A multi‐organ analysis of the role of mTOR in fetal alcohol spectrum disorders

Abstract Alcohol exposure during gestation can lead to fetal alcohol spectrum disorders (FASD), an array of cognitive and physical developmental impairments. Over the past two and a half decades, Mammalian Target of Rapamycin (mTOR) has emerged at the nexus of many fields of study, and has recently been implicated in FASD etiology. mTOR plays an integral role in modulating anabolic and catabolic activities, including protein synthesis and autophagy. These processes are vital for proper development and can have long lasting effects following alcohol exposure, such as impaired hippocampal and synapse formation, reduced brain size, as well as cognitive, behavioral, and memory impairments. We highlight recent advances in the field of FASD, primarily with regard to animal model discoveries and discuss the interaction between alcohol and mTOR in the context of various tissues, including brain, placenta, bone, and muscle, with respect to developmental alcohol exposure paradigms. The current review focuses on novel FASD research within the context of the mTOR signaling and sheds light on mechanistic etiologies at various biological levels including molecular, cellular, and functional, across multiple stages of development and illuminates the dichotomy between the different mTOR complexes and their unique signaling roles.


| INTRODUCTION
Fetal alcohol spectrum disorders (FASD) is an overarching term that encompasses a range of developmental outcomes exhibited in children exposed to alcohol in utero. 1 FASD can present differently in every child, however, intellectual and/or behavioral impairments are always reported. The most severe of these disorders is Fetal Alcohol Syndrome (FAS), which is characterized by facial dysmorphology, growth restriction, and central nervous system/neurodevelopmental abnormalities. 2 As a result, the US Surgeon General and the American Academy of Pediatrics have issued an advisory to abstain from any alcohol when considering pregnancy and throughout pregnancy. 3,4 Although the exact mechanism of FASDs remains elusive, the Mechanistic Target of Rapamycin (mTOR) has emerged as a key control node in a network that responds to metabolic signals and has been implicated in various tissue abnormalities following alcohol pathology. mTOR is one of six phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family members, which are implicated in a variety of cellular functions anywhere from DNA damage sensing and repair to cell cycle progression and arrest. [5][6][7] More specifically, mTOR is a 289-kDa serine/threonine protein kinase that makes up the catalytic subunit of two individual complexes, known as mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). These complexes each have distinctive substrates and functions, in addition to unique accessory proteins and sensitivity to rapamycin. 6,8,9 It is important to note that rapamycin directly inhibits mTORC1, while it only indirectly inhibits the rapamycin-insensitive counterpart in mTORC2. 10 mTORC1 plays a pivotal role in bringing multiple cellular signals together to gauge the availability of nutrients, growth factors, and energy to promote cellular growth and catabolic activity during stress. mTORC1 maintains tight regulation over translation, as this is among the most energy-and resource-intensive cellular processes. 6 It is also critical that cellular building blocks are not broken down again before they can be incorporated into their respective processes, therefore it is crucial that mTOR regulates and suppresses catabolic autophagy. 11 By blocking autophagy, cells are allowed to accumulate proteins and organelles, even if they are redundant or damaged. Not much is known about mTORC2 and its relation to these processes, however recent studies have shown that mTORC2 may promote mTORC1 activity. 6,12 To our knowledge, this is the first review that examines the effects of gestational alcohol exposure on the mTOR signaling pathway. In this review, we analyze studies that examine mTOR, alcohol, and their relation to pregnancy. Our review presents a detailed analysis of various tissues implicated in FASD and potential areas of study including brain, placenta, bone, and skeletal muscle.

