Elsevier

Cellular Signalling

Volume 26, Issue 2, February 2014, Pages 383-397
Cellular Signalling

Select 3′,5′-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain

https://doi.org/10.1016/j.cellsig.2013.10.007Get rights and content

Highlights

  • Each PDE exhibits a unique expression pattern in brain that is highly conserved.

  • PDE11A, PDE8A, and PDE1C increase and PDE7A, PDE4D, and PDE1B decrease with age.

  • Age-related changes in PDEs may affect pGluR1, pCaMKII, TARPs and H3K27me3 signals.

  • Thus, aging is associated with changes in PDE expression and compartmentalization.

  • These results suggest the effects of certain PDE inhibitors may change with age.

Abstract

3′,5′-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26 months) versus young (5 months) Sprague–Dawley rats, with select experiments replicated in old (9 months) versus young (2 months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.

Introduction

Cyclic nucleotides (cAMP and cGMP) are critical intracellular signaling molecules that are required for the proper development and function of the brain (c.f., [1], [2], [3], [4]). Altered cyclic nucleotide signaling has been described in the aged brain [5], [6], [7], [8], [9], [10] and in patients with Alzheimer's disease (AD) [11], [12], [13], [14], [15], [16], [17], [18], [19]. Therapeutic drug targets that selectively restore aberrant cyclic nucleotide signaling (i.e., in brain regions affected by disease) without affecting normal cyclic nucleotide signaling (i.e., in brain regions or the periphery unaffected by disease) may relieve disease-related symptoms without causing unwanted side effects. As such, identification of molecules within the cyclic nucleotide signaling cascades that exhibit differential expression patterns across brain regions is of critical therapeutic importance.

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 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) [20], [21], [22]. PDEs are the only known enzymes to degrade cyclic nucleotides, making them integral regulators of intracellular signaling [23], [24]. There are 11 families of PDEs (PDE1–11) that are encoded by 21 genes [23]. Each of the 21 PDE isoforms shows a unique tissue expression profile across the body, making each highly druggable candidate a unique target capable of modulating separable physiological processes [21]. Thus, selectively targeting PDEs with small molecules or biologics may be one way to modulate cyclic nucleotides in a brain region-specific manner.

Numerous PDE inhibitors are currently under development by the pharmaceutical industry across multiple indications, particularly for cognitive decline associated with aging and/or neuropsychiatric diseases such as Alzheimer's disease [3], [25]. Despite the interest in PDEs as drug targets, very little has been done to characterize how the expression of PDE isoforms may change across the lifespan. A handful of studies have reported changes in expression of select PDEs early in development (e.g., PDE2A, PDE4A, PDE5A, PDE9A, and PDE11A) [26], [27], [28]. Behavioral pharmacology studies suggest these dynamic changes in PDE expression may continue late into adulthood. For example, PDE4 inhibition improves prefrontal cortex-dependent working memory in young monkeys while impairing it in old monkeys [25], [29]. This age-dependent effect of PDE4 inhibition on working memory in primate appears to be related to an increase in cAMP-mediated disinhibition of specific prefrontal cortex neurons [29] that may, in part, be related to decreases in cortical PDE4 expression that occur with age [30], [31]. Thus, if we are to harness the therapeutic potential of PDEs, we must first understand the “when and where” of PDE expression.

Expression patterns across brain regions have been independently reported for most PDE families using various techniques (recently reviewed in detail elsewhere, [3], [32]), but only one study to date has conducted a head to head comparison of all PDE families using the same technique [33]. Lakics et al. [33] mapped mRNA expression of each PDE isoform across several human brain regions and human peripheral tissues using quantitative reverse transcription-polymerase chain reaction (qPCR). This study confirmed that although any given tissue expresses more than one PDE isoform, no two PDE isoforms are expressed in the exact same pattern across tissues. Unfortunately, qPCR does not offer insight into subregional distribution (e.g., cell-layer specific distribution in cortex), and it is not clear if the expression pattern identified in human tissues extends to preclinical species most often used to study phosphodiesterase biology (i.e., mice and rats). As such, we build upon the observations of Lakics and colleagues by using qPCR and in situ hybridization to comparatively map mRNA expression of mouse and rat PDE isoforms across the central nervous system. We reveal that PDE expression patterns in rodent brain are largely consistent with those previously reported in human. In addition, we show for the first time that select PDE isoforms exhibit brain region-specific changes in expression from early to late adulthood that correlate with alterations in downstream signaling events. Together, our results provide novel insights into brain region-specific and age-related differences in the compartmentalization of cyclic nucleotide signaling.

Section snippets

Subjects

Both mice and rats were used for studies herein. Male CD-1 (n = 6, for PCR) and CF-1 mice (n = 4, for in situ hybridization) (Charles River, Wilmington, MA) were ordered at 8–10 weeks of age and allowed to acclimate in-house for 1 week prior to tissue collection. Male and female BALB/cJ mice (n = 18 for Western blots) were bred onsite with breeders obtained from Jackson Laboratories (Bar Harbor, ME). Matings were staggered such that 9 month-old (“old”) and 7–8 week-old BALB/cJ mice (“young”) could be

Mapping PDE expression in the central nervous system of rodents

To directly compare expression of multiple PDE isoforms across various brain regions and the spinal cord, we used qPCR (RQ and StdC in mice and RQ in rats) and in situ hybridization (Fig. 1, Fig. 2, Fig. 3, Fig. 4). All probes were designed to detect all splice variants for a single PDE gene (see Methods). In general, semi-quantitative results obtained by qPCR-RQ (Fig. 1), qualitative results obtained by in situ hybridization (Fig. 2, Fig. 3), and quantitative results obtained by qPCR-StdC (

Discussion

We establish here, for the first time, that a select minority of PDE isoforms exhibit brain region-specific changes in expression from early to late adulthood (Fig. 5). Further, we demonstrate that PDE expression patterns are conserved between rats and mice (Fig. 1, Fig. 2, Fig. 3), and expression patterns in rodents are similar to those previously reported in humans [33]. Our results show that PDEs are dynamically regulated across the lifespan, which must be taken into account when considering

Disclosure statement

All authors were previously, or currently are, full-time employees of Pfizer, Inc. MPK is a member of the Clinical Advisory Board for Asubio Pharmaceuticals. All procedures used in this study were in accordance with the NIH Guidelines for Care and Use of Laboratory Animals (Pub 85–23) and were approved by the Institutional Animal Care and Use Committees of Pfizer and USC.

Acknowledgments

The authors thank Youping Huang for assistance with statistical analyses. This work was funded by Pfizer, a Research Starter Grant from the PhRMA Foundation (MPK), as well as start-up funds, an ASPIRE award and an RDF grant from the University of South Carolina (MPK).

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  • Cited by (0)

    1

    Co-first authors.

    2

    Current affiliation: Koltan Pharmaceuticals, Inc., 300 George St, Suite 530, New Haven, CT 06511, USA.

    3

    Current affiliation: Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008, USA.

    4

    Current affiliation: New York University, 2800 Victory Boulevard, Staten Island, NY 10314, USA.

    5

    Current affiliation: University of Connecticut Health Science Center, 263 Farmington Avenue, Farmington, CT 06030, USA.

    6

    Current affiliation: Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115 USA.

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