Research reportImmunohistochemical localization of PDE10A in the rat brain
Introduction
Cyclic nucleotides play an important role as second messengers in the CNS, serving to regulate a wide variety of neuronal functions. Intracellular concentrations of cAMP and cGMP are modulated by the rate of synthesis by adenylyl and guanylyl cyclases, and of equal importance, by the rate of degradation by a variety of phosphodiesterases (PDEs). Multiple PDE isozymes have been identified over the past decade [26] with 22 unique gene products organized into 11 distinct families according to structural, functional and kinetic properties. Mapping the differential expression of the PDEs constitutes an important early step in elucidating their function.
PDE10A is the single member of one of the newest PDE gene families. Initial characterization in several laboratories [8], [18], [27] indicates that PDE10A is a dual-substrate phosphodiesterase, hydrolyzing cAMP and cGMP with apparent Km values of 0.05 and 3.0 μM, respectively. Northern blot and dot blot studies by all three groups indicated PDE10A expression in brain and Fujishige et al. [6] noted high expression in human brain in two particular regions, the caudate n. and putamen. This group subsequently showed using in situ hybridization that PDE10A mRNA is highly localized within the rodent brain to the caudate n. and olfactory tubercle. These brain regions form the core of the basal ganglia, a system of interconnected nuclei that process cortical information in the context of dopaminergic signaling to regulate motoric, appetitive, and cognitive processes. Thus, the localization of PDE10A within these structures begins to define possible physiological functions for this enzyme.
In order to determine the localization of PDE10A protein within the brain, we produced a monoclonal antibody that recognizes an epitope within the N-terminal domain of this protein. We used this antibody to develop a map of rat brain PDE10A protein expression using immunohistochemical and Western blotting techniques and compared the pattern of protein expression with that of mRNA expression. This analysis reveals that the enzyme is expressed at different levels, and with different subcellular distributions throughout the brain, giving further insight into possible functions.
Section snippets
Isolation and cloning of rat PDE10A cDNA
The protein coding sequence of human PDEA10A (GenBank AB020593) was used to search the National Center for Biotechnology Information (NCBI) Expressed Sequence Tag (EST) Database using the Basic Local Alignment Search Tool (BLAST; [1]). The amino acid sequence predicted by one rat EST (H32734) was homologous to an internal portion of the human protein and was highly homologous to a portion of the sequence for mouse PDE10A (GenBank AF110507). We used this information plus 5′ and 3′ Rapid
Cloning and expression of recombinant rat PDE10A protein
Using 5′ RACE, we identified rat PDE10A clones with three different N-termini. The sequences of two of these match sequences described by Fujishige et al. as PDE10A2 and PDE10A3 [7]. The N-terminal amino acids of the third clone, which is the sequence we expressed, are MSG; the remainder of the protein (LTKED…RKVDD) is identical to the two previously identified splice variants. This clone apparently represents an additional N-terminal splice variant of rat PDE10A. An anti-Tetra His antibody
Discussion
The aim of the present study was to develop a map of the distribution of PDE10A protein and mRNA in the brain, as a further step towards elucidating the function of this newly identified phosphodiesterase. Using a monoclonal antibody 24F3.F11 to the N-terminal sequence of PDE10A, we detected high levels of the enzyme in the medium spiny neurons of the caudate n., n. accumbens and olfactory tubercle. In these neuronal populations, PDE10A appears to be distributed throughout all of the neuronal
Acknowledgements
We acknowledge Dr. V. Bedian for production of the monoclonal antibody, J. Jirka and S. Hawrylik for protein production, K.F. Fennell for providing the N-terminal PDE10A fragment, and the DNA Sequencing Laboratory at Pfizer, Groton for sequencing support.
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