Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.
To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type–specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.
Brain function is absolutely dependent on an appropriate supply of energy. A shortfall in supply-as occurs, for instance, following stroke-can lead rapidly to irreversible damage to this vital organ. While the consequences of pathophysiological energy depletion have been well documented, much less is known about the physiological energy dynamics of brain cells, although changes in the intracellular concentration of adenosine triphosphate (ATP), the major energy carrier of cells, have been postulated to contribute to cellular signaling. To address this issue more closely, we have investigated intracellular ATP in cultured primary cortical astrocytes by time-lapse microscopy using a genetically encoded fluorescent sensor for ATP. The cytosolic ATP sensor signal decreased after application of the neurotransmitter glutamate in a manner dependent on both glutamate concentration and glutamate transporter activity, but independent of glutamate receptors. The application of dopamine did not affect ATP levels within astrocytes. These results confirm that intracellular ATP levels in astrocytes do indeed respond to changes in physiological activity and pave the way for further studies addressing factors that affect regulation of ATP. V C 2017 Wiley Periodicals, Inc.
Astrocytes are a glial cell type, which is indispensable for brain energy metabolism. Within cells, the NADH/NAD redox state is a crucial node in metabolism connecting catabolic pathways to oxidative phosphorylation and ATP production in mitochondria. To characterize the dynamics of the intracellular NADH/NAD redox state in cortical astrocytes Peredox, a genetically encoded sensor for the NADH/NAD redox state, was expressed in cultured cortical astrocytes as well as in cortical astrocytes in acutely isolated brain slices. Calibration of the sensor in cultured astrocytes revealed a mean basal cytosolic NADH/NAD redox ratio of about 0.01; however, with a broad distribution and heterogeneity in the cell population, which was mirrored by a heterogeneous basal cellular concentration of lactate. Inhibition of glucose uptake decreased the NADH/NAD redox state while inhibition of lactate dehydrogenase or of lactate release resulted in an increase in the NADH/NAD redox ratio. Furthermore, the NADH/NAD redox state was regulated by the extracellular concentration of K , and application of the neurotransmitters ATP or glutamate increased the NADH/NAD redox state dependent on purinergic receptors and glutamate uptake, respectively. This regulation by K , ATP, and glutamate involved NBCe1 mediated sodium-bicarbonate transport. These results demonstrate that the NADH/NAD redox state in astrocytes is a metabolic node regulated by neuronal signals reflecting physiological activity, most likely contributing to adjust astrocytic metabolism to energy demand of the brain.
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