versus DHIPP are consistent with their differences in anatomical connectivity to extrahippocam-pal structures. Whereas the VHIPP is highly interconnected with the olfactory bulb and cortices, prefrontal cortex, amygdala, hypo-thalamus, and the shell of the nucleus accumbens, the DH IPP is highly interconnected with sensory-related cortices, thalamic subre-gions, and the core of the nucleus accumbens (c.f., [71, 72]). This segregation of function and connectivity between the VHIPP and DHIPP applies to each of the hippocampal subfields (CA1, CA2, CA3, CA4, DG, subiculum) and can be easily visualized when mapping expression of numerous gene products [72]. PDE11A is one such gene with topographically differentiated expression, with expression in CA1 and subiculum of DHIPP being minimal versus 3-fold higher expression in VHIPP (Fig. 4). 5.2. PDE11A is Required for Intact Brain Function We have shown that adult PDE11A KO mice exhibit a constel-lation of deficits that are consistent with VHIPP dysfunction [19]. PDE11A KO phenotypes included deficits in social behaviors (so-cial odor recognition memory and social avoidance), increased exploration of a novel open field, and increased sensitivity to loco-motor effects of the glutamate N-methyl-D-aspartate receptor an-tagonist MK-801. In addition, PDE11A KO mice showed enlarged lateral ventricles and increased functional activation of ventral CA1 (as per increased Arc mRNA). The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemi-cal dysregulation observed around the glutamate -amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, includ-ing decreased levels of phosphorylated-GluR1Ser845 and the proto-typical transmembrane AMPA-receptor–associated proteins star-gazin (2) and 8 [19]. In contrast, PDE11A KO mice exhibit nor-mal behavior in prepulse inhibition of acoustic startle, contextual and cued fear conditioning, acute dopaminergic challenge, as well as a battery of anxiety- and depression-related behaviors. It is not surprising that deletion of PDE11A leaves some VHIPP behaviors intact (e.g., sensorimotor gating, contextual fear conditioning) be-cause PDE11A is not expressed in all VHIPP subfields (Fig. 4), and it is not the only PDE expressed in VHIPP (c.f., [7, 8]). Thus, defi-cits exhibited by PDE11A KO mice are relatively specific and im-pact a subset of behavioral domains regulated by the VHIPP. Given that inactivating PDE11A mutations have been linked to select adrenocortical tumors in humans (see below), it is important to point out that our PDE11A KO mice do not show any adrenal tu-mor pathology at 1 year of age [19], consistent with the fact that we do not observe any PDE11A expression in the mouse adrenal gland (Figs. 5 and 6). Thus, neurocognitive deficits noted in our PDE11A KO mice are most likely due to the loss of PDE11A signaling within the brain as opposed to indirect effects of adrenal dysfunc-tion. It is not yet known if the impairments exhibited by adult PDE11A KO mice are due to the acute loss of PDE11A signaling in adulthood and/or the chronic loss of PDE11A during development. That said, PDE11A is minimally expressed early in development [19], and we have recently shown that PDE11A mRNA and protein expression continues to increase in the hippocampus well into late adulthood [50]. Together, these studies suggest that the role of PDE11A in brain function evolves across the lifespan. Unfortunately, very few studies to date have studied PDE11A in samples taken from patients with psychiatric disease. PDE11A expression was reported as increased in the cerebellum of patients with bipolar disorder [15]. We are unable to detect PDE11A ex-pression in cerebellum taken from healthy humans or rodents, sug-gesting that a disease state may drive ectopic expression of PDE11A outside of its normal boundaries. Supporting this conten-tion is our recent finding that aged rodents appear to exhibit low levels of PDE11A mRNA expression outside of the hippocampus, specifically in the striatum and cerebellum [50]. S elect gen etic as-sociation studies h ave exam ined PDE11A in the context o f disease and medication response. Preliminary studies examining a total of 752 SNPs have linked several individual single nucleotide poly-morphisms (SNPs) in PDE11A (e.g., rs7585543, P = 4 x 10-5) and a 3 SNP sliding haplotype in PDE11A (P = 6 x 10-5) to lithium re-sponsiveness in patients with bipolar disorder [16, 76, 77]. Interest-ingly, prediction software suggests that PDE11A may be phos-phorylated by AKT and glycogen synthase kinase (GSK) (Fig. 3), two enzymes that are required for the behavio ral effects of lithium (c.f., [78]). In one study of Mexican-American individuals, the PDE11A SNP rs3770018 was associated with major depressive disorder (MDD; P = 5 x 10-4). Further, the PDE11A SNP rs1880916 was nominally associated (P 0.05) with antidepres-sant-response in this group of MDD patients, which were treated with either fluoxetine or desipramine (remission versus non-remission), and the PDE11A SNP rs3770018 was associated with antidepressant response in the fluoxetine-treated group only [17]. A follow-up study conducted in a group of Mexican-American indi-viduals, further identified 3 PDE11A global haplotypes that were associated with the diagnosis of MDD and 1 PDE11A global haplo-type that was associated with antidepressant response to either fluoxetine or desipramine [79]. Findings from the Sequenced Treatment Alternatives for Depression (STAR*D) trial also suggest that variation in PDE11A may be associated with MDD [80]. STAR*D did not, however, find an association between rs1880916 and antidepressant response to citalopram [80]. Similarly, Perlis and colleagues did not find an association between either rs1880916 or rs3770018 and antidepressant response to duloxetine [21]. Most recently, the M878V mutation was found to occur significantly more frequently in a Utah pedigree of high-risk suicide victims relative to Utah non-suicide controls and a non-Utah suicide pedi-gree [81]. As noted above in Section 4, in vitro studies suggest that the M878V mutation diminishes function of PDE11A [27, 28]. Clearly, more patient-based studies are needed to understand how the function of PDE11A may be altered in the context of brain dis-eases. 406 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly Although patient data directly implicating PDE11A itself is lacking, there are a large number of studies reporting VHIPP dys-function [82-103] as well as cyclic nucleotide dysregulation [104-140] in patients with neuropsychiatric disease, particularly schizo-phrenia and Alzheimer’s disease. Certainly, a PDE11A-targeted therapeutic would be well positioned to modulate VHIPP function and to restore aberrant cyclic nucleotide signaling therein. As such, we review here findings implicating VHIPP dysfunction as well as cyclic nucleotide dysregulation in neuropsychiatric disease so that we may glean insight into what potential a PDE11A-targeted thera-peutic might hold. 5.3. VHIPP Deficits in Neurological/Psychiatric Disease Unfortunately, only a fraction of research examining human hippocampal functioning differentiates VHIPP versus DHIPP. Of those studies focusing on the anterior hippocampus in various pa-tient populations, the four most prevalent diseases of focus are sei-zure/epilepsy (~33% of studies), schizophrenia (~18% of studies), Alzheimer’s Disease (AD; ~13% of studies), and mood disorders (~7% of studies). Interestingly, abnormalities in cAMP and cGMP signaling have also been consistently reported in patients with schizophrenia [104-132] and Alzheimer’s disease [133-140]. Our preliminary data suggests that PDE11A KO mice show no differ-ence with regard to pentylenetetrazol-induced seizure susceptibility but PDE11A KO mice do exhibit a number of phenotypes that are consistent with psychiatric disease. Therefore, we will review a number of human findings linking dysfunction of the VHIPP to schizophrenia, AD, and mood disorders as well as one in patients with Cushing Syndrome (a disease to which PDE11A inactivating mutations have been linked), in order to understand the potential of PDE11A as a therapeutic target. 5.3.1. VHIPP Deficits and Schizophrenia Studies examining the VHIPP consistently note deficits in pa-tients with schizophrenia relative to control subjects. Several stud-ies have noted a smaller or deformed VHIPP in patients with schizophrenia [82-87], but see [141]. Other studies have noted al-terations in gene expression [88, 89], functional connectivity [90, 91], and functional activation of the VHIPP in patients with schizo-phrenia [92-94]. Patients with schizophrenia also exhibit select deficits in DHIPP, but most studies examining endpoints in both the VHIPP and DHIPP report more severe deficits in the VHIPP [82, 83, 85, 86, 88, 89, 92], but see [85, 95]. It is not yet known if ab-normalities in the VHIPP are the cause or consequence of disease symptomatology; however, the extent of pathology in this region has been correlated with symptom severity in patients with schizo-phrenia [85, 86, 93, 95, 96], but see [142]. 5.3.2. VHIPP Deficits and Mood Disorders Relative to schizophrenia, far fewer studies have examined the VHIPP of patients with mood disorders (i.e., bipolar disorder [BPD], MDD, and anxiety disorders). Smaller VHIPPs have been noted in patients with BPD [143]. Despite the fact that the VHIPP may be smaller, Timm staining suggests that neuronal sprouting may actually be increased in the VHIPP of patients with BPD [144]. No change was noted in the size of the VHIPP in patients with post-traumatic stress disorder, although smaller DHIPPs were noted in these patients [145]. Greater activation of the VHIPP has been noted in fem ale patients remitted for MDD when successfully encoding positive, but not negative or neutral words [146]. In con- Fig. (6). PDE11A protein appears to be expressed most robustly in the hippocampus (PDE11A4) and spleen (PDE11A1) of female mice. Tissue from PDE11A wild-type (WT) and knockout (KO) mice was processed by Western blot as described in Figure 5. PDE11A WT hippocampus (HIPP) and purified human PDE11A4 (hPDE11A4) were loaded on each gel as a positive control and tissue from PDE11A KO mice was loaded for each organ as a negative control. In general, many non-specific bands are noted in most tissues examined. Though many of these bands correspond to the predicted weights of the various PDE11A isoforms, they do not disappear in tissue from the PDE11A KO, confirming they are non-specific in nature. As observed in Figure 4, PDE11A4 is specifically expressed in hippocampus of female PDE11A WT mice but not female KO mice—no other tissues examined expressed PDE11A4 in female mice. Light bands corresponding to the predicted weight of PDE11A3 (~80 kDa) and PDE11A1 (~50 kDa) are present in the spleen of the female WT mouse but not the female KO. Together, these results suggest that PDE11A expression is quite restricted in the female body. L. INTEST—large intestine; GASTROC—gastrocnemius; PLANT—plantaris. