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Comparative Study
. 2006 Mar 8;26(10):2814-9.
doi: 10.1523/JNEUROSCI.5060-05.2006.

D-aspartate regulates melanocortin formation and function: behavioral alterations in D-aspartate oxidase-deficient mice

Alex S Huang  1 , Anne Beigneux, Zachary M Weil, Paul M Kim, Mark E Molliver, Seth Blackshaw, Randy J Nelson, Stephen G Young, Solomon H Snyder
Affiliations
Comparative Study

D-aspartate regulates melanocortin formation and function: behavioral alterations in D-aspartate oxidase-deficient mice

Alex S Huang et al. J Neurosci. .

Abstract

D-aspartate, an abundant D-amino acid enriched in neuroendocrine tissues, can be degraded by D-aspartate oxidase (Ddo). To elucidate the function of D-aspartate, we generated mice with targeted deletion of Ddo (Ddo(-/-)) and observe massive but selective augmentations of D-aspartate in various tissues. The pituitary intermediate lobe, normally devoid of D-aspartate from endogenous Ddo expression, manifests pronounced increases of immunoreactive D-aspartate in Ddo(-/-) mice. Ddo(-/-) mice show markedly diminished synthesis and levels of pituitary proopiomelanocortin/alpha-MSH, associated with decreased melanocortin-dependent behaviors. Therefore, Ddo is the endogenous enzyme that degrades D-aspartate, and Ddo-enriched organs, low in D-aspartate, may represent areas of high turnover where D-aspartate may be physiologically important.

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Figures

Figure 1.
Figure 1.
Targeted inactivation of Ddo. A, Ddo−/− mice were created by targeted removal of exon 3 via homologous recombination with a neomycin resistance gene (neo), introducing a frame shift. Southern blot was performed with the 5′ probe of BamHI-digested genomic DNA (12.8 kb band in the Ddo+ allele; 4.3 kb band in the Ddo− allele). tk, Thymidine kinase. B, Northern blot of total RNA with a Ddo exon 2–4 probe. The arrowhead denotes potential truncated Ddo transcript. C, Western blot of P2 fraction lysate. Cyto. C, Cytochrome c.
Figure 2.
Figure 2.
Ddo−/− mice display elevated d-aspartate levels. A, HPLC amino acid analysis of 4-week-old kidney d-aspartate (Ddo+/+ vs Ddo+/−, p = 0.021; Ddo+/− vs Ddo−/−, p = 0.008). B, Four-month-old kidney d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.004). C, Four-month-old brain d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.0009). D, Four-month-old adrenal d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.00001). E, Four-month-old ovarian d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.000004). F, Four-month-old testicular d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.0001) and l-aspartate (Ddo+/+ vs Ddo−/−; p = 0.013). *p < 0.05; **p < 0.01; ***p < 0.001; comparison with two-tailed Student’s t test; data are expressed as mean ± SEM.
Figure 3.
Figure 3.
Elevated d-aspartate in Ddo−/− mice pituitary intermediate lobe is associated with diminished melanocortin levels. A, Ddo in situ hybridization (n = 2). B, d-Aspartate immunohistochemistry (n = 4). C, D, POMC in situ hybridization in the pituitary (C; n = 3) and arcuate nucleus (D; n = 2). E, POMC Western blot of crude pituitary lysate (n = 2). F,G, Crude pituitary lysate dot blot (F; n = 4) and immunofluorescence (G; n = 2) of α-MSH. Scale bars, 100 μm. a, Anterior lobe; i, intermediate lobe; p, posterior lobe.
Figure 4.
Figure 4.
Ddo−/− mice display diminished melanocortin-dependent behaviors. A, Body mass measured at indicated ages (2 months, p = 0.03; 7 months, p = 0.007; 12 months, p = 0.004). B, Latency of male mouse to mount (p = 0.01) or intromit (p = 0.025) estrous CD-1 female. C, D, Time spent autogrooming during resident–intruder aggression test (C; n = 8 pairs; p = 0.04) and elevated plus maze test (D; p = 0.02). *p < 0.05; **p < 0.01; comparison by two-tailed t test; data are expressed as mean ± SEM. Sig., Significance.
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