Akt2 modulates glucose availability and downstream apoptotic pathways during development.

Glucose is the primary energy substrate for eukaryotic cells and the predominant substrate for the brain. Studies suggest that glucose serves an additional role in the regulation of cellular functions, including viability. Zebrafish is a tractable system for defining the cellular and molecular mechanisms perturbed by impaired glucose transport and metabolism. Previously, we demonstrated a critical role for the facilitative glucose transporter, Glut1, in the regulation of embryonic brain development. In this study, we aim to identify mediators in this Glut1-sensitive process by investigating the role of the antiapoptotic kinase, Akt2. Results show that abrogating expression of akt2 causes a phenotype strikingly similar to that observed when glut1 expression is inhibited. akt2-deficient embryos exhibit increased neuronal apoptosis, impaired glucose uptake, and death by 72 h postfertilization. Similar to what was observed in the glut1 morphants, inhibiting the expression of the proapoptotic protein, bad, in the context of impaired akt2 expression results in the inhibition of apoptosis and rescue of the morphant embryos. Intriguingly, overexpression of glut1 in the akt2 morphants was also able to rescue these embryos. Quantitative reverse transcription-PCR analysis revealed decreased glut1 transcript expression in akt2 morphant embryos. Taken together, these data suggest that Akt2 modulates glucose availability by regulating Glut1 expression at the transcript level. These data support a role for akt2 in an integrative pathway directly linking glucose, Glut1 expression, and activation of apoptosis and demonstrate the dependence of akt2 on glucose availability for the maintenance of cellular viability, particularly in the central nervous system.

GLUT1 deficiency links nutrient availability and apoptosis during embryonic development.

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.

Alpha-synuclein protects naive but not dbcAMP-treated dopaminergic cell types from 1-methyl-4-phenylpyridinium toxicity.

The pre-synaptic protein, alpha-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that alpha-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP+), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the beta-amyloid peptide, A-beta. Protection from MPP+ was directly correlated with the preservation of mitochondrial function. Specifically, alpha-synuclein rescued cells from MPP+ mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, alpha-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wild-type alpha-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for alpha-synuclein may change during development.