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 407 trast, reduced theta activity was observed in the VHIPP during a task in which depressed patients exhibited impaired spatial naviga-tion [147]. Although this study registered an overall decrease in VHIPP theta activity during impaired spatial nav igation, it was the theta activity in the DHIPP that correlated with individual perform-ance in this task [147]. Finally, increased expression of brain-derived neurotrophic factor has been measured specifically in DG of the VHIPP of patients with MDD, BPD, and schizophrenia who were being treated with antidepressants at the time of death [148]. Thus, despite the well-establish ed role of the VHIPP in emotional processing, the evidence implicating the VHIPP in the pathophysi-ology of mood disorders is relatively limited to date. 5.3.3. VHIPP Deficits and AD VHIPP deficits associated with Alzheimer’s Disease (AD) are similar to those noted above for patients with schizophrenia. Pa-tients with AD, mild cognitive impairment, or frontotemporal de-mentia, show atrophy of the VHIPP [97, 98]. Pyramidal cell loss may be in part to blame for the atrophy as losses were noted in CA1 of patients with AD [99]. In terms of functional changes, individu-als presymptomatic for familial AD show heightened activation of the right VHIPP during encoding of a novel-face-name associations task [100]. Importantly, the extent of anatomical and functional deficits observed in the VH IPP appears to correlate with severity of episodic memory deficits in patients with AD [98, 101-103]. 5.3.4. VHIPP Deficits and Cushing Syndrome Because several reports have found PDE11A inactivating muta-tions in patients with adrenocortical tumors ([27, 29-32] but see [33, 149]), it is also worth noting that one study has examined the anterior hippocampus in patients with Cushing Syndrome. Maheu and colleagues [150] found heightened functional activation of the VHIPP during encoding of an emotional-faces recognition test in patients with Cushing Syndrome. It is interesting to note that PDE11A KO mice show deficits in social odor recognition memory and heightened activation of ventral CA1 [19]. Although studies examining overall hippocampal function have correlated deficits in Cushing Syndrome patients with dysregulated cortisol levels [151-154], a potential role for aberrant PDE11A signaling within the VHIPP itself has not been ruled out. Unfortunately, no studies to date have characterized hippocampal function in patients genotyped with PDE11A inactivating mutations. Thus, it is possible that hip-pocampal deficits observed in patients with Cushing Syndrome may be related to the indirect effects of dysregulated cortisol level as well as the direct effect of PDE11A inactivation within the hippo-campus. 5.4. Cyclic Nucleotide Signaling and Neurological/Psychiatric Disease A wealth of studies have associated cyclic nucleotide dysfunc-tion with neuropsychiatric disease. Interestingly, this literature re-volves mostly around schizophrenia and AD—as did literature im-plicating dysfunction of the VHIPP (see above). Because PDE11A regulates cAMP and cGMP levels, we believe it is important to review here eviden ce implicating these cyclic nucleotides in brain disorders to understand the therapeutic potential PDE11A may hold. 5.4.1. Cyclic Nucleotide Signaling in Schizophrenia A majority of studies examining cyclic nucleotides in patients with psychiatric disease focus on cAMP, while only a few report results related to cGMP. Reduced levels of cAMP production have been consistently noted in platelets, but not leukocytes, of patients with schizophrenia [104-112]. Though a peripheral measure, defi-cits in cAMP production in platelets have correlated with symptom severity in patients with schizophrenia [107, 113, 114]. This sug-gests that cAMP production deficits in platelets mirror cAMP ab-normalities in brain and, thus, might prove a useful biomarker for selecting patients most likely to respond to a cAMP-targeted thera-peutic. While some studies have reported elevated levels of cAMP [113, 115, 116], and decreased levels of cGMP in CSF of patients with schizophrenia [117, 118], others have found no differences in basal levels [155]. Discrepancies among CSF studies may be re-lated to medication history [115, 117, 118, 156-159]. One study has linked olfactory dysfunction in patients to cAMP signaling deficits in the olfactory bulb [119]. Like measures of cAMP production in platelets, it is possible that testing for olfactory dysfunction with cAMP-dependent odorants could be used as a biomarker to predict those patients most likely to benefit from a cAMP-targeted thera-peutic. Finally, cAMP binding sites were diminished in cytosolic fractions, but not membrane fractions, from several brain regions of patients with bipolar disorder [120]. Interestingly, lithium appears to increase levels of the catalytic subunit of PKA in cytosolic frac-tions, but not membrane fractions [160]. Understanding that dis-eases may be associated with cyclic nucleotide deficits only in se-lect subcellular compartments underscores the importance of identi-fying how the subcellular localization of PDE11A is controlled under various conditions. Gs stimulates adenylyl cyclase (AC) to form cAMP. Increased efficacy of Gs been noted in the striatum and leukocytes of patients with schizophrenia [121, 122]. Interestingly, the increased Gs effi-cacy in leukocytes correlated with symptom severity in patients with schizophrenia [122] and was reversed with antipsychotic treatment [123]. The T393C polymorphism in Gs has been geneti-cally linked to deficit schizophrenia in an Italian population sample, such that the 393TT genotype, which increases mRNA expression [161], was observed significantly more in these patients (37.1%) relative to controls (22.8%) [124]. State-dependent changes in ex-pression and/or efficacy of Gs have also b een repeatedly measured in peripheral and central measures from patients with mood disor-ders, such that mania and panic disord er are asso ciated with in-creased Gs whereas depression and suicide are associated with decreased levels [125-132] but see[162]. It is important to note that increased expression of Gs in brain is known to increase cAMP levels in striatum, but decrease cAMP levels in cortex and hippo-campus—the latter due to a compensatory upregulation of PDE activity [163-166]. While the striatal increases in cAMP appear to mediate the Gs-induced increase in anxiety-related behaviors, it is the decreased cortical cAMP levels that appear to be responsible for Gs-induced sensorimotor gating and cognitive deficits [163-166]. These types of brain region-specific alterations in cyclic nucleotide signaling underscore the need for therapeutic targets that are capa-ble of modulating isolated circuits and specific subcellular com-partments—targets such as PDE11A. 5.4.2. Cyclic Nucleotide Signaling in Alzheimer’s Disease and Mild Cognitive Impairment Although fewer studies have examined cyclic nucleotide signal-ing in AD than in psychiatric disease, a number of interesting and consistent findings have implicated a loss of cAMP tone. In contrast to results obtained from patients with schizophrenia, patients with AD exhibit decreased expression and efficacy of Gs signaling in brain and fibroblasts [133, 134] as well as decreased AC expression and activity in brain [135-137]. Decreased expression of guanylate cyclase h as also been noted in reactive astrocytes of AD p atients [138]. Interestingly, one study showed that reductions in basal and stimulated AC activity in hippocampus correlated with the extent of amyloid plaques (though not neuritic plaques or tangles) [139]. While cyclic nucleotide tone appears to be diminished within brain tissue from AD patients, the opposite effect may be in play within the microvasculatu re. Increases in stimulated AC activity, cAMP levels, and PKA activity have been noted in cerebral microvessels from AD patients [167-169]. This may explain why cAMP levels have been noted as elevated in CSF of AD patients [140]. Interest-ingly, in this study, CSF cAMP levels correlated with CSF tau pro-tein levels. Together, these studies support a role for dysregulated cyclic nucleotide signaling in the pathophysiology of AD and sug- 408 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly gest a therapeutic able to increase cAMP levels specifically in the brain might prove beneficial. Taken together, we believe studies reviewed here support a role for PDE11A in brain function and the consideration of PDE11A as a therapeutic target for neuropsychiatric disease. Our studies in PDE11A knockout mice show that PDE11A is required for intact brain function [19] and genetic association studies in humans are beginning to suggest that mutations in PDE11A impact neuropsy-chiatric disease risk [17, 79, 80] and/or responsiveness to neuropsy-chiatric medications ([16, 17, 76, 77, 79], but see [21, 80]). Much more developed is the body of literature implicating both VHIPP dysfunction [82-103] as well as cyclic nucleotide dysregulation [104-140] in the pathophysiology of neuropsychiatric disease, par-ticularly schizophrenia and Alzheimer’s disease. Given the ana-tomical localization and dual-substrate specificity of PDE11A, a PDE11A-targeted therapeutic would be well positioned to address this VHIPP dysfunction and to restore aberrant cyclic nucleotide signaling within the VHIPP. Of course, it is important to remember that the VHIPP does not exist in isolation. Thus, it is possible that a PDE11A-targeted therapeutic might also impact symptomatology associated with dysfunction of other brain regions that receive pro-jections from the VHIPP, including the prefrontal cortex, nucleus accumbens, hypothalamus and amygdala (c.f., [71, 72]). Clearly, it will be important for future studies to determine wheth er or not alterations in PDE11A expression, localization, and/or catalytic activity are found in brain tissue from patients with neuropsy-chiatric disease. Even if PDE11A itself is not changed in the brains of patients, a PDE11A-targeted drug may still hold therapeutic potential by compensating for insults upstream or downstream of PDE11A (e.g., at the AMPA receptor). 