Branched Chain Amino Acid Metabolism in Developmental Brain Injury: Putative Mechanisms and Therapeutic Potential

Millions of children are born annually with acquired or perinatal developmental brain injury and consequent neurodevelopmental disability [1]. Developmental brain injuries are caused by exposure to multiple prenatal or perinatal forms of injury, including inflammation and hypoxia. Unfortunately, there are limited targeted interventions beyond supportive care. Given the number of affected children around the world, there is a pressing need to uncover the molecular mechanisms causing perinatal brain injury and to rigorously test potential therapeutic interventions that will improve clinical outcomes for these vulnerable populations.

Developmental brain injuries can be diagnosed at different stages of gestation, including perinatal complications (e.g., hypoxia or intrauterine infections) that display early signs of neurological dysfunction or diagnosed prenatally (e.g., some intrauterine hemorrhages or fetal growth restriction). Despite variable etiology, the long-term sequelae of developmental brain injuries include reduced brain volume and white matter injury. The frequency of these similar features between mechanisms of injury suggests the possibility of both a common etiology and a common therapeutic response to attenuate injury. The disruption of normal metabolism is a putative etiologic factor in developmental brain injuries. While efforts are ongoing, there are currently no serum, urine, or cerebrospinal fluid (CSF) biomarkers that reproducibly identify the cause of developmental brain injury. This diagnostic gap underscores the urgency of gaining a comprehensive understanding of altered brain metabolism in developmental brain injury to identify metabolites that may serve as diagnostic or prognostic biomarkers. To realize this goal, it is essential to bear in mind that the metabolism of the neonatal brain differs greatly from that of older children and adults. Glucose is the primary energy substrate of the adult brain, but both the fetal and infantile brain rely heavily on energy derived from ketone bodies [2]. In fact, the uptake of ketone bodies in the infant brain is at least 4 times higher than that in adults [3, 4] and accounts for up to 25% of basal energy requirements [5]. Elucidating the contribution of various metabolic pathways that are crucial to energy substrates in the injured neonatal brain could reveal new potential mechanisms for therapeutic interventions.

Surprisingly little attention has focused on the possibility that developmental brain injuries may result from disruptions in branched chain amino acid (BCAA) metabolism. The three BCAAs – leucine, isoleucine, and valine – are essential compounds that cannot be synthesized de novo. In addition to being utilized for protein synthesis, BCAAs can also be catabolized to tricarboxylic acid (TCA) cycle intermediates, and in the case of leucine, to acetoacetate, a ketone body (Fig. 1a). BCAAs also act as signaling molecules as leucine activates the mTOR cascade signaling [6‒9] to promote anabolic cellular processes. Interestingly, supplementation of BCAAs has recently been identified as a potential therapy in traumatic brain injury (TBI), and both preclinical animal models and small clinical trials have demonstrated improvements in neurocognitive outcomes following TBI [10‒13]. While it is unclear exactly how supplementation of BCAAs contributes to improved outcomes, it is currently thought that benefits from BCAAs may be either through the anabolic effect of excess BCAA content, or that BCAAs may replenish the balance of neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA). Given the relative ease and apparent safety of treatment (i.e., altering BCAA content in infant formula), its utility should be considered in the context of early interventions for developmental brain injuries. This review aimed to broadly survey the involvement of BCAA metabolism in hypoxic and inflammatory mechanisms of developmental brain injury, considering the putative contribution to injury by disrupted BCAA metabolism and how such insight might be translated into targeted therapeutic interventions.

The initial step of BCAA catabolism is mediated by BCAA transaminase, BCAT1 (or BCATc) in the cytosol and by the mitochondrial isoform BCAT2 (or BCATm). BCAT1 and BCAT2 show distinct whole-body expression patterns – BCAT1 is highly expressed in the nervous system [14‒19] while BCAT2 is high in peripheral tissues [14]. Within the brain, BCAT1 is primarily confined to neurons, whereas BCAT2 is highly expressed in astrocytes [20]. BCAT1 and BCAT2 reversibly transfer the amino group from BCAAs to alpha-ketoglutarate (α-KG) to form glutamate and the branched chain ketoacids (BCKAs). The BCKAs are then irreversibly catabolized by branched chain ketoacid dehydrogenase (BCKDH), the activity of which is inhibited by branched chain ketoacid kinase (BCKDK) and, conversely, activated by a Mg²⁺/Mn²⁺ dependent protein phosphatase (PPM1K). A series of downstream reactions funnels BCAA-derived carbons into the TCA cycle as acetyl-CoA or succinyl-CoA (Fig. 1). Notably, transamination by BCAT1 or BCAT2 can occur both in the “forward” direction (i.e., BCAA + α-KG → BCKA + glutamate) and the “reverse” direction (i.e., BCKA + glutamate → BCAA + α-KG) (Fig. 1a). BCAT enzymes are equilibrium enzymes, meaning that the relative concentrations of their products and reactants drive the direction of transamination. This process is distinct from the catabolism of the BCAA carbon or the oxidation of the BCAAs to the TCA cycle intermediates, which is irreversible following decarboxylation of the BCKAs (Fig. 1b).

