Dysregulation of glutamine transporter SNAT1 in Rett syndrome microglia: a mechanism for mitochondrial dysfunction and neurotoxicity

“Microglia regulate neurodevelopment by actively surveilling and responding to brain microenvironments (Davalos et al., 2005; Nimmerjahn et al., 2005; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012) and could be strongly influenced by epigenetic mechanisms.”

“Microglia regulate neurodevelopment by actively surveilling and responding to brain microenvironments (Davalos et al., 2005; Nimmerjahn et al., 2005; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012) and could be strongly influenced by epigenetic mechanisms.”

“Microglia regulate neurodevelopment by actively surveilling and responding to brain microenvironments (Davalos et al., 2005; Nimmerjahn et al., 2005; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012) and could be strongly influenced by epigenetic mechanisms.”

“Microglia regulate neurodevelopment by actively surveilling and responding to brain microenvironments (Davalos et al., 2005; Nimmerjahn et al., 2005; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012) and could be strongly influenced by epigenetic mechanisms.”

“Cellular oxygen consumption is a specific and informative indicator of mitochondrial function (Rolo et al., 2009; Brand and Nicholls, 2011).”

“The other possibility is a metabolic reprogramming to favor aerobic glycolysis over oxidative phosphorylation, called the Warburg effect, which is known to be driven by L-Gln catabolism in cancer cells (Wise et al., 2008; Samudio et al., 2009).”

“MECP2 encodes methylCpG-binding protein 2 (MeCP2), an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription (Amir et al., 1999; Chahrour et al., 2008).”

“, 2013); and (2) mCAT, a plasmid that has been shown to direct the expression of catalase only in the mitochondria (Schriner et al., 2005; Dai et al., 2011).”

“Adrian Bird’s laboratory (Guy et al., 2001) were obtained from The Jackson Laboratory.”

“Microglia regulate neurodevelopment by actively surveilling and responding to brain microenvironments (Davalos et al., 2005; Nimmerjahn et al., 2005; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012) and could be strongly influenced by epigenetic mechanisms.”

“To further investigate whether MeCP2 deficiency or SNAT1 overexpression affects the activation status of microglia, we quantified several established markers for the “classical activation” (M1) and “alternative activation” (M2) states by qPCR (Colton, 2009).”

“To test whether enhanced mtROS production as a result of SNAT1 overexpression leads to mitochondrial dysfunction in microglia, we used two mitochondria-targeted approaches using the following: (1) SS-31, a cell-permeable, mitochondriatargeted peptide antioxidant (Zhao et al., 2004) able to concentrate 1000- to 5000-fo…”

“Although it is likely that mtROS, especially hydroxyl rad- icals, reduce ATP production by their effects on components of the electron transport chain (Zhang et al., 1990), it remains to be elucidated how mtROS enhance abnormal glutamate production in MDM. Nonetheless, our data indicate that…”

“We and others previously showed a role of microglial abnormalities in RTT in mouse models (Maezawa and Jin, 2010; Derecki et al., 2012).”

“Epigenetic mechanisms play an important role in the brain’s capability to adapt to environmental challenges (Borrelli et al., 2008).”

“…in inner mitochondrial membrane and help scavenge hydroxyl radicals and peroxynitrite and possibly HOCl and peroxyl radicals (Gruber et al., 2013); and (2) mCAT, a plasmid that has been shown to direct the expression of catalase only in the mitochondria (Schriner et al., 2005; Dai et al., 2011).”

“This toxicity was blocked by MeAIB, a paradigm analog inhibitor of system A transporters (Mackenzie and Erickson, 2004), suggesting that the toxicity was mediated by excessive L-Gln uptake.”

“One novel mode of such epigenetic influence is bioenergetics (Wallace and Fan, 2010).”

“Some such compounds have recently moved into clinical trials and could, in principle, benefit girls with RTT (Gruber et al., 2013; Dai et al., 2014).”

“At elevated levels, intracellular L-Gln could damage mitochondria via its metabolism in mitochondria to ammonia and glutamate, causing cell toxicity or functional aberrations (Albrecht and Norenberg, 2006).”

“…gene mutation (Amir et al., 1999; Levitt and Campbell, 2009) and MeCP2 regulation is also abnormal in a large subgroup of autistic individuals (Samaco et al., 2005; Moretti and Zoghbi, 2006; Nagarajan et al., 2008; Levitt and Campbell, 2009; Ramocki et al., 2009), our studies may implicate a…”

“Primary microglia cultures were prepared from mixed glia cultures established from newborn mice with the “shaking-off” method as described previously (Suzumura et al., 1987).”