6. POSSIBLE ROLE FOR PDE11A IN INFLAMMATION A handful of disparate studies have emerged that suggest PDE11A may play a role in regulating inflammatory processes. The PDE11A SNP rs11684634 was genetically linked (P = 8.9 x 10-7) to allergic asthma in a population of patients with early-onset persis-tent asthma [34]. Importantly, these authors attempted to replicate their finding by querying PDE11A SNPs in 5 other existing genome wide association studies of asthma and found a nominally signifi-cant association (P 0.05) with at least 1 PDE11A SNP in 3 out of the 5 cohorts [34]. Interestingly, these same cohorts previously demonstrated an association between asthma and PDE4D [170], and the PDE4 inhibitor roflumilast is currently used in the clinic for chronic obstructive pulmonary disease. A separate genome wide association study identified a link between the PDE11A SNP rs4893980 and whether or not individuals infected with M. tubercu-losis went on to develop extrapulmonary tuberculosis or remained asymptomatic [35]. Studies in rodents support a functional role for PDE11A in cell typ es relevant to inflammation and immun e func-tion. Witwicka and colleagues found that PDE11A mRNA expres-sion was relatively robust in rat peritoneal exudate macrophages (PEMs), but negligible in resident peritoneal macrophages [36]. Following 24 hours of ‘resting’ culture, PDE11A mRNA expres-sion in PEMs fell to below the limit of detection; however, incuba-tion of those PEMs with the bacterial endotoxin lipopolysaccharide (LPS) for an additional 24 hours significantly reactivated PDE11A mRNA expression [36]. Thus, PDE11A may play a role in modulat-ing the inflammatory responses of macrophages. Bazhin and col-leagues found that mouse regulatory T cells express significantly lower levels of PDE11A mRNA than do activated conventional T cells [37]. These authors also found that regulatory T cells exhibit significantly lower levels of total PDE activity relative to conven-tional activated T cells, and suggest this lower level of PDE activity is primarily accounted for by lower levels of PDE8A and PDE11A catalytic activity [37]. This suggests that PDE11A may play a role in regulating the ability of regulatory T cells to suppress antigen-specific immune responses by activated conventional T cells. In summary, genetic findings in humans and functional studies in rodents support a role for PDE11A in inflammation and immune function; however, the exact mechanism by which PDE11A may impact this aspect of physiology (e.g., regulating cytokine release, modulating susceptibility to bacterial invasion, etc.) remains to be determined. 7. PROGRESS IN THE DEVELOPMENT OF PHARMA-COLOGICAL TOOLS 7.1. PDE11A Activators Using an in vitro FRET-based assay, Jager and colleagues screened compounds for the ability to bind to and initiate a confor-mational change in a fragment of PDE11A4 that included both GAF domains [43]. Both cG MP and the cGMP analog Rp-8-pCPT-PET-cGMPS (but not cAMP) bound to the PDE11A4 GAF domain construct [43]. Cyclic GMP and the cGMP analog Rp-8-pCPT-PET-cGMPS were then tested for their ability to modulate catalytic activity of full-length PDE11A4. Whereas cGMP failed to stimulate PDE11A catalytic activity, Rp-8-pCPT-PET-cGMPS produced a ~4-5-fold increase in PDE11A-mediated hydrolysis of both cAMP and cGMP [43]. Importantly, Rp-8-pCPT-PET-cGMPS increased catalytic activity of not only recombinant human PDE11A4 but also native PDE11A4 enriched from mouse hippocampus [43]. Of note, Rp-8-pCPT-PET-cGMPS does not bind to the GAF domains of PDE5A or PDE2A when tested in a similar FRET assay, suggesting this compound may exhibit some degree of specificity with regard to targeting PDEs [171]. Interestingly, the authors note in the dis-cussion of the paper (data not shown) that Rp-8-pCPT-PET-cGMPS was incapable of stimulating PDE11A catalytic activity when the 196 amino acids upstream of the PDE11A4 GAF-A domain were deleted [43]. Clearly, these results have important implications for future drug screening efforts aimed at identifying PDE11A com-pounds. These results suggest it will be important to screen com-pound libraries against full-length PDE11A, as opposed to some truncated version of the protein. Though the study by Jager and colleagues serves as important proof that PDE11A activators can be identified, Rp-8-pCPT-PET-cGMPS is likely to have limited use as a tool PDE11A activator because it is well known to have signifi-cant off-target activities, including inhibition of PKG and cGMP-gated ion channels (see product insert for Rp-8-pCPT-PET-cGMPS on www.biolog.de). 7.2. PDE11A Inhibitors A screen to identify PDE11A inhibitors has also been con-ducted [172]. Ceyhan et al used a yeast-based high-throughput assay to screen a large chemical library (~200, 000 compounds) for potential PDE11A inhibitors [172]. The primary yeast-based screen yielded 1143 hits. Following triage and counterscreens, 99 com-pounds moved on to characterization in secondary assays to assess selectivity versus all other PDE families. Four potent and selective PDE11A inhibitors were ultimately identified: BC11-15, BC11-19, BC11-28, and BC11-38 [172]. In an enzymatic assay, IC50s of the 4 compounds ranged from 0.11 to 0.33 M. BC11-15 and BC11-19 showed some off-target activity in their ability to inhibit other PD E families at much higher concentrations ( 25 M and 10 M, re-spectively), but BC11-28 and BC11-38 showed no off target activi-ties at concentrations 100 M. Thus, BC11-28 and BC11-38 dem-onstrated 350-fold selectivity for PDE11 versus all other PDEs. Despite the ability of all four compounds to potently inhibit PDE11A cGMP hydrolytic activity in both the yeast-based and enzyme assays, only BC11-38 inhibited PDE11A cAMP hydrolytic activity in a cell-based assay employing H295R cells, a human adrenocortical tumor cell line that endogenously expresses PDE11A. Importantly, BC11-38 had no effect in HeLa cells, which do not express PDE11A. The authors suggest that the failure of BC11-15, BC11-19, and BC11-28 to increase cAMP levels in H295R cells may be related to differences in the cell culture media between the yeast-based assay and the mammalian cell culture as- Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 409 say (e.g., reduced solubility of compounds, greater non-specific binding of compounds to proteins in the cell culture media, etc.) [172]. An additional possibility, discussed at greater length below, is that certain compounds may be functionally selective in their ability to inhibit cAMP-PDE11A versus cGMP-PDE11A catalytic activity. Full-length PDE11A4 was used in this screening (as op-posed to an isolated catalytic domain), so it is not yet known whether BC11-38 works by competing for the substrate binding pocket or through an allosteric binding site [172]. The screens con-ducted in cell culture show that BC11-38 does cross th e cell mem-brane; however, it is not yet known if BC11-38 crosses the blood brain barrier. BC11-38 is now commercially available, but no in-formation is yet available regarding its pharmacokinetic profile and no studies have yet reported the effect of this drug in vivo. It re-mains to be seen whether BC11-38 will prove a useful tool to ad-minister to whole animals, but BC11-38 at the very least stands as a proof that PDE11A can be inhibited with a high degree of selectiv-ity versus other PDEs. 7.3. What can PDE5A Inhibitors Teach us about PDE11A Function? The above mentioned developments relied on screens of chemi-cal libraries to identify compounds that target PDE11A, but it should also be possible to rationally design drugs targeting this enzyme. The PDE5A inhibitor tadalafil is a relatively potent inhibi-tor of PDE11A with reported IC50 values for inhibition of cGMP hydrolysis ranging from 50nM to 73nM [48, 173]. By systemati-cally altering tadalafil, Mohamed et al demonstrated trends between certain structural modifications and altered preference for PDE11A versus PDE5A [173, 174]. Specifically, selectivity for PDE5A over PDE11A was enhanced by decreasing the size of the tadalafil ter-minal tetracycle ring and increasing the bulkiness of N-alkyl sub-stituents on the ring [174]. Although the authors of that study in-tended to discover modifications that could improve PDE5A selec-tivity, the report points to the possibility that the same approach could be taken to develop derivatives with a greater selectivity for PDE11A over PDE5A. Indeed, a recent report has combined what is known about how various PDE5 inhibitors as well as BC11-38 inhibit PDE11A with what is known about PDE5A crystal structure to make in silico predictions about PDE11A enzyme-inhibitor in-teractions, with the hope of furthering efforts to identify PDE11A-selective compounds. Although selective PDE11A inhibitors have yet to be tested in animals, it is possible that some in vivo effects of PDE5A inhibitors may be related to off-target inhibition of PDE11A. Of the clini-cally-available PDE5A inhibitors, tadalafil most potently inhibits PDE11A (IC50 73 nM) and may do so at clinically-relevant doses because tadalafil is only ~7 -fold selective for PDE5A versus PDE11A [48, 175-177]. Certainly, preclinical studies have used doses of tadalafil (15 mg/kg p.o.) that are high enough produce brain exposures that cover the IC50 for PDE11A several fold [178]. Sildenafil is also somewhat potent at PDE11A (IC50 3.8 M), but is ~1000-fold more selective for PDE5A versus PDE11A [48, 49]. Though sildenafil is not likely to inhibit PDE11A with doses used in the clinic [48], the same cannot be said for preclinical studies conducted in rodents. Importantly, sildenafil and tadalafil both cross the blood brain barrier [179, 180]. In a direct comparison, tadalafil and sildenafil (each 10 mg/kg/day i.p. x 14 days) both attenuated social deficits but not hyperactivity exhibited by rats bred to exhibit depression-like behaviors (Flinders Sensitive Line rats) [180]. In a separate study, subchronic administration (10 mg/kg/day p.o. x 14 days) of tadalafil reduced the effects of mater-nal separation in rats as measured by decreased apoptosis in the hippocampus and a decrease in immobile time in the forced swim test [181]. Similarly, tadalafil and sildenafil (15 mg/kg/day p.o. x 10 weeks) improved hippocampus-dependent learning and attenu-ated hippocampal tau phosphorylation in a mouse model of Alz-heimer’s Disease [178]. The effects of tadalafil and sildenafil on the hippocampus are particularly suggestive of an off-target PDE11A effect because PDE11A is expressed strongly in the rat hippocam-pus, while PDE5A expression in hippocampus is minimal at best [182-186] (Fig. 7). Of course, it is possible that the central effects of PDE5A inhibitors may be related to general changes in blood flow as opposed changes in cyclic nucleotide signaling directly within brain cells. With regard to sperm motility, sildenafil in-creased sperm motility in infertile human males whereas tadalafil had the opposite effect [175]. This effect of tadalafil is consistent with the observation that PDE11A knockout mice have a decrease in forward motility of sperm with no decrease in overall motility, viability or fertility [56, 176]. Definitive experiments in PDE5A or PDE11A KO mice are needed to determine if the in vivo effects of PDE5A inhibitors, particularly those in the hippocampus, are re-lated to PDE5A and/or PDE11A inhibition. 