Reductions in hypothalamic Gfap expression, glial cells and alpha-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

Background: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Galphas, termed XLalphas, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. Results: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive alpha-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3rd ventricle, while alpha-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were, unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. Conclusion: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since alpha-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections. 6 wildtype and 6 Gnasxlm+/p- RNA isolates were used, which were pooled in pairs in equal concentrations prior to rRNA removal using the RiboMinus kit (Life Technologies)背景：小鼠下丘脑的神经元与胶质细胞分化在出生时并未完成，而是会在出生后的前两周持续进行。营养状态与瘦素（Leptin）缺乏可影响神经元投射与胶质细胞模式的成熟，且在肥胖小鼠模型中可观察到下丘脑胶质增生。Gnasxl是基因组印记Gnas基因座的可变转录本，其编码信号蛋白Galphas的变体XLalphas，该蛋白在下丘脑的特定区域表达。Gnasxl缺陷型小鼠会表现出出生后生长迟缓与营养不足，而存活的成年个体则维持消瘦与高代谢状态，并伴随交感神经系统（sympathetic nervous system, SNS）活性增强。目前尚未有研究探究该基因敲除对下丘脑神经网络的影响。结果：对Gnasxl缺陷型小鼠下丘脑进行RNA测序分析，结果显示成年个体样本中的胶质纤维酸性蛋白（Glial fibrillary acidic protein, GFAP）表达显著下调。组织学分析证实，仅在下丘脑区域，GFAP阳性胶质细胞的数量出现减少。该减少仅见于成年组织样本，而出生后各个阶段的下丘脑组织并未发现差异，这表明成年Gnasxl缺陷型小鼠的这种变化是对其早期生长表型与高代谢状态的适应性改变。尤为显著的是，大量GFAP阳性α-室管膜细胞（alpha-tanycytes）及其突起出现丢失，这些细胞与突起构成了第三脑室内侧及背侧区域的室管膜层；而沿正中隆起（median eminence, ME）与漏斗隐窝分布的α-室管膜细胞则未受影响。该变化同时伴随波形蛋白（Vimentin）与巢蛋白（Nestin）表达的局部下调。下丘脑胶质溶质转运蛋白的RNA水平未发生改变，这提示剩余的星形胶质细胞与室管膜细胞可能存在代偿性上调。结论：Gnasxl缺陷并不会直接影响下丘脑的胶质细胞发育，因为Gnasxl仅在神经元中表达，且GFAP阳性星形胶质细胞与室管膜细胞在出生后早期阶段表现正常。成年下丘脑中GFAP阳性细胞的丢失，似乎是Gnasxl缺陷型小鼠出生后营养不足、低血糖以及持续高代谢与消瘦状态所导致的结果，这与肥胖小鼠模型中观察到的胶质增生现象形成鲜明对比。由于α-室管膜细胞同时作为成年神经祖细胞发挥功能，本研究结果或许提示Gnasxl缺陷型小鼠的下丘脑结构存在进一步的发育异常，可能包括神经元组成与投射异常。本次实验共使用6只野生型小鼠与6只Gnasxlm+/p-缺陷型小鼠的RNA分离样本，在使用RiboMinus试剂盒（Life Technologies）去除核糖体RNA之前，将样本按等浓度两两混合。