“This overproduction could be reduced by restriction of L-Gln in the medium (Maezawa and Jin, 2010).”

“The other possibility is a metabolic reprogramming to favor aerobic glycolysis over oxidative phosphorylation, called the Warburg effect, which is known to be driven by L-Gln catabolism in cancer cells (Wise et al., 2008; Samudio et al., 2009).”

“…in a large subgroup of autistic individuals (Samaco et al., 2005; Moretti and Zoghbi, 2006; Nagarajan et al., 2008; Levitt and Campbell, 2009; Ramocki et al., 2009), our studies may implicate a novel mechanism for autism where mitochondrial dysfunction also plays a role (Giulivi et al.,…”

“To test whether this is the consequence of SNAT1 overexpression, we transfected BV-2 cells with a SNAT1 expression plasmid vector (Varoqui et al., 2000).”

“…et al., 1999; Levitt and Campbell, 2009) and MeCP2 regulation is also abnormal in a large subgroup of autistic individuals (Samaco et al., 2005; Moretti and Zoghbi, 2006; Nagarajan et al., 2008; Levitt and Campbell, 2009; Ramocki et al., 2009), our studies may implicate a novel mechanism for…”

“The astrocyte cultures were prepared from newborn mice as described previously and cultured for 14 d in vitro before being used for experiments (Maezawa et al., 2009).”

“Mice with MECP2 knock-out also show mitochondrial dysfunction in the skeletal muscle (Gold et al., 2014) and abnormal mitochondrial ultrastructure in the dendrite (Belichenko et al., 2009).”

“However, a MeCP2 deficiency-initiated molecular pathway to account for microglial abnormalities in RTT has not been reported, despite the significant role of microglia in RTT (Maezawa and Jin, 2010; Derecki et al., 2012, 2013; Schwartz et al., 2013).”

“…microglia is overproduction of glutamate, to a degree that may cause low levels of neurotoxicity manifested as pruning of synapses and dendrites (Hutchins and Barger, 1998; Barger et al., 2007), clearly shown in our current data and previous studies (Maezawa and Jin, 2010; O’Driscoll et al., 2013).”

“…regulation is also abnormal in a large subgroup of autistic individuals (Samaco et al., 2005; Moretti and Zoghbi, 2006; Nagarajan et al., 2008; Levitt and Campbell, 2009; Ramocki et al., 2009), our studies may implicate a novel mechanism for autism where mitochondrial dysfunction also plays a…”

“…expression studies revealed gene alterations in RTT human and mouse samples (upregulation of ubiquinolcytochrome c reductase core protein 1 and downregulation of cytochrome c oxidase subunit 1) that may lead to mitochondrial respiratory chain defects (Kriaucionis et al., 2006; Gibson et al., 2010).”

“AAGGACA-3 /5 -TGAGTAGCAGCCTTGGAATG-3 ; and IGF-I, 5 TGGATGCTCTTCAGTTCGTG-3 /5 -CACAATGCCTGTCTGAGGTG-3 (Lee et al., 2013; Moreno et al., 2014; Tang et al., 2014).”

“Given that L-Gln taken up by cells is quickly accumulated in mitochondria and hydrolyzed by mitochondrial glutaminase to glutamate (Matés et al., 2009), the upregulation of SNAT1 in MDM may contribute to overproduction of glutamate.”

“9), which leads to production of mtROS (Jayakumar et al., 2004; Svoboda and Kerschbaum, 2009; Albrecht et al., 2010).”

“…(Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased oxidative stress (Sierra et al., 2001; De Felice et al., 2009).”

“6A,C,D), a mitochondrial complex I inhibitor (Giordano et al., 2012), suggesting that the L-Gln stimulatory effect was not caused by some extra-mitochondrial action.”

“As expected, neither SS-31 nor mCAT reduced MitoSox levels (data not shown) because SS-31 is unlikely to react directly with superoxide (Gruber et al., 2013) and mCAT acts on hydrogen peroxide, downstream of superoxide.”

“AAGGACA-3 /5 -TGAGTAGCAGCCTTGGAATG-3 ; and IGF-I, 5 TGGATGCTCTTCAGTTCGTG-3 /5 -CACAATGCCTGTCTGAGGTG-3 (Lee et al., 2013; Moreno et al., 2014; Tang et al., 2014).”