| mTOR architechture
mTORC1 is made up of three core components: mTOR, Target of rapamycin complex subunit LST8 (mLST8), and its defining scaffold protein Regulatory-Associated Protein of mTOR (RAPTOR). 6 This RAPTOR scaffold facilitates the mTORC1 accessory factor, Proline-Rich AKT Substrate 40 kDa (PRAS40), which behaves as an mTORC1 inhibitor alongside DEP-domain-containing mTOR-interacting protein (DEPTOR). RAPTOR is also necessary for intracellular localization of mTORC1 and plays a role in recruiting substrates by binding to respective TOR signaling motifs. While the core of mTORC2 also includes mTOR and mLST8, it is instead defined by the scaffold protein Rapamycin-Insensitive Companion of mTOR (RICTOR), which binds to Mammalian Stress-activated MAP Kinaseinteracting Protein 1(mSIN1), DEPTOR, and protein associated with rictor 1 or 2 (PROTOR1/2). 6

| mTORC1 signaling pathway and protein synthesis
To increase the production of proteins, lipids, nucleotides, and ATP, while maintaining a balance with catabolic activity, mTORC1 must phosphorylate the appropriate substrates ( Figure 1). mTORC1 directly promotes protein synthesis by ultimately phosphorylating the Eukaryotic Translation Initiation Factor 4Ebinding Proteins (4E-BPs), as well as Ribosomal Protein S6 Kinase 1 (S6K1). Along with Phosphoinositidedependent Kinase (PDK), which phosphorylates the activation loop (Thre229), mTORC1 phosphorylates S6K1 on its hydrophobic motif (Thre389), which stimulates its kinase activity. 13,14 mTORC1 and S6K1 in turn amplify RNA Polymerase I and RNA Polymerase III activity F I G U R E 1 Downstream targets of mTORC1 signaling. Activation of mTORC1 initiates a downstream cascade that enhances the production of proteins, lipids, nucleotides, and other macromolecules. In tandem with this anabolic paradigm, mTORC1 inhibits catabolic processes, such as autophagy and lysosome biogenesis. via phosphorylation of the Upstream Binding Factor (UBF) 15

| mTORC1 signaling pathway and autophagy
Normally mTORC1 phosphorylates, and in turn inhibits Unc-51-like Autophagy-activating Kinase 1 (ULK1) 22 at Ser757 and Autophagy-related 13 (ATG13) 23 at Ser355. These important effectors of autophagy induction, in tandem with 200-kDA FAK family kinase-interacting protein (FIP200) and Autophagy Related 101 (ATG101), 24,25 spur formation of the autophagosome. However, during nutrient deprivation, inhibition of mTORC1 allows these molecules to resume autophagosome initiation, as well as permit the nuclear translocation of the Transcription Factor EB (TFEB) 26 and the Transcription Factor E3 (TFE3) 27 to coordinate lysosomal biogenesis. mTORC1 facilitates both protein synthesis and autophagy via the aforementioned signaling pathways, with little overlap aside from environmental cues. Herein we will review alcohol's effect on mTOR machinery in various tissues within the context of binge alcohol exposure, fetal development, and elucidate future directions.

ALCOHOL AND mTOR ON BRAIN DEVELOPMENT
The mTOR signaling pathway is closely associated with normal fetal and postnatal brain development and is also necessary for growth, survival, and differentiation of neural stem cells. 10 Normal postnatal development of brain size is regulated by mTOR signaling and mice lacking mTOR exhibit smaller brain size with fewer neurons and neural progenitors. 28 Exposure to alcohol during development alters expression of proteins involved in both neuronal growth and differentiation via the mTOR signaling pathway, and can lead to oxidative stress. 29 Herein, we will discuss how mTOR signaling, translation dysregulation, and neuroprotection mediated by autophagy can occur in multiple brain regions such as the cortex, hippocampus, and cerebellum following prenatal alcohol exposure.