7.4. Potential for Functional Selectivity of PDE11A Modulators Functional selectivity is a term used to indicate that a given protein has the potential to activate one of several second messen-ger cascades depending on what ligand binds the protein of interest. This concept is most commonly used in terms of receptor pharma-cology; however, there are hints in the literature to suggest the same concept may be at play with dual-specificity PDEs. As reviewed above, PDE11A hydrolyzes cAMP and cGMP equally well; how-ever, the concentration at which a given compound will inhibit each of those hydrolytic activities is not always equal. The pan-PDE inhibiting compounds IBMX, zaprinast and dipyrimadole inhibit the cAMP- and cGMP-hydrolyzing activity of PDE11A with equal potency [11]. Some PDE5A-preferring inhibitors, \"8”, \"28”, and \"29” also inhibit both the cAMP- and the cGMP-hydrolyzing activ-ity of PDE11A with equal potency; however, this is not always the case [174]. Tadalafil inhibits the cAMP-hydrolyzing activity of PDE11A with an IC50 that is 5.9-fold higher than that required to inhibit the cGMP-hydrolyzing activity of PDE11A [174, 177]. Similarly, the PDE5A-preferring inhibitors \"45”, \"14”, \"11”, \"XXI”, \"25”, and \"33” inhibit the cAMP-hydrolyzing activity of PDE11A with IC50s 9.5-, 8.7-, 6.5-, 4.5-, 2.2-, and 2-fold higher, respectively, than those required to inhibit the cGMP-hydrolyzing activity of PDE11A [173, 174, 177]. Whether the differential activ-ity of these compounds in regulating the cAMP- versus cGMP-hydrolyzing activity of PDE11A reflects something inherent about PDE11A (i.e., the cGMP-hydrolyzing activity is easier to inhibit) or something inherent to PDE5A-preferring inhibitors (i.e., the fact that they were originally developed to target a cGMP-specific PDE) remains to be determined. As noted above, the PDE11A-preferring inhibitors BC11-15, BC11-19, and BC11-28 all inhibited the cGMP-hydrolyzing activity of PDE11A in an enzyme and yeast-based assay but failed to inhibit the cAMP-hydrolyzing activity of PDE11A in a mammalian cell culture paradigm [172]. It will be of interest to future studies to determine if BC11-15, BC11-19, and BC11-28 can inhibit the cAMP-hydrolyzing activity of PDE11A in a cell-free enzymatic assay to determine if the failure of these com-pounds in mammalian cell culture was truly related to media arti-facts or, in fact, reflected functional selectivity in their ability to only inhibit the cGMP-hydrolyzing activity of PDE11A. The PDE11A-preferring inhibitor BC11-38 was able to inhibit both the cGMP- and the cAMP-hydrolyzing activities of PDE11A; however, these endpoints were measured in different assays so IC50s cannot be directly compared. If functional selectivity is possible for dual-specificity PDEs, it would offer yet another mechanism we could employ to more selectivity target specific pools of cyclic nucleo-tides. 8. PROPERTIES OF AN IDEAL DRUG TARGET There are several properties that the pharmaceutical industry believes an ‘ideal’ drug target should have [187]. The first set of properties an ideal drug target should have is related to the strength in rationale for the target, and the second set of properties is related 410 Current Pharmaceutical Design, 2015, Vol. 21, No. 3 Michy P. Kelly to the logistics involved in running a drug discovery and develop-ment program. We will discuss PDE11A in the context of each of these desired properties. 8.1. Strength in Rationale The first 4 properties that the pharmaceutical industry believes an ideal drug target should have are related to the strength in ration-ale of the target. First, a target should be disease modifying or have proven function in the disease or disease-relevant tissue [187]. Studies in PDE11A knockout mice certainly point to an important role of PDE11A in function of the ventral hippocampus [19]. As reviewed in detail above, dysfunction both in cyclic nucleotide signaling and in the function of the anterior hippocampal formation (human analog of rodent ventral hippocampus) have been repeat-edly implicated in diseases such as schizophrenia and Alzheimer’s disease. In addition, a handful of studies have now emerged impli-cating PDE11A itself in human neuropsychiatric disease and neu-ropsychiatric medication responsiveness. Further, several papers suggest that decreased function of PDE11A may be a contributing factor in the pathophysiology associated with specific types of ad-renal hyperplasias and tumors. Second, a target should have limited function outside of the physiological process of interest and, third, knockout studies should predict a favorable side effect profile. In our own studies [19] as well as those of another laboratory [56], PDE11A KO mice were largely normal and healthy outside of the subtle phenotypes related to neurocognitive deficits and sperm ca-pacitance, respectively. In our studies, histopathology carried out to 1 year of age found no gross abnormalities in any tissue [19]. The fact that PDE11A knockout mice appear relatively normal is likely due to the restricted nature of PDE11A tissue expression, which leads up to property four. Forth, ideally, a target of interest will show minimal expression outside of the tissue of interest. This fea-ture is particularly critical for neuroscience drug targets. Because of the blood-brain-barrier, it is challenging to get drugs into the brain. Thus, quite often, drug levels are far higher in the periphery than they are in the brain. This drastically increases the potential for side effect liability related to peripheral organs. Thus, an ideal neurosci-ence drug target would show minimal to no expression outside of the brain. Even within th e brain, it is better that a target be as re-stricted as possible to the neural circuit of interest in order to avoid central side effects (e.g., seizure, sedation, etc.). In this regard, PDE11A ranks quite high. As noted above, we find PDE11A ex-pression within the brain is negligible outside of the hippocampal formation. Further, our own studies (Figs. 5 and 6) and a review of the literature (Table 1) suggests that relatively few tissues outside of the brain express PDE11A. Thus, we believe the rationale for PDE11A as a candidate drug target is growing in terms of both efficacy and potential side effect profiles. 8.2. Logistical Realities The final 3 properties that the pharmaceutical industry believes an ideal drug target should have are related to the logistics involved in running an actual drug discovery and development program. Fifth, the target should be ‘druggable’ and ‘assayable’. This means that it is important to have a functional endpoint that can be modu-lated by small molecules or biological entities and that can be measured in a relatively high-throughput assay for screening large scale libraries. Phosphodiesterases in general have been long pur-sued by the pharmaceutical industry because they are a very drug-gable and assayable family of enzymes. This is underscored by the fact that several PDE inhibitors targeting different PDE families have successfully made it to market (e.g., PDE5 inhibitors: silde-nafil, vardenafil, tadalafil; PDE4 inhibitor: roflumilast; PDE3 in-hibitors: cilostazol, milrinone; non-specific PDE inhibitors: theo-phylline and dipyrimadole) [3]. Indeed, as reviewed above, the Hoffman laboratory at Boston College recently published results of a medium-throughput screen that successfully identified PDE11A inhibitors, proving this PDE family is druggable and assayable [172]. Sixth, a biomarker should be available that can reliably ver-ify target engagement in the tissue of interest. It has been estab-lished in clinical trials that CSF cyclic nucleotide levels can be used as a surrogate marker to verify activ ity of PDE inhibitors in brain. Further, in patients with schizophrenia, deficits in cAMP produc-tion in platelets have correlated with symptom severity [107, 114] and olfactory deficits have been tied to cAMP dysfunction in the olfactory bulb [119]. Thus, either endpoint might prove a useful biomarker for selecting patients most likely to respond to a cAMP-targeted therapeutic, particularly in the case of schizophrenia. Measuring plasma cortisol might also prove a useful biomarker to validate target engagement in the adrenal gland of humans, as PDE11A inhibition increases cortisol/corticosterone production in cell-based assays [172]. Seventh, a target need s to have room to maneuver in terms of unique intellectual property space. Little chemical equity has been published to date for PDE11A. The fact that PDE11A is a realistic candidate for drug development increases the value of understanding its biological function. 9. LOOKING FORWARD Over the past 13 years we have learned much about PDE11A function, but many questions remain. Identifying in which tissues and cell types and at what age PDE11A functions will be key to considering how PDE11A can be pursued as a drug target. The \"when and where” of PDE11A function is particularly important with regards to its role in brain function, given that PDE11A ex-pression dramatically increases in the hippocampus throughout the lifespan [19, 50]. It will be of interest to future studies to determine if such age-related increases in expression extend to other organs in which PDE11A is expressed. Understanding the developmental epochs during which PDE11A function is most critical will impact whether PDE11A can be considered as a mechanism to reverse symptoms in adults or as a prophylactic mechanism to prevent de-velopment/onset of disease. It is equally critical that we gain an understanding of how phosphorylation (or other post-translational modifications) and/or oligomerization influence PDE11A localiza- Fig. (7). PDE5A, the PDE most closely related to PDE11A, is enriched in the cerebellum. Autoradiographic in situ hybridization conducted in 3-5 month-old rats and mice shows robust expression of PDE5A mRNA in the cerebellum, particularly in the Purkinje cell layer. A thionin stained section is shown to the left of the autoradiographs to show what structures are present at this level of the brain. Note, the hippocampus is included in these sections, but no staining of PDE5A mRNA appears in that region of the autoradiographs. Brightness and clarity adjusted to enhance graphical clarity. Mouse image enlarged to a greater extent than the rat image. Does Phosphodiesterase 11A (PDE11A) Hold Promise Current Pharmaceutical Design, 2015, Vol. 21, No. 3 411 tion and function. Protein-protein binding and post-translational modifications (e.g., palmitoylation) are the two key mechanisms by which PDEs are localized to subcellular compartments and, thus, cyclic nucleotide microdomains are created [4, 188, 189]. From a drug target identification perspective, GAF domains—the domains where protein-protein interactions occur—are highly interesting because they are found in no other mammalian proteins other than PDEs [3]. Understanding the function of the intramolecular signals that regulate PDE11A subcellular localization may enable more sophisticated drug development efforts that target specific localiza-tions of the enzyme instead of its total catalytic activity, which may become necessary as we learn more that only specific subcellular microdomains are affected by a given disease (e.g., [120]). Finally, we need to gain clarity on the signaling mechanisms that lie up-stream and downstream of PDE11A, and how downstream effects of PDE11A modulation may differ between acute and chronic ma-nipulations. For example, while chronic genetic deletion of PDE11A triggers a decrease in phosphorylation of the AMPA re-ceptor subunit GluR1 [19], the acute effects of PDE11A inhibitors on this endpoint have yet to be established. Clearly more studies examining PDE11A in patient tissue are needed to drive a therapeu-tic indication for a PDE11A modulator, but we believe the data to date strongly support the potential of PDE11A as a future therapeu-tic target. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS The authors would like to thank Mr. Christopher Roller and Ms. Geetanjali Pathak for technical assistance and Mr. Alexander Hart-man for helpful discussion, editorial feedback, and assistan ce with artwork for (Figs. 1 and 2). This work was funded by start-up funds from the University of South Carolina School of Medicine and a Research Starter Grant from the PhRMA Foundation (MPK). ABBREVIATIONS AA = Amino acids ABL = Ableson leukemia oncogene cellular ho-molog AC = Adenylyl cyclase AD = Alzheimer’s Disease AIMAH = ACTH-independent macronodular adrenal hyperplasia AMPA = Amino-3-hydroxy-5-methyl-4-isozazolepropionic AT = Adrenal tumor AUR = Aurora Kinase; B58C = British 1958 Birth Cohort BP = Base pairs BPD = Bipolar disorder CAMK = Ca2+/calmodulin-dependent protein kinases CBLM = Cerebellum CDK = Cyclin-dependent kinase CK1e- = Casein kinase 1, epsilon CRE = cAMP-response element CS = Cushing Syndrome CSK = Carboxyl-terminal Src kinase DHIPP = \"Dorsal-posterior” hippocampal formation DMPK = dystrophia myotonica protein kinase DYRK = Dual Specificity YAK1-Related Kinase E. ADIPOSE = Epididymal adipose EPH = Ephrin Kinase EPIDIDYM = Epididymis FHS = Framingham Heart Study GAF-B domain = cGMP binding PDE, Anabaena adenylyl cyclase and E. co li FhlA domain GASTROC = Gastrocnemius GFRs = Growth Factor Receptor Tyrosine Kinases GRK = G-protein coupled receptor kinase GSK = Glycogen synthase kinase hPDE11A4 = Purified human PDE11A4 HSPDE11A = Homo sapiens PDE11A gene JAK A = Janus-family tyrosine kin ase KIS = Kinase interacting with stathm in KO = Knockout L. INTEST = Large intestine LPS = Lipopolysaccharide M. ADIPOSE = Mesentery adipose MAPK = Mitogen-activated protein kinase MAS = McCune-Albright Syndrome MDD = Major depressive disorder MET = Mesenchymal epithelial transition factor transmembrane tyrosine kinase MP = Management program PC = Prostate cancer PDE11A = Phosphodiesterase 11A PDEs = Phosphodiesterases PEK = Pancreatic eukaryotic initiation factor 2a-subunit kinase PEMs = Peritoneal exudate macrophages PFC = Prefrontal cortex PIKK = Phosphatidylinositol 3-kinase-related pro-tein kinase PKA = Protein kinase A, a cAMP-activated kinase PKB = Protein kinase B (also known as AKT, which is not an acronym) PKC = Protein Kinase C PKG = Protein kinase G, a cGMP-activated kinase PLANT = Plantaris PPNAD = Primary pigmented nodular adrenocortical disease PT = Pituitary tumor RET = Rearranged during transfection tyrosine Kinase RSK = Ribosomal protein S6 kinase S. 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J Clin Endocrinol Metab 2011; 96: E135-40. Received: April 2, 2014 Accepted: August 25, 2014 Citations (29)References (201)... Four transcript isoforms designated PDE11A1-4, which result from different transcription initiation sites and alternative splicing, encode different PDE11A isoforms with unique N-terminal domains. Germline mutations, expression changes, and functional alterations in PDE11A have been linked to brain function, tumorigenesis, and in ammation [20,23,24]. For example, PDE11A is required for intact brain function across the lifespan [25], and studies have suggested that variations in PDE11A may be associated with bipolar disorder, depression and other psychiatric diseases [26,27]. ...... The p.Arg202His variant is only present in the PDE11A4 isoform, whereas the p.Leu756Gln variant is present in all PDE11A isoforms. PDE11A isoforms are expressed in a tissue-speci c manner [24]. In a previous study, Kelly et al. found that PDE11A4 is highly expressed in hippocampal areas but not in any of the other tissues examined. ...Two Novel PDE11A Genetic Variants Increase Tau Phosphorylations in Early-onset Alzheimer s DiseasePreprintFull-text availableAug 2020 Wei Qin Aihong ZhouXiumei ZuoJianping JiaBackground: Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and has become a major health issue. However, a large number of genetic risk factors remain undiscovered.Methods: To identify novel risk genes and better understand the molecular pathway underlying AD, whole-exome sequencing (WES) was performed in 215 early-onset AD (EOAD) patients and 55 unrelated healthy controls of Han Chinese ethnicity. Subsequent direct sequencing was performed in 4962 individuals to validate the selected rare mutations. Computational annotation and in vitro functional studies were performed to evaluate the role of candidate mutations in EOAD and the underlying mechanisms.Results: We identified two rare missense mutations in the phosphodiesterase 11A (PDE11A) gene, resulting in p.Arg202His, and p.Leu756Gln, in individuals with EOAD. Both mutations are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation, and classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at T181, S404, S202, S416, S214, S396 and AT8 epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cAMP levels, protein kinase A (PKA) activation, and cAMP response element-binding protein (CREB) phosphorylation. Additionally, pretreatment with a PKA inhibitor (H89) suppressed PDE11A mutation-induced p-Tau formation.Conclusions: Our results demonstrate that both PDE11A mutations and PDE11A knockdown increase Tau phosphorylation through the cAMP/PKA pathway, suggesting that PDE11A is a novel risk gene for AD. This study provides insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.ViewShow abstract... Four transcript isoforms designated PDE11A1-4, which result from different transcription initiation sites and alternative splicing, encode different PDE11A isoforms with unique N-terminal domains. Germline mutations, expression changes, and functional alterations in PDE11A have been linked to brain function, tumorigenesis, and in ammation [20,23,24]. For example, PDE11A is required for intact brain function across the lifespan [25], and studies have suggested that variations in PDE11A may be associated with bipolar disorder, depression and other psychiatric diseases [26,27]. ...... The p.Arg202His variant is only present in the PDE11A4 isoform, whereas the p.Leu756Gln variant is present in all PDE11A isoforms. PDE11A isoforms are expressed in a tissuespeci c manner [24]. In a previous study, Kelly et al. found that PDE11A4 is highly expressed in hippocampal areas but not in any of the other tissues examined. ...Two Novel Pde11a Genetic Variants Increase Tau Phosphorylations in Early-onset Alzheimer s DiseasePreprintFull-text availableJun 2020 Wei Qin Aihong ZhouXiumei Zuo Jianping JiaBackground: Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and has become a major health issue. However, a large number of genetic risk factors remain undiscovered.Methods: To identify novel risk genes and better understand the molecular pathway underlying AD, whole-exome sequencing (WES) was performed in 215 early-onset AD (EOAD) patients and 55 unrelated healthy controls of Han Chinese ethnicity. Subsequent direct sequencing was performed in 620 individuals to validate the selected rare mutations. Computational annotation and in vitro functional studies were performed to evaluate the role of candidate mutations in EOAD and the underlying mechanisms.Results: We identified two rare missense mutations in the phosphodiesterase 11A (PDE11A) gene, resulting in p.Arg202His, and p.Leu756Gln, in 4 individuals with EOAD. Both mutations are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation, and classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at T181, S404, S202, S416, S214, S396 and AT8 epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cAMP levels, protein kinase A (PKA) activation, and cAMP response element-binding protein (CREB) phosphorylation. Additionally, pretreatment with a PKA inhibitor (H89) suppressed PDE11A mutation-induced p-Tau formation.Conclusions: Our results demonstrate that both PDE11A mutations and PDE11A knockdown increase Tau phosphorylation through the cAMP/PKA pathway, suggesting that PDE11A is a novel risk gene for AD. This study provides insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.ViewShow abstract... The PDE11 family of cyclic nucleotide PDEs is also a dual substrate family hydrolyzing both cAMP and cGMP and is encoded by the PDE11A gene (Beavo, 1995;Kelly, 2017). PDE11A contains four different isoforms (PDE11A1-4) of which PDE11A4 is highly expressed in the ventral hippocampal formation (Kelly, 2015) and low levels are noted in the dorsal hippocampus, spinal cord, and dorsal root ganglion (Kelly, 2018b). Outside of the nervous system, PDE11A expression appears to be sparse (Kelly, 2015). ...... PDE11A contains four different isoforms (PDE11A1-4) of which PDE11A4 is highly expressed in the ventral hippocampal formation (Kelly, 2015) and low levels are noted in the dorsal hippocampus, spinal cord, and dorsal root ganglion (Kelly, 2018b). Outside of the nervous system, PDE11A expression appears to be sparse (Kelly, 2015). PDE11A4 is the only PDE whose expression in the brain originates solely from the hippocampal formation (Kelly et al., 2014). ...Genetic manipulation of cyclic nucleotide signaling during hippocampal neuroplasticity and memory formationArticleFull-text availableApr 2020PROG NEUROBIOL M.P. KellyPim R.A. Heckman Robbert HavekesDecades of research have underscored the importance of cyclic nucleotide signaling in memory formation and synaptic plasticity. In recent years, several new genetic techniques have expanded the neuroscience toolbox, allowing researchers to measure and modulate cyclic