“9), which leads to production of mtROS (Jayakumar et al., 2004; Svoboda and Kerschbaum, 2009; Albrecht et al., 2010).”

“AAGGACA-3 /5 -TGAGTAGCAGCCTTGGAATG-3 ; and IGF-I, 5 TGGATGCTCTTCAGTTCGTG-3 /5 -CACAATGCCTGTCTGAGGTG-3 (Lee et al., 2013; Moreno et al., 2014; Tang et al., 2014).”

“…Campbell, 2009) and MeCP2 regulation is also abnormal in a large subgroup of autistic individuals (Samaco et al., 2005; Moretti and Zoghbi, 2006; Nagarajan et al., 2008; Levitt and Campbell, 2009; Ramocki et al., 2009), our studies may implicate a novel mechanism for autism where mitochondrial…”

“…expression studies revealed gene alterations in RTT human and mouse samples (upregulation of ubiquinolcytochrome c reductase core protein 1 and downregulation of cytochrome c oxidase subunit 1) that may lead to mitochondrial respiratory chain defects (Kriaucionis et al., 2006; Gibson et al., 2010).”

“This elevation could be attributable to increased protein levels of glutamine synthetase, an exclusively astrocytic enzyme, in Mecp2 /y astrocytes (Okabe et al., 2012).”

“Patients with RTT showed abnormal mitochondrial morphology (Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased…”

“Starting from our recent ChIP-seq data (Yasui et al., 2013), we identified a novel mechanism, illustrated in Figure 9, that coherently links dysregulation of a direct target of MeCP2, SNAT1, to mitochondrial abnormalities.”

“Cell viability was assayed by MTT (tetrazolium salt) as described previously (Maezawa et al., 2006).”

“…(Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased oxidative stress (Sierra et al., 2001; De Felice et al., 2009).”

“, 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased oxidative stress (Sierra et al.”

“Patients with RTT showed abnormal mitochondrial morphology (Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased…”

“Patients with RTT showed abnormal mitochondrial morphology (Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased…”

“, 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased oxidative stress (Sierra et al.”

“Potentially aggravating this vulnerability, NMR microscopy studies revealed elevated cerebral L-Gln levels in Mecp21lox mice (Ward et al., 2009) and glutamine/Cr ratios in RTT girls (Horská et al., 2009).”

“The well characterized, highly specific polyclonal anti-SNAT1 was described previously (Varoqui et al., 2000; Grewal et al., 2009) and was used in 1:1000 dilution.”

“Patients with RTT showed abnormal mitochondrial morphology (Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased…”

“Potentially aggravating this vulnerability, NMR microscopy studies revealed elevated cerebral L-Gln levels in Mecp21lox mice (Ward et al., 2009) and glutamine/Cr ratios in RTT girls (Horská et al., 2009).”

“In fact, before the identification of the causative RTT genes, mitochondrial abnormalities were suspected as the cause of RTT (Philippart, 1986).”

“System A transporter activities have been well characterized in BV-2 microglia cells (Schilling and Eder, 2004; Van den Eynden et al., 2011).”

“…(Ruch et al., 1989; Wakai et al., 1990; Dotti et al., 1993; Cornford et al., 1994), enzymatic defects of the mitochondrial respiratory chain (Eeg-Olofsson et al., 1989; Coker and Melnyk, 1991; Heilstedt et al., 2002), and increased oxidative stress (Sierra et al., 2001; De Felice et al.,…”

“Primary microglia cultures were prepared from mixed glia cultures established from newborn mice with the “shaking-off” method as described previously (Suzumura et al., 1987).”

“This result also supports the notion that MDM affects brain function not through assuming any conventionally defined activation state.”

“6A; Rolo et al., 2009).”

“System A transporter activities have been well characterized in BV-2 microglia cells (Schilling and Eder, 2004; Van den Eynden et al., 2011).”

“Microglia, the brain resident myeloid-derived cell, are among the most recent cell types implicated in RTT pathogenesis (Derecki et al., 2012; Cronk et al., 2015; Jin et al., 2015; Maezawa and Jin, 2010).”

“Jin et al., 2015 Mecp2, whose mutations cause Rett syndrome, was observed to regulate mitochondrial bioenergetics through a glutamine transporter in microglia.”