| Brain mTORC1 dysregulation in FASD
Alcohol interacts with mTOR to impair protein synthesis in multiple brain regions. In a study, cortical neurons were isolated from Postnatal Day (PND) 14 C57BL/6 mouse fetuses and cultured in 100 mM alcohol for 5 days. 29 These neurons were then used in a microarray consisting of 638 sequence-verified genes to identify pathways significantly modulated following alcohol exposure. Notably, mRNA expression of FKBP rapamycin-associated protein was decreased by 22%, which associates with mTOR to restrict access of mTOR substrates to the complex. 29 Eukaryotic Translation Initiation Factor 4E (eIF4E), which is an mRNA 5′-cap binding protein, was also decreased by 24%, and mitogen-activated protein kinase kinase (MAPKK) decreased by 24% as well, which phosphorylates eIF4E. These results suggest alcohol exposure represses the kinase activity of mTOR and leads to decreased activation of the downstream substrate Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 (4E-BP1). Hypophosphorylated 4E-BP1 in turn competes with Eukaryotic Translation Initiation factor 4G (eIF4G) for a binding site on eIF4E and the resultant eIF4E/4E-BP complexes impair translation by inhibiting binding of eIF4E to the mRNA cap structure, thereby hindering protein synthesis and possibly resulting in apoptosis. 29 We have previously reported there was a strong effect observed in the hippocampal proteome of Sprague-Dawley rats given 6 g/kg alcohol from Gestational Day (GD) 11-20 following a short 4.5 g/kg acclimation period (GD 5-10). Of note, this was the most vulnerable brain region studied in terms of protein changes, with over 600 significantly altered proteins. 30 Many of these dysregulated proteins are regulators of cellular growth and developmental morphogenesis, and are specifically involved in the mTOR signaling pathway. Another study with a similar alcohol exposure paradigm (4.5 g/kg GD 5-10, 6 g/kg  in Sprague-Dawley rats showed that alcohol also dysregulates mTORC1 signaling and alters proteins that complex with mTOR in the fetal hippocampus. 31 Of note, there is a decrease in p-mTOR (Ser2448) and an increase in DEPTOR expression levels in the fetal hippocampus, which can inhibit mTORC1 signaling following alcohol exposure. An increase in total 4E-BP1 expression also infers a dysregulation of mTORC1 signaling, along with an increase in phosphorylation levels of 4E-BP1 and S6K1 (Figure 2), which are phosphorylated downstream of mTORC1 to initiate protein synthesis. 31 Exposure to alcohol over PND4-9, a period in rodent brain development comparable to the third trimester in humans, impairs hippocampal neurodevelopment and reduces normal dendritic spine density, neurogenesis, and long-term potentiation. A study has demonstrated that taurine and choline levels are decreased, whereas glutamate F I G U R E 2 Effect of gestational chronic binge alcohol exposure on mTORC1 signaling in the fetal hippocampus. Immunoblot analysis showed that gestational chronic binge alcohol exposure significantly increased (A) P-4E-BP1 level (↑ 41%; p = .0156) and (B) total 4E-BP1 expression (↑ 20%; p = .0251) in the fetal hippocampi. (C) Immunofluorescence staining shows that P-4E-BP1 was expressed in the oriens layer (OL) of the fetal hippocampus and the representative image shows CA3 field near fimbria (FI). (D) Gestational alcohol exposure significantly increased (↑ 57%; p = .0383) the level of P-p70 S6K, whereas (E) total p70 S6K expression was not different between groups. Data are shown as mean ± SEM and as fold of control. *Significance was established a priori at p < .05. Adapted with permission from Lee et al. 31 (PF-Cont = Pair-fed Control). and glutamine are increased in the dentate gyrus of rats exposed to alcohol between PND4-9 (5 g/kg/day, intragastrically). 32 However, pretreatment with rapamycin directly before the aforementioned alcohol administration (3 and 10 mg/kg, intraperitoneally) protected dentate gyrus cells against changes in concentration of taurine, choline, glutamate, and glutamine induced by neonatal alcohol exposure. These nutrients are crucial for proper neurodevelopment and these results implicate an interaction between alcohol and mTOR in regulation of amino acid concentrations. Of note, adult male rats (PND60) also had the deleterious effects following neonatal alcohol exposure on the dentate gyrus prevented when administered a rapamycin pre-treatment. 32 These discoveries show that exposure to alcohol during development not only disrupts the mTOR signaling pathway, but that these nutrients can in fact be rescued following rapamycin pre-treatment.