nucleotide gradients with high spatiotemporal resolution. Here, we will provide an overview of studies using genetic approaches to interrogate the role cyclic nucleotide signaling plays in hippocampus-dependent memory processes and synaptic plasticity. Particular attention is given to genetic techniques that measure real-time changes in cyclic nucleotide levels as well as newly-developed genetic strategies to transiently manipulate cyclic nucleotide signaling in a subcellular compartment-specific manner with high temporal resolution.ViewShow abstract... Proof of principle for such an approach first emerged with studies using dominant-negative PDEs (DN-PDEs), which are catalytically inactive mutants that would displace their endogenous PDE. Using specific DN-PDE4 isoforms, in vitro studies have successfully altered perinuclear cAMP signalling 240 , β-arrestin-dependent desensitization of the β2-adrenergic receptor 241,242 , growth control of prostate cancer cells 193 , prostanoid receptor-mediated cAMP signalling 243 , glucagon-like peptide-1 release 244 and cAMP gradients at the centrosome 245 . DN-PDE4 tools have also yielded beneficial effects in vivo. ...... Brain (hippocampal formation), spinal cord dorsal horn (specifically in somatostatin + neurons) and dorsal root ganglia: PDE11A4 Brain: neuronal cell bodies, dendrites and axons. ~50% in cytosol, ~25% in nuclear fractions and ~25% between the soluble and Seminal vesicles, salivary glands pancreas, pituitary and liver: PDE11A (isoform not specified) (7,8,36,(243)(244)(245) [Note: reports of tissue expression highly inconsistent, c.f., (36). Most reliable included] insoluble membranes (no expression in glia) ...Therapeutic targeting of 3′,5′-cyclic nucleotide phosphodiesterases: inhibition and beyondArticleAug 2019Nat Rev Drug Discov George S BaillieGonzalo S. Tejeda M.P. KellyPhosphodiesterases (PDEs), enzymes that degrade 3 ,5 -cyclic nucleotides, are being pursued as therapeutic targets for several diseases, including those affecting the nervous system, the cardiovascular system, fertility, immunity, cancer and metabolism. Clinical development programmes have focused exclusively on catalytic inhibition, which continues to be a strong focus of ongoing drug discovery efforts. However, emerging evidence supports novel strategies to therapeutically target PDE function, including enhancing catalytic activity, normalizing altered compartmentalization and modulating post-translational modifications, as well as the potential use of PDEs as disease biomarkers. Importantly, a more refined appreciation of the intramolecular mechanisms regulating PDE function and trafficking is emerging, making these pioneering drug discovery efforts tractable.ViewShow abstract... Interestingly, PDE4A/B/D isozymes exhibit both distinct patterns of distribution in the brain and differences in subcellular localization. Gene knock-out (KO) studies in mice support their differential roles in behavior, including anxiety versus memory [42][43][44]. ...Selective inhibition of PDE4B Reduces Binge Drinking in Two C57BL/6 SubstrainsArticleFull-text availableMay 2021 Christopher Leonardo Jimenez Chavez Camron D Bryant Melissa A Munn-Chernoff Karen K SzumlinskiCyclic AMP (cAMP)-dependent signaling is highly implicated in the pathophysiology of alcohol use disorder (AUD), with evidence supporting the efficacy of inhibiting the cAMP hydro-lyzing enzyme phosphodiesterase 4 (PDE4) as a therapeutic strategy for drinking reduction. Off-target emetic effects associated with non-selective PDE4 inhibitors has prompted the development of selective PDE4 isozyme inhibitors for treating neuropsychiatric conditions. Herein, we examined the effect of a selective PDE4B inhibitor A33 (0-1.0 mg/kg) on alcohol drinking in both female and male mice from two genetically distinct C57BL/6 substrains. Under two different binge-drinking procedures, A33 pretreatment reduced alcohol intake in male and female mice of both substrains. In both drinking studies, there was no evidence for carry-over effects the next day; however, we did observe some sign of tolerance to A33′s effect on alcohol intake upon repeated, intermittent, treatment (5 injections of 1.0 mg/kg, every other day). Pretreatment with 1.0 mg/kg of A33 augmented sucrose intake by C57BL/6NJ, but not C57BL/6J, mice. In mice with a prior history of A33 pretreat-ment during alcohol-drinking, A33 (1.0 mg/kg) did not alter spontaneous locomotor activity or ba-sal motor coordination, nor did it alter alcohol s effects on motor activity, coordination or sedation. In a distinct cohort of alcohol-naïve mice, acute pretreatment with 1.0 mg/kg of A33 did not alter motor performance on a rotarod and reduced sensitivity to the acute intoxicating effects of alcohol. These data provide the first evidence that selective PDE4B inhibition is an effective strategy for reducing excessive alcohol intake in murine models of binge drinking, with minimal off-target effects. Despite reducing sensitivity to acute alcohol intoxication, PDE4B inhibition reduces binge alcohol drinking, without influencing behavioral sensitivity to alcohol in alcohol-experienced mice. Furthermore, A33 is equally effective in males and females and exerts a quantitatively similar reduction in alcohol intake in mice with a genetic predisposition for high versus moderate alcohol preference. Such findings further support the safety and potential clinical utility of targeting PDE4 for treating AUD.ViewShow abstract... The p.Arg202His variant is only present in the PDE11A4 isoform, whereas the p.Leu756Gln variant is present in all PDE11A isoforms. PDE11A isoforms are expressed in a tissue-specific manner (26). In a previous study, Kelly (27) found that PDE11A4 is highly expressed in hippocampal areas but not in any of the other tissues examined. ...Exome sequencing revealed PDE11A as a novel candidate gene for early-onset Alzheimer s diseaseArticleFull-text availableApr 2021HUM MOL GENET Wei Qin Aihong ZhouXiumei Zuo Jianping JiaTo identify novel risk genes and better understand the molecular pathway underlying Alzheimer’s disease (ad), whole-exome sequencing (WES) was performed in 215 early-onset ad (EOAD) patients and 255 unrelated healthy controls of Han Chinese ethnicity. Subsequent validation, computational annotation and in vitro functional studies were performed to evaluate the role of candidate variants in EOAD. We identified two rare missense variants in the phosphodiesterase 11A (PDE11A) gene in individuals with EOad. Both variants are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation, and are classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of ad patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at multiple epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cAMP levels, protein kinase A (PKA) activation, and cAMP response element-binding protein (CREB) phosphorylation. Additionally, pretreatment with a PKA inhibitor (H89) suppressed PDE11A variant-induced Tau phosphorylation formation. This study offers insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.ViewShow abstract... Studies of adrenal, prostate and testicular cancer have suggested that PDE11A variants may represent susceptibility modifiers rather than direct and sufficient causes of these neoplasms [63]. This gene may play a key role also in spermatogenesis and fertilization potential, as suggested by observation that Pde11a knockout mice displayed reduced sperm concentration, rate of forward progression, percentage of live spermatozoa and increased premature/ spontaneous capacitance [17]. ...PDE11A gene polymorphism in testicular cancer: sperm parameters and hormonal profileArticleFull-text availableMar 2021J Endocrinol InvestigFabiana Faja Federica FinocchiT. Carlini Francesco LombardoPurposeTesticular germ cell tumours (TGCTs) is the most common malignancy among young adult males. The etiology is multifactorial and both environmental and genetic factors play an important role in the origin and development of TGCT. Genetic susceptibility may result from the interaction of multiple common and low-penetrance genetic variants and one of the main candidate genes is PDE11A . Many PDE11A polymorphisms were found responsible for a reduced PDE activity in TGCT patients, who often also display impaired hormone and sperm profile. The aim of this study was to investigate testicular function and PDE11A sequence in testicular cancer cases.MethodsSemen analysis was performed in 116 patients with unilateral and bilateral sporadic TGCTs and in 120 cancer-free controls. We also investigated hormone profile and PDE11A polymorphisms using peripheral blood samples.ResultsOur data revealed that TGCT patients showed lower testosterone levels, higher gonadotropins levels and worse semen quality than controls, although the mean and the medians of sperm parameters are within the reference limits. PDE11A sequencing detected ten polymorphisms not yet associated with TGCTs before. Among these, G223A in homozygosity and A288G in heterozygosity were significantly associated with a lower risk of testicular tumour and they displayed a positive correlation with total sperm number.ConclusionsOur findings highlight the key role of PDE11A in testis and suggest the presence of an underlying complex and fine molecular mechanism which controls testis-specific gene expression and susceptibility to testicular cancer.ViewShow abstract... The p.Arg202His variant is only present in the PDE11A4 isoform, whereas the p.Leu756Gln variant is present in all PDE11A isoforms. PDE11A isoforms are expressed in a tissue-speci c manner [26]. In a previous study, Kelly et al. found that PDE11A4 is highly expressed in hippocampal areas but not in any of the other tissues examined. ...Exome Sequencing Revealed PDE11A as a Novel Candidate Gene for Early-Onset Alzheimer s diseasePreprintFull-text availableNov 2020 Wei Qin Aihong ZhouXiumei Zuo Jianping JiaBackground: Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and has become a major health issue. However, a large number of genetic risk factors remain undiscovered.Methods: To identify novel risk genes and better understand the molecular pathway underlying AD, whole-exome sequencing (WES) was performed in 215 early-onset AD (EOAD) patients and 55 unrelated healthy controls of Han Chinese ethnicity. Subsequent direct sequencing was performed in 4962 individuals to validate the selected rare mutations. Computational annotation and in vitro functional studies were performed to evaluate the role of candidate mutations in EOAD and the underlying mechanisms.Results: We identified two rare missense mutations in the phosphodiesterase 11A (PDE11A) gene, resulting in p.Arg202His, and p.Leu756Gln, in individuals with EOAD. Both mutations are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation, and classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at T181, S404, S202, S416, S214, S396 and AT8 epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cAMP levels, protein kinase A (PKA) activation, and cAMP response element-binding protein (CREB) phosphorylation. Additionally, pretreatment with a PKA inhibitor (H89) suppressed PDE11A mutation-induced p-Tau formation.Conclusions: Our results demonstrate that both PDE11A mutations and PDE11A knockdown increase Tau phosphorylation through the cAMP/PKA pathway, suggesting that PDE11A is a novel risk gene for AD. This study provides insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.ViewShow abstract... In the dorsal horn, cyclic nucleotide signaling contributes to pain-related neuronal plasticity, and other members of the phosphodiesterase family have been directly implicated in the pathogenesis of inflammatory and neuropathic pain (Kallenborn-Gerhardt et al., 2014). Previous studies have detected Pde11a transcript and PDE11A protein in the bulk analyses of the spinal cord, but its cellular expression pattern is unknown (Kelly, 2015). Accordingly, we selected Pde11a for further examination. ...Transcriptional Profiling of Somatostatin Interneurons in the Spinal Dorsal HornPreprintFull-text availableNov 2017 Alexander ChamessianMichael Young Yawar J QadriThomas Van de VenThe spinal dorsal horn (SDH) is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain. Identifying the transcriptomic profile of SST neurons could elucidate the unique genetic features of this population and enable selective analgesic targeting. To that end, we combined the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) method and Fluorescence Activated Nuclei Sorting (FANS) to capture tagged SST nuclei in the SDH of adult male mice. Using RNA-sequencing (RNA-seq), we uncovered more than 13,000 genes. Differential gene expression analysis revealed more than 900 genes with at least 2-fold enrichment. In addition to many known dorsal horn genes, we identified and validated several novel transcripts from pharmacologically tractable functional classes: Carbonic Anhydrase 12 (Car12), Phosphodiesterase 11A (Pde11a), Protease-Activated Receptor 3 (F2rl2) and G-protein Coupled Receptor 26 (Gpr26). In situ hybridization of these novel genes revealed differential expression patterns in the SDH, demonstrating the presence of transcriptionally distinct subpopulations within the SST population. Pathway analysis revealed several enriched signaling pathways including cyclic AMP-mediated signaling, Nitric Oxide Synthase signaling, and voltage-gated calcium channels, highlighting the importance of these pathways to SST neuron function. Overall, our findings provide new insights into the gene repertoire of SST dorsal horn neurons and reveal several candidate targets for pharmacological modulation of this pain-mediating population.ViewShow abstract... PDE11A is an enzyme uniquely enriched in the hippocampus that breaks down cyclic AMP and cyclic GMP equally well. Some studies 49,50 show that phosphodiesterase genes are strongly linked with vulnerability to antidepressant treatment response and major depression disorder. PDE11A KO mice also exhibit significantly higher levels of the pro-inflammatory cytokine IL-6 relative to BALB/ cJ and WT mice, respectively. ...Understanding the genetic aspects of resistance to antidepressants treatmentArticleFull-text availableJul 2020EUR REV MED PHARMACO Ali M Alqahtani Chidambaram KumarappanV KumarR SrinivasanMajor depression disorder (MDD) is an extremely prevalent disorder and is expected to be the second leading cause of disease burden by 2020 according to the World Health Organisation (WHO). Moreover, this disease burden is predicted to rise in the next 20 years. Antidepressant medications are vital in the therapy of major depression. However, approximately 30-60% of patients treated with current antidepressant drugs fail to attain remission of depressive symptoms leading to drug resistance. Such patients account for a disproportionately great burden of disease, as supported by cost, augmented disability, and suicidal incidents. Antidepressants resistance remains to challenge mental health care professionals, and more relevant research relating newer medications is necessitated to enhance the quality of life of patients with depression. Enhancement in response rates continue the major challenge in antidepressant research, thus a wealth of potentials still exists concerning the antide-pressant resistance for the management of major depression. However, the mechanisms causing resistance to antidepressant treatment remain unknown. Hence, clinical and basic research in understanding the fundamental mechanism of antidepressant resistance should remain a key priority. One potential source accounting for these differences in treatment outcome is genetic variations. The pharmacological mechanisms behind antidepressant response are only partly known but genetic factors play a significant role. Future research of risk factors should assist to advance the understanding of the mechanisms underlying drug resistance in mood disorders and contribute to progress their therapeutic management. Thus, psychiatrists could rely on more effective approaches to treat depressive episodes, reducing the incidence of further drug resistance. This review critically summarises the author s view on many aspects of treatment resistance, specific genetic biomarkers, potential strategies and clinical relevance from both clinical and preclini-cal studies in drug resistance to antidepressant therapies. Finally, this will allow us to suggest possible recommendations and innovative treatment strategies improve therapeutic outcomes in managing antidepressant resistance.ViewShow abstractShow more.Cyclic GMP in the CSF of patients with schizophrenia before and after neuroleptic treatmentArticleFull-text availableDec 1976Psychopharmacology Richard P EbsteinJ BiedermanRanan Rimón Robert H BelmakerCerebrospinal fluid (CSF) cyclic GMP may derive from central cholinergic neurotransmission. Measurement of CSF cyclic GMP may allow evaluation of possible implications of the dopaminergic hyperactivity in schizophrenia proposed by the dopamine hypothesis. The CSF cyclic GMP levels in 27 drug-free schizophrenic patients was measured and compared to that in 9 psychiatrically-healthy individuals. The mean CSF cyclic GMP level of the schizophrenic patients was 23 per cent lower than that of the control group, but this difference did not attain statistical significance. In addition the CSF cyclic GMP levels in a group of 10 schizophrenic patients were compared before and after 2 months of neuroleptic treatment. The mean level of cyclic GMP rose 50 per cent after treatment with phenothiazines (P less than 0.05). These results could indicate some tendency for decreased activity of central cholinergic neurons in schizophrenia as well as a restored dopaminergic-cholinergic balance after neuroleptic treatment.ViewShow abstractGenetic risk factors in two Utah pedigrees at high risk for suicideArticleFull-text availableNov 2013 Hilary Coon Todd Darlington Richard Pimentel Douglas D GrayWe have used unique population-based data resources to identify 22 high-risk extended pedigrees that show clustering of suicide over twice that expected from demographically adjusted incidence rates. In this initial study of genetic risk factors, we focused on two high-risk pedigrees. In the first of these (pedigree 12), 10/19 (53%) of the related suicides were female, and the average age at death was 30.95. In the second (pedigree 5), 7/51 (14%) of the suicides were female and the average age at death was 36.90. Six decedents in pedigree 12 and nine in pedigree 5 were genotyped with the Illumina HumanExome BeadChip. Genotypes were analyzed using the Variant Annotation, Analysis, and Search program package that computes likelihoods of risk variants using the functional impact of the DNA variation, aggregative scoring of multiple variants across each gene and pedigree structure. We prioritized variants that were: (1) shared across pedigree members, (2) rare in other Utah suicides not related to these pedigrees, (3)  5% in genotyping data from 398 other Utah population controls and (4) 5% frequency in publicly available sequence data from 1358 controls and/or in dbSNP. Results included several membrane protein genes (ANO5, and TMEM141 for pedigree 12 and FAM38A and HRCT1 for pedigree 5). Other genes with known neuronal involvement and/or previous associations with psychiatric conditions were also identified, including NFKB1, CASP9, PLXNB1 and PDE11A in pedigree 12, and THOC1, and AUTS2 in pedigree 5. Although the study is limited to variants included on the HumanExome BeadChip, these findings warrant further exploration, and demonstrate the utility of this high-risk pedigree resource to identify potential genes or gene pathways for future development of targeted interventions.ViewShow abstractMutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complexArticleFull-text availableSep 2000J CLIN INVESTMairead Casey Carl VaughanJie HeCraig T BassonCardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.ViewShow abstractExploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivativesArticleFull-text availableSep 2012EUR J MED CHEM Nermin Salah Amal AliShreen M El-Nashar Ashraf H. AbadiViewNeuroanatomy of Social Behaviour: An Evolutionary and Psychoanalytic PerspectiveBookApr 2018Ralf-Peter BehrendtViewMutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complexArticleJan 2001J CLIN INVESTMairead Casey Carl VaughanJie HeCraig T BassonViewReduced cAMP-signal transduction in postmortem hippocampus of demented old people.ArticleJan 1988T G Ohm Jürgen Bohl Björn LemmerViewSelect 3 ,5 -cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brainArticleOct 2013 M.P. Kelly Wendy O Adamowicz Susan E Bove Robin J KleimanViewCyclicAMP production by polymorphonuclear leukocytes in psychiatric disordersArticleFeb 1989BIOL PSYCHIATPhilip D. Kanof Emil F CoccaroC A JohnsKenneth L. DavisThe cyclic adenosine monophosphate (cAMP) responses to histamine, prostaglandin-E1, and isoproterenol in polymorphonuclear leukocytes from drug-free normal controls and patients with schizophrenia or major depressive disorder were compared. These three groups of subjects did not differ in their cAMP responses to receptor activation. Exacerbated and remitted patients with either schizophrenia or major depressive disorder did not differ in their cAMP responses. The data indicate that in polymorphonuclear leukocytes, the cAMP responses to activation of histamine H2, prostaglandin-E1, or betaadrenergic receptors are neither state-independent nor state-dependent markers for schizophrenia or major depressive