Reyes Samantha T, Mohajeri Sanaz, Krasinska Karolina, Guo Scarlett G, Gu Meng, Pisani Laura, Rosenberg Jarrett, Spielman Daniel M, Chin Frederick T
Department of Radiology, Stanford University, Stanford, CA, United States.
Stanford University Mass Spectrometry Laboratory, Stanford University, Stanford, CA, United States.
Front Mol Neurosci. 2020 Dec 18;13:612685. doi: 10.3389/fnmol.2020.612685. eCollection 2020.
Fragile X syndrome (FXS) is the leading monogenetic cause of autism spectrum disorder and inherited cause of intellectual disability that affects approximately one in 7,000 males and one in 11,000 females. In FXS, the gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABA subunits. Therefore, FMRP loss adversely impacts the neuronal firing of the GABAergic system which creates an imbalance in the excitatory/inhibitory ratio within the brain. Current FXS treatment strategies focus on curing symptoms, such as anxiety or decreased social function. While treating symptoms can be helpful, incorporating non-invasive imaging to evaluate how treatments change the brain's biology may explain what molecular aberrations are associated with disease pathology. Thus, the GABAergic system is suitable to explore developing novel therapeutic strategies for FXS. To understand how the GABAergic system may be affected by this loss-of-function mutation, GABA concentrations were examined within the frontal cortex and thalamus of 5-day-old wild type and knockout mice using both H magnetic resonance imaging (H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice and evaluate whether H-MRS is suitable to capture regional GABA concentration differences at the front end of the critical cortical period where abnormal neurodevelopment occurs due to FMRP loss is first detected. H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and knockout mice. To substantiate the results of our H-MRS studies, LC-MS/MS was also performed on brain homogenates from age-matched mice. We found significant changes in GABA concentration between the frontal cortex and thalamus within each mouse from both wild type and knockout mice using H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and knockout mice by LC-MS/MS, validating that FMRP loss directly affects the GABAergic system. Thus, these new findings support the need to develop an effective non-invasive imaging method to monitor novel GABAergic strategies aimed at treating patients with FXS.
脆性X综合征（FXS）是自闭症谱系障碍的主要单基因病因，也是智力残疾的遗传性病因，影响约七千分之一的男性和一万一千分之一的女性。在FXS中，该基因沉默，阻止脆性X智力低下蛋白（FMRP）的表达，而FMRP直接靶向多个GABA亚基的mRNA转录本。因此，FMRP缺失对GABA能系统的神经元放电产生不利影响，导致大脑内兴奋/抑制比例失衡。目前的FXS治疗策略侧重于缓解症状，如焦虑或社交功能下降。虽然治疗症状可能有所帮助，但采用非侵入性成像来评估治疗如何改变大脑生物学特性，可能有助于解释哪些分子异常与疾病病理相关。因此，GABA能系统适合用于探索开发针对FXS的新型治疗策略。为了解功能丧失突变如何影响GABA能系统，利用氢磁共振成像（H-MRS）和液相色谱-串联质谱（LC-MS/MS）检测了5日龄野生型和基因敲除小鼠额叶皮质和丘脑内的GABA浓度。我们的目标是开发一种适用于新生小鼠的可靠扫描方法，并评估H-MRS是否适合在关键皮质期前端捕捉区域GABA浓度差异，在此阶段，由于FMRP缺失首次检测到异常神经发育。H-MRS对野生型和基因敲除小鼠额叶皮质和丘脑内的GABA浓度进行了定量。为证实我们H-MRS研究的结果，还对年龄匹配小鼠的脑匀浆进行了LC-MS/MS检测。利用H-MRS和LC-MS/MS，我们发现野生型和基因敲除小鼠各自的额叶皮质和丘脑之间GABA浓度存在显著变化。通过LC-MS/MS在野生型和基因敲除小鼠的相同区域也检测到显著的GABA水平，证实FMRP缺失直接影响GABA能系统。因此，这些新发现支持开发一种有效的非侵入性成像方法以监测旨在治疗FXS患者的新型GABA能策略的必要性。
