GABA Measurement in a Neonatal Fragile X Syndrome Mouse Model Using 1H-Magnetic Resonance Spectroscopy and Mass Spectrometry

Abstract

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 Fmr1 gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABAA 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 Fmr1 knockout mice using both 1H magnetic resonance imaging (1H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice in vivo and evaluate whether 1H-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. 1H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and Fmr1 knockout mice. To substantiate the results of our 1H-MRS studies, in vitro 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 Fmr1 knockout mice using 1H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and Fmr1 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.

Fragile X Syndrome as an interneuronopathy: a lesson for future studies and treatments

“…In human patients, a reduction of the GABA Amediated intracortical inhibition associated to an increase of intracortical circuit excitability was reported (Morin-Parent et al, 2019). Moreover, a decreased GABA concentration in the frontal cortex and thalamus of neonatal PND 5 Fmr1 KO mice was found (Reyes et al, 2020). In line with the reduced excitability showed by INs, also the availability of GABA is decreased at PND 21 in the Fmr1 KO amygdala, due to a decline in the number of inhibitory synapses and a reduced expression of GAD65/67, a rate-limiting enzyme for GABA synthesis (Olmos-Serrano et al, 2010; Figure 1).…”

“…However, to advance the research in the field several aspects could be taken into account to design future studies: I. To date, most of the studies have characterized FXS INs in adult mice (Olmos-Serrano et al, 2010;Paluszkiewicz et al, 2011b;Arbab et al, 2018;Goel et al, 2018;Kokash et al, 2019;Lee et al, 2019;Reinhard et al, 2019;Lovelace et al, 2020;Yang et al, 2020;Pouchelon et al, 2021;Kalinowska et al, 2022), while only a few studies have taken in consideration interneuronal impairment during the critical window of postnatal development (Nomura et al, 2017;Castagnola et al, 2020;Reyes et al, 2020;Rais et al, 2022). It would be interesting to study and compare various ages in Fmr1 KO mice, which are associated to an altered function of INs through the different steps of neurodevelopment.…”

“…II. The different brain areas have been studied differently: more attention has been paid to cortex (Selby et al, 2007;Gibson et al, 2008;Paluszkiewicz et al, 2011b;Patel et al, 2013;He et al, 2014;Nomura et al, 2017;Goel et al, 2018;Wen et al, 2018a,b;Lee et al, 2019;Pirbhoy et al, 2020;Reyes et al, 2020;Pouchelon et al, 2021) compared to other brain regions, such as the hippocampus (Arbab et al, 2018;Reinhard et al, 2019;Hwang et al, 2022), leading to missing molecular and behavioral information to understand the physiopathology of FXS. III.…”

From circuits to behavior: Amygdala dysfunction in fragile X syndrome

“…FMRP is widely expressed in inhibitory INs ( Olmos-Serrano et al, 2010 ) and many functional elements of γ-aminobutyric acid (GABA) neurotransmission are dysregulated in the context of loss of FMRP ( Paluszkiewicz et al, 2011a ). As early as P5, GABA concentrations are altered in the frontal cortex and thalamus in Fmr1 KO mice ( Reyes et al, 2020 ). Functional GABA A receptors are heteropentamers whose unique receptor subunit composition determines their pharmacologic and physiologic properties ( Hevers and Luddens, 1998 ; Rudolph and Mohler, 2006 ).…”