disorder.ViewShow abstractMedical Treatment of Cushing s DiseaseArticleJan 2013 Richard A Feelders Leo HoflandContext: Cushing s disease (CD) is associated with serious morbidity and, when suboptimally treated, an increased mortality. Although surgery is the first-line treatment modality for CD, hypercortisolism persists or recurs in an important subset of patients. Considering the deleterious effects of uncontrolled CD, there is a clear need for effective medical therapy.Objective: In this review, we discuss molecular targets for medical therapy, efficacy, and side effects of the currently used drugs to treat hypercortisolism and focus on recent developments resulting from translational and clinical studies.Evidence acquisition: Selection of publications related to the study objective was performed via a PubMed search using relevant keywords and search terms.Main findings: Medical therapy for CD can be classified into pituitary-directed, adrenal-blocking, and glucocorticoid receptor-antagonizing drugs. Recent studies demonstrate that somatostatin receptor subtype 5 (sst(5)) and dopamine receptor subtype 2 (D(2)) are frequently (co-)expressed by corticotroph adenomas. Pituitary-directed therapy with pasireotide and cabergoline, targeting sst(5) and D(2), respectively, is successful in approximately 25-30% of patients. Adrenal-blocking drugs can be effective by inhibiting steroidogenic enzyme activity. Finally, the glucocorticoid receptor antagonist mifepristone induces clinical and metabolic improvement in the majority of patients. Each drug can have important side effects that may impair long-term treatment. Generally, patients with moderate to severe hypercortisolism need combination therapy to normalize cortisol production.Conclusion: Medical therapy for CD can be targeted at different levels and should be tailored in each individual patient. Future studies should examine the optimal dose and combination of medical treatment modalities for CD.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectcAMP Signaling, PKA and Hippocampal Function Ted Abel Robbert Havekes Steven J Siegel[...]Kevin MerkelEach neuron contains thousands of synapses, each of which needs to be independently modifiable by experience. Work in the Abel lab suggests that localized signaling mediated by scaffolding proteins generates synapse-specific activation of biochemical pathways that mediate long-term memory storage. Beginning as a postdoctoral fellow, Dr. Abel pioneered genetic approaches to define the role of the cAMP signaling and protein kinase A in hippocampal synaptic plasticity and memory. More recently, his lab has focused on spatial compartmentalization of PKA signaling via a large family of A-kinase anchoring proteins (AKAPs), which restrict PKA and other signaling molecules to specific subcellular locations such as the plasma membrane where they interact with receptors, adenylyl cyclases, and ion channels. This research includes computational approaches to model spatially restricted signaling along with genetic, imaging and pharmacological approaches. ... [more]View projectChapterA Role for Phosphodiesterase 11A (PDE11A) in the Formation of Social Memories and the Stabilization...September 2017 · Advances in Neurobiology M.P. KellyThe most recently discovered 3′,5′-cyclic nucleotide phosphodiesterase family is the Phosphodiesterase 11 (PDE11) family, which is encoded by a single gene PDE11A. PDE11A is a dual-specific PDE, breaking down both cAMP and cGMP. There are four PDE11A splice variants (PDE11A1-4) with distinct tissue expression profiles and unique N-terminal regulatory regions, suggesting that each isoform could be ... [Show full abstract] individually targeted with a small molecule or biologic. PDE11A4 is the PDE11A isoform expressed in brain and is found in the hippocampal formation of humans and rodents. Studies in rodents show that PDE11A4 mRNA expression in brain is, in fact, restricted to the hippocampal formation (CA1, possibly CA2, subiculum, and the adjacently connected amygdalohippocampal area). Within the hippocampal formation of rodents, PDE11A4 protein is expressed in neurons but not astrocytes, with a distribution across nuclear, cytoplasmic, and membrane compartments. This subcellular localization of PDE11A4 is altered in response to social experience in mouse, and in vitro studies show the compartmentalization of PDE11A4 is controlled, at least in part, by homodimerization and N-terminal phosphorylation. PDE11A4 expression dramatically increases in the hippocampus with age in the rodent hippocampus, from early postnatal life to late aging, suggesting PDE11A4 function may evolve across the lifespan. Interestingly, PDE11A4 protein shows a three to tenfold enrichment in the rodent ventral hippocampal formation (VHIPP; a.k.a. anterior in primates) versus dorsal hippocampal formation (DHIPP). Consistent with this enrichment in VHIPP, studies in knockout mice show that PDE11A regulates the formation of social memories and the stabilization of mood and is a critical mechanism by which social experience feeds back to modify the brain and subsequent social behaviors. PDE11A4 likely controls behavior by regulating hippocampal glutamatergic, oxytocin, and cytokine signaling, as well as protein translation. Given its unique tissue distribution and relatively selective effects on behavior, PDE11A may represent a novel therapeutic target for neuropsychiatric, neurodevelopmental, or age-related disorders. Therapeutically targeting PDE11A4 may be a way to selectively restore aberrant cyclic nucleotide signaling in the hippocampal formation while leaving the rest of the brain and periphery untouched, thus, relieving deficits while avoiding unwanted side effects.Read moreChapterFull-text availablePDE11AJanuary 2016 M.P. KellyView full-textArticleFull-text availablePDE11A negatively regulates lithium responsivitySeptember 2016 · Molecular Psychiatry M.P. Kelly Gautam pathak PathakM.J. Agostino[...]K BisharaLithium responsivity in patients with bipolar disorder has been genetically associated with Phosphodiesterase 11A (PDE11A), and lithium decreases PDE11A mRNA in induced pluripotent stem cell-derived hippocampal neurons originating from lithium-responsive patients. PDE11 is an enzyme uniquely enriched in the hippocampus that breaks down cyclic AMP and cyclic GMP. Here we determined whether ... [Show full abstract] decreasing PDE11A expression is sufficient to increase lithium responsivity in mice. In dorsal hippocampus and ventral hippocampus (VHIPP), lithium-responsive C57BL/6J and 129S6/SvEvTac mice show decreased PDE11A4 protein expression relative to lithium-unresponsive BALB/cJ mice. In VHIPP, C57BL/6J mice also show differences in PDE11A4 compartmentalization relative to BALB/cJ mice. In contrast, neither PDE2A nor PDE10A expression differ among the strains. The compartment-specific differences in PDE11A4 protein expression are explained by a coding single-nucleotide polymorphism (SNP) at amino acid 499, which falls within the GAF-B homodimerization domain. Relative to the BALB/cJ 499T, the C57BL/6J 499A decreases PDE11A4 homodimerization, which removes PDE11A4 from the membrane. Consistent with the observation that lower PDE11A4 expression correlates with better lithium responsiveness, we found that Pde11a knockout mice (KO) given 0.4% lithium chow for 3+ weeks exhibit greater lithium responsivity relative to wild-type (WT) littermates in tail suspension, an antidepressant-predictive assay, and amphetamine hyperlocomotion, an anti-manic predictive assay. Reduced PDE11A4 expression may represent a lithium-sensitive pathophysiology, because both C57BL/6J and Pde11a KO mice show increased expression of the pro-inflammatory cytokine interleukin-6 (IL-6) relative to BALB/cJ and PDE11A WT mice, respectively. Our finding that PDE11A4 negatively regulates lithium responsivity in mice suggests that the PDE11A SNPs identified in patients may be functionally relevant.View full-textArticleA phosphodiesterase 11 (Pde11a) knockout mouse expressed functional but reduced Pde11a: Phenotype an...October 2020 · Molecular and Cellular EndocrinologyIsaac LevyEva Szarek Andrea Gutierrez Maria[...] Constantine StratakisPhosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cyclic adenosine and guanosine mono-phosphate (cAMP and cGMP, respectively). Increased cAMP signaling has been associated with adrenocortical tumors and Cushing syndrome. Genetic defects in phosphodiesterase 11A (PDE11A) may lead to increased cAMP signaling and have ... [Show full abstract] been found to predispose to the development of adrenocortical, prostate, and testicular tumors. A previously reported Pde11a knockout (Pde11a-/-) mouse line was studied and found to express PDE11A mRNA and protein still, albeit at reduced levels; functional studies in various tissues showed increased cAMP levels and reduced PDE11A activity. Since patients with PDE11A defects and Cushing syndrome have PDE11A haploinsufficiency, it was particularly pertinent to study this hypomorphic mouse line. Indeed, Pde11a-/- mice failed to suppress corticosterone secretion in response to low dose dexamethasone, and in addition exhibited adrenal subcapsular hyperplasia with predominant fetal-like features in the inner adrenal cortex, mimicking other mouse models of increased cAMP signaling in the adrenal cortex. We conclude that a previously reported Pde11a-/- mouse showed continuing expression and function of PDE11A in most tissues. Nevertheless, Pde11a partial inactivation in mice led to an adrenocortical phenotype that was consistent with what we see in patients with PDE11A haploinsufficiency.Read moreArticleIdentification of Biologically Active PDE11-Selective Inhibitors Using a Yeast-Based High-Throughput...January 2012Ozge Ceyhan Kivanc Birsoy Charles S HoffmanThe biological roles of cyclic nucleotide phosphodiesterase 11 (PDE11) enzymes are poorly understood, in part due to the lack of selective inhibitors. To address the need for such compounds, we completed an ~200,000 compound high-throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay, and identified 4 potent and selective PDE11 inhibitors. One compound, along with two ... [Show full abstract] structural analogs, elevates cAMP and cortisol levels in human adrenocortical cells, consistent with gene association studies that link PDE11 activity to adrenal function. As such, these compounds can immediately serve as chemical tools to study PDE11 function in cell culture, and as leads to develop therapeutics for the treatment of adrenal insufficiencies. Our results further validate this yeast-based HTS platform for the discovery of potent, selective, and biologically active PDE inhibitors.Read moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? 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