| Brain mTORC2 signaling disruptions in FASD
Few studies have been done to elucidate mTORC2 and its role in FASD pathology, however sufficient ground work has been laid to warrant further exploration of the mTORC1 and mTORC2 dichotomy. For example, a deep-sequencing analysis of fetal cortexes obtained from C57BL/6J mice exposed to alcohol (2.9 g/kg, GD7-17intraperitoneal injections) revealed 105 out of 109 identified differentially expressed genes were targets of mTORC2. 33 The authors concluded that the dysregulation and subsequent suppression of genetic pathways regulating mitochondrial dysfunction, oxidative phosphorylation, and EIF2 Signaling is unique to the cortex of alcohol-exposed brains and are regulated specifically by mTORC2. Chang et al. also observed that multiple members of the mTORC2 complex were upregulated in the alcohol-exposed fetal cortexes, in addition to downregulation of mTORC2 targets which are normally negatively regulated by mTORC2. Fetal cerebral cortical neuroepithelial stem cells derived from C57BL/6 mouse fetuses (GD12.5) cultured in alcohol containing medium (0, 160, and 240 mg/dL) also reproduced the suppression of mTORC2 target genes Atp5e and Atp5mf. 33 These genes are notable as they directly impact mitochondrial function and metabolism. In addition to these discoveries, a study by Lee et al. revealed increased expression of RICTOR in the fetal hippocampus following binge-alcohol exposure in Sprague-Dawley dams (4.5-6 g/kg, GD5-21). 31 mTORC2 has a documented role in establishing epigenetic changes in regard to gene functions and can lead to alterations in both metabolism and oxidative respiration following alcohol exposure, 33 however more work needs to be done to elucidate the cooperation between mTORC1 and mMTORC2 with regard to oxidative stress responses.

| Changes in brain autophagic flux attributed to mTOR signaling disruptions in FASD
Alcohol and mTOR interact to regulate autophagy. The process of autophagy, also known as autophagic flux, includes the sequential steps of phagophore nucleation or initiation, elongation and subsequent maturation of cargo sequestration, as well as degradation of the sequestered cargo. 25,34 This tightly regulated pathway is controlled by a group of autophagy-related genes (ATG) and leads to lysosomal degradation and turnover of cytoplasmic organelles and proteins. As a neuroteratogen, exposure to alcohol produces reactive oxygen species (ROS) that can result in oxidative stress in the developing brain. This oxidative stress can cause neuronal death mainly in the form of apoptosis, which has been known to be modulated by autophagy. 34 SH-SY5Y neuroblastoma cells treated with 0.4% alcohol in medium and PND7 mice injected twice subcutaneously with alcohol (2.5 g/kg) over 8 h showed that alcohol-induced LC3 lipidation, a marker for autophagy, 35 was mediated by increasing autophagic flux rather than by inhibiting autophagosome degradation. 34 This suggests that alcohol increased autophagic activity rather than block the fusion of autophagosomes with lysosomes during development. mTOR is a known regulator of autophagy and may be an important pathway in the induction of a cellular self-protective response to alleviate alcoholinduced oxidative stress in the fetal mouse brain. For example, rapamycin, an inhibitor of mTOR and an initiator of autophagy, has been shown to further increase alcoholinduced upregulation of LC3 lipidation, as well as the formation of autophagosomes (Figure 3). 34 Notably, Chen et al. went on to demonstrate that stimulation of autophagic flux by rapamycin decreased alcohol-induced ROS production and also confirmed that antioxidants alone alleviated alcohol-stimulated autophagy. Therefore, mTOR signaling modulation may activate autophagy as a result of alcohol neurotoxicity and is important for maintaining cellular homeostasis in pregnancy. 30