BCAAs are essential amino acids that can only be obtained from a diet and are transported from blood to brain by the large neutral amino acid transporter (LAT1). Leucine is taken up by the brain more rapidly than any other amino acid [21], and the transfer of its amino group to α-KG plays a major role in forming the excitatory neurotransmitter glutamate. Whole-body isotope tracing reveals that BCAAs are highly oxidized to TCA cycle intermediates in skeletal muscle, brown fat, and liver, while contribution to these products by the brain is minimal [22]. Instead, BCAAs are primarily metabolized to donate nitrogen within the brain – at least one-fifth of all glutamate nitrogen is derived from BCAAs in rodent brains [23‒26]. While the contribution to glutamate from BCAAs seems to be slightly lower in human brain cell types [26], BCAA transamination to yield glutamate exceeds BCAA oxidation in human and mouse whole brain slices [26] as well as primary cultured mouse astrocytes [23]. One potential critical role of BCAA metabolism is to facilitate a putative “astrocytic-neuronal BCAA-glutamate cycle,” in which transamination (i.e., BCAAs → BCKAs + glutamate) and reverse transamination (i.e., BCKAs + glutamate → BCAAs) [27, 28] enable the brain to rapidly produce and clear glutamate from synaptic clefts (Fig. 1c). Thus, it is pertinent to consider whether transamination by BCAT enzymes preferentially produces BCAAs or BCKAs in the context of developmental brain injuries that cause glutamate excitotoxicity.

There is increasing evidence suggesting that BCAA metabolism plays a role in immune cell function and response. BCAAs are necessary for immune cell function both in vitro and in vivo, but high BCAA content may impair immune function [29]. Currently, the effect of BCAAs on inflammatory processes and responses in the brain is not yet understood to be either pro- or anti-inflammatory. BCAA and lipopolysaccharide (LPS) administration in rats increases pro-inflammatory and decreases anti-inflammatory cytokines in the hippocampus and cerebral cortex [30]. Acute administration of high doses of BCAAs increases pro-inflammatory cytokines in the hippocampus and cortex of infant rodents, but chronically high levels of BCAAs seem to have the opposite effect in older animals [31]. This difference could be related to the effect of excess BCAAs on microglia, which De Simone et al. [32] have demonstrated to promote an incomplete “M2,” anti-inflammatory phenotype. In this study, high BCAA content promoted phagocytosis in microglia, suggesting that BCAAs may activate the immune response in this cell population. Whether BCAA activation of microglia can ultimately reduce inflammation via promoting phagocytosis within the neonatal or adult brain is not known. However, the differences between inflammatory responses in the neonatal and adult brain responses suggest that studying BCAA metabolism in the context of inflammatory developmental brain injury could reveal pathological processes that impact neurodevelopment.