“…glutamate release that may also be relevant in a pathological context, which include disruption to glutamine metabolism (Maezawa and Lee-Way, 2010; Jin et al., 2015), volume-regulated anion channels (Harrigan et al., 2008) and gap junction hemi-channels (Maezawa and Lee-Way, 2010) that offer…”

“Using this method, 94% of isolated cells are microglial cells, as determined by flow cytometry (26).”

“Microglia were acutely isolated from adult brains without culturing as described (Jin et al., 2015).”

“, 2015), and in isolated Mecp2−/y microglia (Jin et al., 2015) provides further evidence that the absence of MeCP2 leads to impaired mitochondrial function.”

“Glutamine, a highly abundant amino acid in plasma, has been believed not to cross the BBB freely due to multiple transporters (Jin et al. 2015).”

“Microglia were isolated using CD11b-based immunopanning as previously described (Bohlen et al., 2017; Jin et al., 2015).”

“Many studies have demonstrated significant up-regulation of microglial GLS under pathological conditions that include AD, HAND, multiple sclerosis (MS), ALS, Rett syndrome (RTT), and other neurological diseases (Gao et al., 2019; Jin et al., 2015; Tian et al., 2008; Ye et al., 2013).”

“The uptake of glutamine in microglia is conducted via SNAT1 (solute carrier family 38a member 1 protein) [82].”

“The purity of isolated microglia, determined by flow cytometry, was consistently between 94 to 96%.(26) Compared to Wt microglia (Fig.”

“CD11b cells from the infarct area were isolated as previously described.(16,27) Briefly, mice were anesthetized with isoflurane 2, 5, and 8 days after reperfusion MCAO and killed by cervical dislocation.”

“, for example, found that an increase in the expression of Slc38a1 (which codes for a glutamine transporter) caused by Mecp2 deficiency results in a glutamine-dependent decrease in microglia viability and a reduction in brain microglial number through the production of reactive oxygen species (ROS), which causes mitoc…”

“As a consequence, adenosine triphosphate (ATP) production is significantly reduced in MeCP2-deficient microglia, and this ATP reduction leads to a greater potential for the apoptosis of abnormal microglia (Jin et al., 2015) (Figure 2).”

“[10] shown that MeCP2 acts as transcriptional repressor for the glutamine transporter SNAT1.”

“6,7 Fatty acids are metabolized by β -ox- idation within the “mitochondrial ketogenic pathway” (MKP) to generate β -hydroxybutyrate (BHB), a ketone body.”

“Snat1 overexpression or Mecp2 KO reduces the number of microglia, causes mitochondrial dysfunction, and results in over-production of glutamate leading to excitotoxicity [105].”

“Although the precise mechanism mediating adverse microglial effects on neurons is still debated [57,58], excessive release of glutamate [59,60] and inflammatory cytokines have been replicated in different studies [61,62].”

“Interestingly, an increased mitochondrial mass was also reported for Mecp2 microglia of the juvenile mouse brain [78].”

“This is observed in fragile X syndrome (57 – 60), Rett syndrome (61, 62), Angelman syndrome (63 – 65), Phelan-McDermid syndrome (66, 67), and the 22q11 CNV (45).”

“This set of results is consistent with previous findings that the number of microglia in the brain was decreased in Mecp2KO mice (Jin et al., 2015; Cronk et al., 2015b), and indicates that microglia in Mecp2KO mice expressed Cx 3 cr1 at the same level as those in WT mice.”

“…of neurons but also important for the functions of glial subtypes in the brain as well as of many cell types in the immune system, including microglia and macrophages (Maezawa and Jin, 2010; Derecki et al., 2012; O’Driscoll et al., 2013; Cronk et al., 2015; Jin et al., 2015; Cronk et al., 2016).”

“To our surprise we found that Mecp2-null mice progressively lose microglia according to the severity of disease (Cronk et al., 2015), an observation also reported in another recent publication (Jin et al., 2015).”

“However, although microglial MeCP2 knockdown directly caused NMDA receptor-dependent excitotoxic neuronal cell death [61], astrocytic MeCP2 deficiency did not affect the rate of neuronal cell death [62].”

“Mecp2 regulates glutamate synthesis and mitochondria function in microglia by acting as a transcriptional repressor of the major glutamate transporter, SNAT1 (Jin et al., 2015).”

“, 2016), and metabolic alterations regarding glutamate uptake and ROS generation (Jin et al., 2015).”

