Effects of Ethanol and NAP on Cerebellar Expression of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10−12 M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

<p>Abstract</p> <p>Background</p> <p>Methylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition.</p> <p>Methods</p> <p>To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine <it>Mut </it>embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the <it>MUT </it>gene. Enzymatic and expression studies were used to assess the extent of functional correction.</p> <p>Results</p> <p>Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or <it>Mut </it>murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-¹⁴C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes.</p> <p>Conclusion</p> <p>These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.</p

Alteration of gene expression by alcohol exposure at early neurulation

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.</p> <p>Result</p> <p>Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, <it>Sox5, Bhlhe22</it>), neural growth factor genes [<it>Igf1, Efemp1</it>, <it>Klf10 </it>(<it>Tieg), and Edil3</it>], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (<it>Rbp1</it>), and <it>de novo </it>expression of aldehyde dehydrogenase 1B1 (<it>Aldh1B1</it>). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos.</p> <p>Conclusion</p> <p>This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.</p

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype.</p> <p>Methods</p> <p>To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function.</p> <p>Results</p> <p>Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24–48 hours, the methylmalonic acid content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens.</p> <p>Conclusion</p> <p>The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism.</p