Maternal immune activation is caused by illness or viral infection during pregnancy and has been hypothesized to increase the risk of neurodevelopmental disorders like autism and schizophrenia in affected offspring [33‒37]. There are various potential mechanisms through which maternal immune activation may affect neurodevelopment, including cytokine production that crosses the placenta and blood-brain barrier, immune dysregulation, and activation of microglia in the fetal brain. Animal models have demonstrated that BCAA metabolism may be perturbed by maternal immune activation, although the effect on available BCAA content in the fetal brain is mixed. In 1 study of pregnant rats, polyinosinic:polycytidylic acid (poly(I:C)) administration decreased maternal plasma and placental BCAA content, yet BCAA levels in the fetal brain were unchanged [38]. However, another study concerning poly(I:C) infection in pregnant rats noted significant downregulation of leucine and isoleucine in fetal brains, with no changes to amino acid transporter mRNA levels in the placenta or fetal brain [39]. Maternal immune activation in non-human primates, induced by a derivative of poly(I:C), poly-ICLC, upregulated BCAAs and intestinal samples and fecal matter from fetuses, but not in CSF [40]. Cord blood from pregnant humans infected with mild cases of COVID-19 had increased levels of leucine and isoleucine [41], but the effect on the fetal brain was not measured. These conflicting results concerning the effect of maternal immune activation on BCAA content in the fetal brain suggest BCAA levels are likely not a sensitive biomarker for maternal immune activation, but there is potential that alterations to BCAA metabolism by maternal immune activation could play a role in disrupting neurodevelopment in the fetus. While BCAA content was unchanged by poly(I:C)-induced maternal immune activation in the fetal rat brain, metabolic labeling suggested that infection at E15 significantly decreased maternofetal transport of leucine [38]. In the same study, infection at E21 increased leucine transport [38], suggesting that the timing of illness may influence maternofetal BCAA exchange dynamics, and consequently may reflect or induce alterations in BCAA metabolism in the fetal brain. In one study, Bcat1 was significantly upregulated in the fetal brain 24 h after poly(I:C) administration at E14, but not Bcat2 [39]. While the authors speculate that this could indicate increased BCAA catabolism, it is pertinent to consider the reversibility of the enzyme, especially since Bckdk, a downstream regulator of BCAA oxidation, is not significantly upregulated at this time [39]. In fact, Bcat1 could increase BCAA content by preferentially catalyzing reverse transamination if BCKAs are not oxidized by Bckdh after enhanced conversion of BCAAs to BCKAs. If so, the decrease in maternofetal BCAA transport observed at this timepoint [38] could be compensatory in nature. In addition, which cell types drive the upregulation of Bcat1 in the fetal brain is unclear. For example, it is unknown whether microglia, like macrophages, expresses high levels of BCAT1 [42]. Given these possibilities, and the fact that maternal immune activation may have different effects at different stages of gestation, stable isotope tracing studies and parsing out metabolic contributions from different cell types in animal models will be crucial for understanding how maternal infection affects BCAA metabolism in the fetal brain.

Intrauterine inflammation, also known as chorioamnionitis, is characterized by the infection and inflammation of intrauterine tissues that surround the fetus, including the chorion, amnion, placenta, and even amniotic fluid. Both maternal immune activation and intrauterine inflammation expose the fetus to a relatively intense immune response, but the latter perturbation greatly increases the risk for preterm delivery [43], and consequent heightened risk for neurodevelopmental disorders, as well as cognitive, sensory, and motor impairments [44‒46]. In a model of intrauterine inflammation that involves LPS injection into the uterus of pregnant mice, the concentration in the fetal brain of leucine, isoleucine, and valine is increased 6 h after injection [47, 48]. However, these differences disappear at 48 h after injection [47, 48], suggesting that the effect of intrauterine inflammation on fetal brain BCAA metabolism is transient. Notably, downstream BCAA metabolites increase in both the placenta and fetal brain [47, 48], which may indicate that LPS-induced intrauterine inflammation increases BCAA catabolism. If so, higher BCAA concentrations in the placenta and fetal brain would imply increases in maternofetal BCAA transport, although this has not yet been investigated.

These observations, made in animal models of maternal immune activation and intrauterine inflammation, demonstrate a link between BCAA metabolism and inflammatory developmental brain injury in the fetal brain. While alterations in BCAA metabolism have been noted in maternal immune activation models utilizing poly(I:C) and intrauterine injections of LPS, it is pertinent to note that different models of maternal immune activation exist. Thus, it may be important to consider that the kind of infection could induce distinct metabolic profiles in fetal brains. While both maternal immune activation and intrauterine inflammation animal models suggest potential alterations in BCAA catabolism (Fig. 2), BCAA transamination and oxidation remain to be investigated thoroughly using stable isotope tracing. Moreover, whether these alterations promote or combat inflammation in the fetal brain is unclear. Elucidating the effect of BCAAs on neuroinflammation in the context of inflammatory developmental brain injuries is critical to assess how modulation of BCAA levels in the neonatal brain could yield therapeutic benefit or further exacerbate injury.