| mTOR & neurodevelopment dysregulation following alcohol exposure
mTOR not only regulates autophagic machinery as a means to reduce the burden of oxidative stress brought on by alcohol, but mTOR is also a crucial signaling pathway for dendritic pruning. Dendritic pruning is an essential part of normal brain development, selectively removing inappropriate connections that form during the early stages of neuronal growth. 36 A binge alcohol paradigm, in which adolescent PND30-43 C57BL/6 mice were given daily intraperitoneal injections (3 mg/kg), reported a greater number of dendritic spines in dentate gyrus and granule cells, suggestive of reduced pruning in the hippocampus. 36 Additional 3D analysis of spines performed using z-stacks, noted an increase in thin spines in females and stubby spines in males. This result was accompanied by lower levels of LC3-II, implicating autophagy is responsible for dendritic pruning. 36 The addition of rapamycin returned the number of dendritic spines to a normal density, as well as restored normal morphology, resulting in hippocampal dendritic spines similar to those in normal development. This shows that alcohol induces changes in mTOR signaling, which in turn mediates autophagic activity, and can result in improper dendritic pruning and neurodevelopment.
Another study cultured cortical microvessels on PND2 from NMRI mice in medium with 50 mM alcohol in the presence or absence of 200 nM rapamycin. 37 Girault et al. confirmed a reduction in mTOR signaling along with an upregulation of LC3 lipidation following alcohol treatment in mouse brain endothelial cells. They also showed that autophagy is responsible for protection in both astrocytes and neurons, and suggest that this neuroprotection provided by autophagy may result from mitophagy, or specifically the removal of damaged mitochondria. Notably, the reduction in mTOR signaling was correlated with a reduction in neuronal length, which indicates altered vascular plasticity, as rapamycin prevented this effect. 37 This work demonstrates that proper mTOR signaling is likely important for maintaining autophagy, vascular plasticity and therefore proper neurodevelopment.

| mTOR linked to cognition, behavior, and memory impairments following alcohol exposure
In addition to the interaction between mTOR and autophagy as an important driver of ROS clearance, synaptic pruning, and appropriate vascular plasticity following alcohol exposure in neurodevelopment, autophagy is implicated in the maintenance of long-term cognition and memory function as well. During memory formation, mTOR is activated in multiple regions of the brain, however a critical control step in synaptic plasticity and memory formation is the regulation of hippocampal protein synthesis. 38,39 In this context, behavioral tests including Hebb-Williams mazes, novel object recognition, and passive avoidance tests were conducted in C57BL/6 mice exposed to alcohol from PND30-43 (3 g/kg). 36 Interestingly, these mice required significantly more time to complete each maze, and in fact, failed to recognize novel objects, displayed a significantly shorter discrimination index on the object recognition task, and also a shorter latency during passive avoidance tests compared with those in both saline-treated and rapamycin-treated groups. Rapamycin restored low cAMP-responsive element-binding protein (CREB) levels, a cellular transcription factor implicated in neuronal plasticity and long-term memory formation in the hippocampus, following alcohol exposure. 36 Other studies done in alcohol-exposed PND4-9 Wistar rats (5 g/ kg) confirmed cognitive impairments, spatial learning impairments, and long-term recognition memory impairments, and showed that pre-treatment with rapamycin (3 and 10 mg/kg) ameliorated these effects. 32,40 Lopatynska-Mazurek et al. attributed these cognitive impairments to overall challenges in spatial processing and changes in biomarkers within the DG, 32 all of which were reversed in rapamycin-treated rats. Taken together, these results suggest alcohol can lead to cognitive alterations and poor retention in memory tasks, and supports the contention that mTOR regulates synaptic plasticity, memory storage, and cognition.