“In support of these assumptions, increased energy expenditure and intensified ATP turnover were brought to light in neonatal hippocampal Mecp2−/y neurons [59], and a mitochondrial energy-wasting status has been proposed for RTT [60].”

“…Whole cell patch clamp Loss of FMRP: ↑Cortical activity, ↑Locomotor activity, ↓Social novelty preference and memory acquisition deficits; Corrected by Fmr1 reactivation Jin et al., 2021 Fmr1−/y iPSC from patients with FXS differentiated into astrocytes RNA sequencing, immunocytochemistry.”

“Importantly, upregulation of SLC38A1 due to the lack of methylation at its promoter was observed in peripheral blood in MDD [33] and in microglia in Rett syndrome [34].”

“Increased SNAT1 activity within the glutamate synthesis pathway occurs due to a lack of MeCP2-mediated repression [103].”

“Later, MECP2 has been found to act as a transcriptional repressor of the major glutamine transporter SNAT1 (also known as SLC38A1), leading to glutamate overproduction in Mecp2-null microglia [9].”

“MeCP2 down regulation or SNAT1 over expression in microglia resulted in mitochondrial dysfunction and neurotoxicity due to overproduction of glutamate (Jin et al. 2015).”

“MeCP2 is expressed in microglia and related mononuclear phagocytes and regulates their functions, but the pathological role of microglia in RTT remains controversial (Maezawa and Jin, 2010; Derecki et al., 2012; Wang et al., 2015; Horiuchi et al., 2017; Schafer et al., 2016; Jin et al., 2015; Cronk et al., 2015).”

“This agrees with studies that reported a decrease in brain ATP synthesis [33–35], but while knowledge was mostly limited to whole brain analysis, we have studied bioenergetic function both between different brain areas and at two different time-points through the phenotype progression.”

“Mecp2-null microglia also contributed to these pathologically high extracellular glutamate levels by upregulation of glutaminase and the glutamine transporter SNAT1 (Jin et al., 2015; Maezawa & Jin, 2010).”

“…genes, proteins, and enzyme activities, and reduced ATP level have also been documented in RTT condition (Kriaucionis et al. 2006; Pecorelli et al. 2013; Gold et al. 2014; Valenti et al. 2017; De Filippis et al. 2015; Li et al. 2013; Saywell et al. 2006; Gibson et al. 2010; Jin et al. 2015).”

“Of note, previous in vitro studies suggested that microglia from Mecp2-/y mice are responsible for the over-production of glutamate [70], which exerts a neurotoxic effect on dendrites and synapses [70,71].”

“Upregulated SNAT1 (encoded by the gene SLC38A1) and glutaminase (GLS) proteins in microglia of Mecp2 - null male have been reported to contribute to the superfluous glutamates release under the Mecp2 - deficient condition and contribute to neurotoxicity (36, 37).”

“elevated lactate and pyruvate in blood and cerebral spinal fluid (26), as well as markers of oxidative damage (27) that are also seen in mouse models (28, 29).”

“However, microglia express the glutamine transporter SNAT1 (Jin et al. 2015; Yamada et al. 2019) and possess the necessary cellular machinery to me-tabolize glutamine (Bernier, York, Kamyabi, et al. 2020).”

“In neurons and glial cells of Rett syndrome cerebral organoids, mitochondrial length and area were reduced compared to those in the control group, and the alteration was particularly significant in glial cells [205–207].”

“Strong evidence suggests that microglia could be a common effector in RTT, supporting the microglia hypothesis discussed above (Maezawa and Jin 2010; Derecki et al. 2012; Jin et al. 2015).”

“92 Upregulation of the transporter was sufficient to increase glutamine uptake and glutamate production and induce toxicity in microglia secondary to the elevated glutamine levels.”

“This discrepancy was caused by the imbalance of Gln/Glu ratios and deregulation of Gln homeostasis, resulting in mitochondrial dysfunction and microglial neurotoxicity [9].”

“Our experiments also showed increased of expression of Snat1 , which encodes a microglial-specific glutamine transporter and is required for glutamine metabolism 43 .”

“However, mice with an overexpression of the glutamine transporter SLC38A1 constitutively produced and excreted glutamate into the interstitial space, resulting in neurotoxicity [18 ].”

“A decade ago, molecular mechanisms were identified linking MeCP2 to the regulation of bioenergetic pathways in microglia (Jin et al., 2015).”