Compromised oxygen supply to the fetus induces prenatal hypoxia, which is a significant cause of brain injury and neurodevelopmental disorders like autism and epilepsy. Hypoxia has previously been linked to BCAA metabolism in studies of cancer metabolism. Tumor microenvironments can be hypoxic in poorly vascularized solid tumors, and local hypoxia is important in cancer cell metabolism. For instance, the “Warburg Effect” describes a phenomenon in which cancer cells preferentially produce lactate rather than pyruvate [49‒51], like the shift from aerobic to anaerobic respiration that occurs in a hypoxic setting. BCAA metabolism is modulated by cancer, as plasma BCAA levels [52‒54] are elevated in various cancers, and both BCAT1 [55‒58] and LAT1 [59‒66] are upregulated in tumors and cancer cells. Cancer cells may exploit BCAA metabolism to artificially keep BCAA levels high and promote anabolism by mTOR activation. In addition, evidence suggests that reprogramming BCAA metabolism may also reduce oxidative stress induced by hypoxia, thus promoting tumor survival in hypoxic settings. BCAT1 has been shown to reduce reactive oxygen species (ROS) in cancer cells and human macrophages [55, 67]. In addition, inhibition of bcat-1 in C. elegans neurons increased the oxygen consumption rate [68], suggesting that BCAT1 mediates oxidative stress. Notably, BCAT1 is highly expressed in the central nervous system [14‒19], and recent work in glioblastoma cells suggests that BCAT1 is a hypoxia-inducible factor (HIF) response gene [69]. Its upregulation by hypoxia in various cell types [69‒71] suggests a direct relationship between BCAA metabolism and hypoxic brain injury.

Transient hypoxic events that limit oxygen supply to the fetus at term can cause a perinatal brain injury known as hypoxic ischemic encephalopathy (HIE). HIE can result from placental abruption, uterine rupture, preeclampsia, as well as other complications during birth. Approximately 40% of infants diagnosed with HIE develop a neurodevelopmental disorder [1, 72], suggesting acute hypoxia has dramatic effects on the developing brain. More severe cases of HIE present with large brain lesions, which are commonly studied using the Vannucci model [73], which exposes early postnatal rodent pups to hypoxia after ligation of the carotid artery to induce ischemic injury in the ipsilateral side of the brain. Andiné et al. [74] determined that the excitatory amino acids glutamate and aspartate are increased in the affected hemisphere in this model, and that BCAA also increases in both the intra- and extra-cellular fluid of the cortex. More recently, another group noted increases in valine in the cortex, midbrain, and hippocampus in the same model [75]. Anoxia without ligation of the carotid does not influence leucine levels in the rat brain [76], suggesting that elevations in BCAA concentrations may be related to severity of injury, as hypoxia alone induces milder brain injury in neonatal rodents [73, 77]. Severe hypoxic exposure in fetal lambs increases BCAAs in CSF [78], and newborn pigs exposed to hypoxia have increased concentrations of valine in urine [79]. In addition, leucine [80‒83] and isoleucine [82, 83] are elevated in cord blood by birth asphyxia and HIE. While these findings have been demonstrated in animal models, human studies of HIE indicate that serum proteins serve as biomarkers of hypoxic brain injury [84]. Indeed, a diagnostic “footprint” that integrates multiple metabolites and proteins may be beneficial in identifying HIE from cord blood samples [85]. BCAAs, like other amino acids, are dysregulated by hypoxia in the developing brain. However, the upregulation of BCAAs after hypoxia does not seem to be unique to the brain tissue as BCAAs are elevated in plasma from rats exposed to hypoxia [86]. Overall, HIE seems to elevate BCAA content in the fetal brain, but future work should validate increases in BCAAs after hypoxic incidents, especially in samples from humans.

