Aspartoacylase (ASPA) is an oligodendrocyte-restricted enzyme that catalyzes the hydrolysis of neuronally derived N-acetylaspartate (NAA) to acetate and aspartic acid. ASPA deficiency leads to the fatal childhood autosomal recessive leukodystrophy Canavan disease (CD). Here we demonstrate that the previously described ENU-induced nur7 mouse mutant is caused by a nonsense mutation, Q193X, in the Aspa gene (Aspa nur7 ). Homozygous Aspa nur7nur7 mice do not express detectable Aspa protein and display an early-onset spongy degeneration of CNS myelin with increased NAA levels similar to that observed in CD patients. In addition, CNS regions rich in neuronal cell bodies also display vacuolization. Interestingly, distinct myelin rich areas, such as the corpus callosum, optic nerve, and spinal cord white matter appear normal in Aspa nur7/nur7 mice. Reduced cerebroside synthesis has been demonstrated in CD patients and animal models. To determine the potential relevance of this observation in disease pathogenesis, we generated Aspa nur7/nur7 mice that were heterozygous for a null allele of the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase (Cgt), which is responsible for catalyzing the synthesis of the abundant myelin galactolipids. Despite reduced amounts of cerebrosides, the Aspa nur7/nur7 ;Cgt ϩ/ Ϫ mice were not more severely affected than the Aspa nur7 mutants, suggesting that diminished cerebroside synthesis is not a major contributing factor in disease pathogenesis. Furthermore, we found that myelin degeneration leads to significant axonal loss in the cerebellum of older Aspa nur7 mutants. This finding suggests that axonal pathology caused by CNS myelin defects may underlie the neurological disabilities that CD patients develop at late stages of the disease.

No abstract

Purpose: Colon cancer is a major cause of cancer deaths. Dietary factors contribute substantially to the risk of this malignancy. Western-style diets promote development of azoxymethane-induced colon cancer. Although we showed that epidermal growth factor receptors (EGFR) controlled azoxymethane tumorigenesis in standard fat conditions, the role of EGFR in tumor promotion by high dietary fat has not been examined. Experimental Design: A/J × C57BL6/J mice with wild-type Egfr (Egfr wt ) or loss-offunction waved-2 Egfr (Egfr wa2 ) received azoxymethane followed by standard (5% fat) or western-style (20% fat) diet. As F 1 mice were resistant to azoxymethane, we treated mice with azoxymethane followed by one cycle of inflammation-inducing dextran sulfate sodium to induce tumorigenesis. Mice were sacrificed 12 weeks after dextran sulfate sodium. Tumors were graded for histology and assessed for EGFR ligands and proto-oncogenes by immunostaining, Western blotting, and realtime PCR. Results: Egfr wt mice gained significantly more weight and had exaggerated insulin resistance compared with Egfr wa2 mice on high-fat diet. Dietary fat promoted tumor incidence (71.2% versus 36.7%; P < 0.05) and cancer incidence (43.9% versus 16.7%; P < 0.05) only in Egfr wt mice. The lipid-rich diet also significantly increased tumor and cancer multiplicity only in Egfr wt mice. In tumors, dietary fat and Egfr wt upregulated transforming growth factor-α, amphiregulin, CTNNB1, MYC, and CCND1, whereas PTGS2 was only increased in Egfr wt mice and further upregulated by dietary fat. Notably, dietary fat increased transforming growth factor-α in normal colon. Conclusions: EGFR is required for dietary fat-induced weight gain and tumor promotion. EGFR-dependent increases in receptor ligands and PTGS2 likely drive diet-related tumor promotion. (Clin Cancer Res 2009;15(22):6780-9)

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2 F 1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6 F 2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2 F 4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-A (6.4 F 1.3-fold) and increased phospho-(active) EGFR (5.9 F 1.1-fold), phospho-(active) ErbB2 (2.3 F 0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3 F 0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P < 0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P < 0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies. [Cancer Res 2007;67(2):827-35]