| Activation of autophagic machinery via mTOR
Let us examine multiple ways alcohol has been demonstrated to interact with mTOR to produce a neuroprotective response. At the molecular level, mTORC1 regulates autophagy through modulation of specific proteins. For example, normally mTORC1 phosphorylates ATG13 and ULK1/2, which in turn inhibits the activity of the ATG13-ULK1/2-RB1CC1/FIP200-ATG101 complex and prevents the activation of autophagy. 34 However, alcohol can inhibit phosphorylation of two main mTORC1 substrates: 4E-BP1 and S6K1. It was also reported that rapamycin and alcohol together produced greater dephosphorylation of S6K1 and 4E-BP1 than did treatment of cells with either rapamycin or alcohol alone. Both rapamycin and a knockdown of mTOR by siRNA each offered neurons protection against alcohol-induced apoptosis and show the importance of the mTORC1 pathway in alcohol-induced autophagy and neurotoxicity. 41 Another potential mechanism for activation of autophagy following alcohol exposure is via adenosine 5′-monophosphate-activated protein kinase (AMPK), which is upstream of mTORC1 and also plays a critical role in regulating autophagy in neurons. 41 Normally, the activation of AMPK inhibits mTORC1 activity through the phosphorylation of the mTORC1-associated proteins RAPTOR and Tuberous Sclerosis 2 (TSC2), thereby activating autophagy. AMPK is activated via oxidative stress, which is a requirement for ROS-stimulated autophagy, and indicates that alcohol can activate autophagy by regulating AMPK in neuronal cells. 41 These results support the role of mTOR-regulated autophagy in oxidative stress relief, alterations in vascular plasticity, synaptic spine changes, along with memory and cognitive dysfunction in multiple brain regions induced by binge-like exposure to alcohol. It was also shown that inhibition of mTOR via rapamycin or knockdown of mTOR offers neuroprotection against alcohol-induced cell death, further supporting that the mTOR pathway is involved in alcohol-induced autophagy and cell death. 41 Therefore, it is likely that alcohol modulates autophagy in the brain by inhibiting the mTOR pathway. Taken together, rapamycin may be a promising pharmacological intervention against developmental alterations associated with binge alcohol exposure and may provide future studies with further upstream and downstream pharmacological targets.

| IMPLICATIONS OF mTOR SIGNALING IN ALCOHOL-EXPOSED PLACENTA
The mTOR signaling pathway is directly regulated in the placenta by sensing oxygen and nutrient availability. 42 Among nutrients, chronic alcohol use has a targeted negative effect on folate transport through the placenta. 43 Low gestational folate levels are associated with impaired placental mTOR signaling, which can mediate an inhibition of placental nutrient transporter expression, leading to conditions like intrauterine growth restriction (IUGR). 44 Similarly, in baboons, fetal weight and mTORC1 signaling were both directly correlated with maternal folate levels. 45 In humans, the mTORC1 complex is highly expressed in the placental syncytiotrophoblast and decreased mTOR signaling in the placenta was associated with fetal growth restriction. 42 Interestingly, human placental mTOR signaling was observed to increase in cases of large for gestational age fetuses. 45 So, the importance of both mTOR potentiation and disruption in the placenta should not be underestimated.
In comparing appropriate for gestational age and IUGR placentas, several downstream signaling proteins of mTORC1 have been analyzed for their phosphorylation states. 45 4EBP1 was not differently phosphorylated, while S6K1 was significantly so, indicating that alcohol's influence on intrauterine growth restriction may occur via the S6K1 component of the activated mTORC1 downstream signaling cascade. 45 Interestingly, in human IUGR cases not limited to alcohol exposure, overall mTOR protein expression in IUGR placentas was found to be 51% higher compared to appropriate for gestational age placentas, 45 potentially indicating a self-correcting feedback mechanism to boost activated mTOR signaling. mTOR is a major signaling component in the cell migration processes of first-trimester extravillous trophoblasts, a type of differentiated placental cell critical for developing the placental-uterine interface. 46 Administration of growth factors which activate mTOR through activation of upstream AKT and PI3K resulted in an increase in the motility of the trophoblast. 46 Alcohol treatment (20-40 mM for 24 h) on a first-trimester extravillous trophoblast cell line SGHPL4 was found to reduce proliferation, as well as inhibit amino acid uptake at 40 mM alcohol concentrations. 46 Later in development, alcohol negatively affects placental uptake of glucose and folic acid, leading to nutrient deficiencies in the placenta and fetus. 46 Others have noted particular structural deficits. For example, alcohol consumption during pregnancy does not appear to significantly alter placental weight, even as birth weights and placental-to-birth weight ratios are significantly affected. 47 As the placenta is a crucial interface point for the fetus to receive nutrients, altering its mTOR pathway inflicts consequential effects on the developing fetus, even if the placenta itself is only moderately transformed.