The mechanisms behind elevations in brain BCAA concentration after acute hypoxia should be thoroughly elucidated before BCAA supplementation or restriction is considered a potential intervention. BCAA concentrations increase in organotypic mouse brain slices exposed to hypoxia in vitro. This increase was found to be independent of BCAA transporter function [87], suggesting that BCAAs are not being transported into the fetal brain at a higher rate. The authors of the study note that this finding is uniquely interesting in that the BCAAs cannot be synthesized de novo and consider the possibility of a novel synthesis pathway. However, altered metabolic flux of BCAAs during hypoxia could also explain this increase. Untargeted metabolomics of a mouse hippocampal neuronal cell line exposed to hypoxia demonstrated decreases in metabolites downstream of the BCKAs [88], suggesting that BCKA oxidation could be reduced during hypoxia. Importantly, BCKDH is a dehydrogenase that is inhibited by nicotinamide adenine dinucleotide (NADH), which would rise in hypoxic conditions where the electron transport chain is impaired and cannot convert NADH to NAD+. Given that BCAT1 and BCAT2 are reversible enzymes, accumulation of BCKAs could enhance reverse transamination, and contribute to increased BCAA levels (Fig. 2). Notably, this would allow rapid consumption of excess glutamate to α-KG during glutamate excitotoxicity, which can occur in cases of hypoxic developmental brain injury. Alternatively, increased BCAA content could result from enhanced protein breakdown during hypoxia, and excess BCKAs could react with ROS [89] to reduce oxidative stress (Fig. 2). Nevertheless, to remain at a homeostatic concentration of amino acids after hypoxia ceases would require enhanced deamination of the amino acids to increase glutamate. While supplementation of BCAAs or BCKAs could be entertained as a potential therapeutic to regulate glutamate levels after injury, it is important to note that excess BCAAs or BCKAs can have detrimental effects on metabolism in a cell-type-specific manner, such as inhibition of the malate aspartate shuttle in astrocytes caused by BCKA addition [90]. Determining cell-type specific transamination dynamics through stable isotope tracing is a crucial component of assessing the potential benefits of therapeutic intervention for this kind of brain injury.

Perinatal stroke occurs when injury to brain blood vessels causes localized infarction in the brain. Disruption of blood flow and reperfusion can cause cell death in the brain and ultimately cause neurological dysfunction such as cerebral palsy and epilepsy [91‒93]. Stroke is commonly induced by middle cerebral artery occlusion (MCAO) in animal models, and its application in the neonatal period has been utilized to study mechanisms and treatments for perinatal stroke. Permanent MCAO in neonatal mice elevated leucine and isoleucine in the injured hemisphere, which could not be rescued by two different cell therapies [94]. In the case of permanent MCAO, leucine and isoleucine may remain elevated regardless of treatment as blood flow was not restored, and the injured hemisphere may still be hypoxic. In adults, plasma isoleucine levels are associated with the risk of cardioembolic stroke, but not other subtypes of stroke. Additionally, BCAA supplementation contributed to improvements in sarcopenia in adults recovering from stroke, but impact on neurological-related outcomes was not assessed [95]. Given the relationship between hypoxia and BCAA metabolism, alterations in BCAA metabolism within perinatal stroke are likely to be related to acute hypoxia.

In addition to acute hypoxic events like HIE and stroke, chronic fetal hypoxia can impact neurodevelopment and cause brain injury in neonates. Chronic hypoxia during gestation has varied etiologies, including placental insufficiency, living at a high altitude, smoking while pregnant, and congenital heart defects in the fetus. Placental insufficiency is perhaps the best studied of these factors and can cause intrauterine growth restriction (IUGR), a condition in which the fetus is small for gestational age. Uterine artery ligation is commonly used to model placental insufficiency causing IUGR in animal models. Notably, brain-sparing occurs in many cases of IUGR, where nutrients are redirected to support brain growth, resulting in smaller body size but normal head and brain size. However, uterine ligation induces white matter injury, neuronal death, and cognitive and motor deficits in offspring [96], suggesting that chronic hypoxia can induce brain injury in IUGR pregnancies. Uterine artery ligation reduces fetal plasma BCAAs during late gestation in rats [97], and Bckdh activity in skeletal muscle and liver is increased [98]. This could suggest higher BCAA utilization and catabolism in the fetus during chronic hypoxia induced by placental insufficiency. However, Lin et al. [99] suggest that decreased BCAA content in the fetus in multiple animal models could be explained by a reduction in placental-fetal BCAA transport. In fact, BCAA levels from human umbilical cord blood and maternal blood were found to be elevated in IUGR pregnancies [100] and BCAAs were increased in cord blood from selective fetal growth restriction pregnancies, which causes nutrients to be unevenly distributed across monochorionic twins [101]. In the same study, cases of growth restriction-related brain injury did not show differences in BCAAs, nor did BCAA content correlate with neurocognitive outcomes later in childhood [101]. Leucine supplementation has been suggested in treating IUGR [102], but meta-analysis across multiple animal models and human cases of IUGR suggests that BCAA supplementation does not seem to improve fetal growth [103]. The effect of BCAA supplementation on brain injury outcomes associated with placental insufficiency has not yet been investigated. While these would be important for determining if BCAA treatment is beneficial for placental insufficiencies, it may pose a challenge to determine what effects of BCAA metabolism alterations by hypoxia or differences in nutrient exchange and how these separately affect outcomes of IUGR pregnancies. The effects of pregnancy at high altitude, smoking, and fetal congenital heart defects are also associated with developmental brain injury [104‒106]. Given the emerging relationship between hypoxia and BCAA metabolism, investigating these conditions could provide an interesting perspective on how chronic hypoxia and metabolism interplay to affect developmental brain injury. Investigating these etiologies and comparing them to placental insufficiency models would offer an understanding of the relative contribution of hypoxia’s effect on BCAA metabolism or alterations in BCAA maternofetal transport to neonatal brain injury.

