Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase

Abstract

PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.

Fig. 1

(A, B) Localization of PDE10A and DARPP-32 in striatum of cynomolgus monkey. Sections of cynomolgus monkey were stained sequentially with antibodies to PDE10A (green) and DARPP-32 (red) as described in Experimental Procedures. Images were merged and overlap between PDE10A and DARPP-32 is shown in yellow. Two sections are shown: A, magnification 20×; B, magnification 60×. (C) Western blot of striatal extracts from two cynomolgus monkeys (lanes 2 and 3) and rat (lane 4) stained for PDE10A-ir with 24F3.F11. Equal amounts of striatal extracts were loaded. Rat recombinant PDE10A is shown as a comparator (lane 5) and molecular weight markers are in lane 1.

Fig. 2

Localization of PDE10A, nNOS, calretinin, ChAT, and parvalbumin in striatum of cynomolgus monkey. Sections of cynomolgus monkey were stained sequentially with antibodies to PDE10A (A, C-F) and nNOS (B, C), calretinin (D), ChAT (E), parvalbumin (F) as described in Experimental Procedures. Images were merged (C, PDE10A/ nNOS, D, PDE10A/calretinin, E, PDE10A/ChAT, F, PDE10A/parvalbumin) and no overlap was detected between PDE10A and the other makers. In F, lines in the x and y axis mark points of interest. The intensity of the immunoreactive signal at each point along these lines is depicted on the bottom and right hand sides of the figure. Magnification 20×.

Fig. 3

Schematic outline of subcellular fractionation sequence. See Experimental Procedures for full description of different fractions. Red, fractions containing substantial PDE10A-ir, black, fractions with little or no detectable PDE10A-ir.

Fig. 4

Western blot analysis of subcellular fractions from rat striatum with detection using a LiCor Odyssey. Fractions indicated at the top of the figure and antigens indicated on the left are described in Experimental Procedures.

Fig. 5

Western blot analysis of SPM fractions from rat striatum with detection using a LiCor Odyssey. Fractions indicated at the top of the figure and antigens indicated on the left are described in Experimental Procedures.

Fig. 6

Western blot analysis of PDE10A association with the high speed membrane fraction with ECL Detection System. P20, the 100,000×g membrane fraction as indicated in Fig. 3; LB, lysis buffer; Na, 1 M NaCl; T-0.4%, 0.4% Triton X-100; Na₂CO₃,0.1MNa₂CO₃; P, pellet; S, supernatant.

Fig. 7

Localization of PDE10A in rat striatum by immuno-electron microscopy. Gold particles (black dots) represent PDE10A-ir. D, dendrite; P, vesicle-filled presynaptic terminal; S, spine. Blue arrows, PDE10A-ir apposed to dendritic and spine membranes, red arrows, PDE10A-ir within spines not apparently apposed to membranes, yellow arrows, PDE10A-ir at the edges of the PSD.