| ALTERATIONS IN mTOR SIGNALING IN BONES FOLLOWING ALCOHOL EXPOSURE
Outside of pregnancy, more is known about alcohol's teratogenic effects on mTOR in the bones of adult males and nonpregnant females. Broadly speaking, chronic binge drinking has negative effects on bone remodeling, which can lead to loss of bone mass and eventually osteoporosis. 48 Alcohol intake in nonpregnant female mice (50 mM for 3 days) induced apoptosis in osteocytes, promoted osteoclast differentiation, suppressed osteogenesis, as well as increased adipogenic differentiation. 49 In the same study, alcohol exposure led to elevated phosphorylation levels of mTOR and S6K1 in bone marrow mesenchymal stem cells (BMMSCs). Inhibition of the mTOR pathway with rapamycin (1.5 mg/kg/day for 14 days) restored the disruption in BMMSC differentiation induced by heavy alcohol consumption. 49 In another study, rats were given increasing doses of alcohol in their drinking water (from 5-30% w/v) over a six-week period to model chronic alcoholism. 50 Eight out of 10 rats in the alcohol group exhibited severe osteonecrosis of the femoral head. In human bone mesenchymal stem cell culture, alcohol (50 mM) significantly downregulated Osteocalcin (OCN), Collagen 1, and RUNX family transcription factor 2 (RUNX2) expression through activation of the mTOR signaling cascade, as evidenced by significantly increased levels of p-mTOR and p-S6K1 (Ser371). 50 This increased phosphorylation of mTOR was observed in conjunction with an enhanced proliferative capacity of bone mesenchymal stem cells, promoting the development of an age-related phenotype wherein the bone mesenchymal stem cell's capacity for self-renewal and its potential for differentiation are reduced. 50 nM rapamycin and 10 mM betaine treatment reversed the inhibition of osteogenic gene expression brought on by alcohol exposure. 50 Alcohol has also been found to downregulate human dental pulp cell differentiation and mineralization via activation of the mTOR pathway. 50 mM alcohol significantly upregulated the activity of p-mTOR in dental pulp cells, without altering the viability of those cells. 51 The upregulation of the mTOR pathway by alcohol in these cells led to a downregulation of known odontoblastic markers as measured by mRNA levels, including OCN, RUNX2, Dentin Sialophosphoprotein, and Dentin Matrix Protein 1. Rapamycin treatment relieved the inhibition of mRNA expression of those markers. This indicates that alcohol may be harmful to the regenerative capabilities of dental pulp cells, and that this phenotype is likely a result of mTOR signaling potentiation. 51 The growth-restricted phenotype observed in alcoholexposed rat pups is an expected outcome of whole-body skeletal bone growth restriction. Increased potentiation of mTOR activity appears to be the cause of restricted fetal growth, and further research into this connection is merited.

SIGNALING IN MUSCLE FOLLOWING ALCOHOL EXPOSURE
In adult men and nonpregnant women, skeletal muscle protein synthesis is partially inhibited after acute alcohol intoxication. 52 Alcohol exposure affects all muscles, but those effects are most noticeable in type II fast-twitch muscle fibers of chronic alcoholics. 53 Since alcohol has not been shown to downregulate the total number of ribosomes, an effect on translation has been inferred in these tissues. 52 It is also speculated that the downstream kinase functionality of mTORC1 activation is suppressed by alcohol exposure, either directly or indirectly, because the total amount of mTOR core and associated proteins was not changed. 52 Regarding pathway effects, decreases in phosphorylation states in upstream insulin receptor IRS-1 or protein kinase B are not directly involved in alcohol's effect on mTOR phosphorylation and the alcohol-induced decrease of phosphorylation of nearby downstream mTOR pathway components involved in translation within skeletal muscle. 53 Alcohol exposure can also affect mTOR in muscle cells by way of altering how they utilize nutrients. For example, under normal conditions, leucine supplementation enhanced the phosphorylation of eIF4G, S6K1 and mTOR itself, promoting additional protein synthesis. Alcohol treatment appeared to induce a leucine resistance within skeletal muscles, introducing a state where leucine no longer promoted those changes when administered in combination with alcohol. 52 eIF4E-BP1, when exposed to alcohol in the gastrocnemius of rats, was found to reduce the phosphorylation of three key threonine residues by 40% to 80%, leading it to return or remain in its inactive eIF4E•eIF4G complex. 52 Meanwhile, in the heart, an important regulator of the antioxidant response to cardiomyopathy induced by chronic alcohol intake, the protein NRF2, can function as a metabolic switch and regulate downstream targets of mTOR. 54 Interestingly, in both cardiac and skeletal muscle, alcohol consumption (rats fed 12% to 36% of total caloric intake as alcohol, ad libitum 54 ) increases the binding attraction between RAPTOR and DEPTOR, promoting a more closed conformation of the complex, thereby suppressing the activity of mTORC1 and its active downstream components. 55 The effect of chronic alcohol intake on RAPTOR is a phosphorylation on ser-792. This residue is naturally phosphorylated by AMPK, and this phosphorylation leads to an inhibition of mTORC1's downstream pathway components. 55 However, in chronically alcoholfed rats, this ser-792 residue of RAPTOR is phosphorylated without any observable increase in AMPK activity. There is no mechanistic explanation for this effect yet.
Alcohol-derived pathologies have been extensively studied in adults, but the disruption of fetal muscle development by maternal alcohol consumption is a frontier with great potential for breakthroughs in FASD research. This is especially important area of study, as there are significant metabolic differences between fetal and adult skeletal muscle tissues.

| Elevated mTORC2 signaling in myoblasts following alcohol exposure
It is important to note that mTORC2 may also play a role in regulating mTORC1 following alcohol exposure. For instance, alcohol-exposed C2C12 mouse myoblasts (100 mM, 18-24 hours) revealed elevated levels of total RICTOR, mSin1, PRR5, and DEPTOR, which are all mTORC2 complex components. 56 RICTOR was then shown to have decreased binding with DEPTOR and 14-3-3 protein, regulators of metabolic function, 57 as well as an enhanced association with the remaining aforementioned mTORC2 proteins, demonstrating increased mTORC2 activity. 56 To further test RICTOR's role in regulating alcohol-induced changes, cells were transfected with RICTOR shRNA in order to produce RICTOR knockdown cells. Interestingly, RICTOR knockdown cells treated with alcohol revealed that the inhibitory effect on protein synthesis was partially ameliorated, as compared to scrambled shRNA transfected controls. 56 Noting that mTORC1 activity typically decreases following alcohol exposure, 58 this may also signal increased mTORC2 activity and a potential positive feedback loop between these complexes. These studies together represent an early step indicating a homeostatic balance between the two mTOR complexes following alcohol treatment. This balance could prove fruitful in the search for more mechanistic causes within the field of fetal development and maternal alcohol consumption.

| CONCLUSION
In this review we have aggregated supported works that depict mTOR as a clear linchpin in alcohol-impaired fetal development and non-pregnant adult binge-alcohol paradigms. Recent studies have elucidated the structure of this signaling pathway, allowing us to mechanistically peer into how the mTOR signaling cascade transduces cues from binge alcohol exposure into molecular action. mTORC1 in particular contributes to many processes implicated in alcohol pathology, including protein synthesis, autophagy, nutrient transport, and cell proliferation, however little is known about mTORC2 and its role in FASD. We have also considered mTOR as a potential target in various tissues for future FASD studies and have noted mTOR as a possible target for pharmacological intervention.