A relationship between hypoxia and BCAA metabolism has emerged in the field of cancer metabolism, but evidently there is a connection with pathologies caused by hypoxia in the brain as well. In the context of developmental brain injury, acute hypoxia seems to elevate BCAA concentrations in the fetal brain [74, 75], but the mechanism governing this increase is unknown. Chronic models of fetal hypoxia would allow an opportunity to understand how BCAA metabolism may alter in adaptation to long-term oxygen deprivation. It is especially important to understand how these dynamics may regulate glutamate in the injured brain, as well as how they may also contribute to reducing ROS, which would be elevated by hypoxia [107‒109]. While ROS serves as signaling molecules, it has been suggested that excess ROS production in the neonatal brain may contribute to neuronal injury in hypoxic brain injury [110]. The mechanism through which BCAT1 reduces ROS is not yet clear; however, one possibility is that BCAT1 stimulates the production of glutamate, a precursor to ROS scavenger glutathione. In addition, Hillier et al. [111] reported a novel peroxidase activity of BCAT1 in vitro, but BCKAs can also scavenge ROS [89]. Alternative explanations may include a relationship between BCAT1 and antioxidant transcriptional programs mediated by NRF2 [112]. Elucidating the mechanism(s) through which BCAT1 reduces oxidative stress remains an active point of investigation that could potentially reveal an antioxidant role for BCAT1 during hypoxic brain injury.

Therapeutic interventions targeting BCAA metabolic pathways may be attractive strategies for the neonatal population, given the feasibility and relative ease of intervention with medical infant formula. Success in treating inborn errors of metabolism (IEMs) of BCAA metabolism and TBI with dietary BCAA supplementation suggest potential for therapeutic modulation of BCAA content in the period after neonatal brain injury.

Maple syrup urine disease (MSUD) is a rare inborn error of metabolism that results from mutations in a subunit of the BCKDH complex (BCKDHA, BCKDHB, DBT) or PPM1K [113]. Loss of function mutations impair the ability to catabolized BCKAs, and thus, MSUD is characterized by dangerous elevations in BCAA levels. Left untreated, MSUD causes cerebral edema, encephalopathy, and death in the first few weeks or months of life [114]. Those affected present with lesions scattered throughout the brain with a propensity for white matter abnormalities [115‒119]. Fortunately, the advent of medical formula with restricted BCAA content has significantly prolonged the lives of individuals with MSUD. However, even with dietary restriction of BCAAs, patients are at a greater risk of developing neuropsychiatric illness [120, 121]. While MSUD is characterized by the inability to catabolize the BCAAs, another IEM called BCKDK deficiency causes unchecked BCAA catabolism and is associated with an autism-like phenotype [122]. Pathogenic mutations in BCKDK cause heightened activity of the BCKDH complex, leading to chronically low BCAA levels. While it was originally suggested that BCAA supplementation could potentially improve neurobehavioral and neurodevelopmental outcomes [123, 124], these studies use small cohorts and lack control groups. It has been recently demonstrated that reducing the activity of the DBT subunit of the BCKDH complex could yield beneficial effects in BCKDK deficiency [125]. Cobalamin C (cblC) deficiency affects the catabolism of valine and isoleucine downstream of the BCKDH complex and also causes a neurodevelopmental phenotype in the fetus that can be alleviated by nutritional supplementation. cblC converts vitamin B12 to adenosylcobalamin, a cofactor essential for the conversion of methylmalonyl-CoA to succinyl-CoA, which is a process that enables entry of valine and isoleucine carbon into the tricarboxylic acid cycle. Arising from mutations in the MMACHC gene, cblC deficiency causes intellectual disability, hydrocephalus, and visual impairment [126]. Fortunately, prenatal identification and hydroxocobalamin supplementation during pregnancy prevented intellectual and visual complications in one case [127], suggesting that early identification and prenatal supplementation can be effective in preventing adverse neurodevelopmental outcomes. Prenatal and early postnatal treatment by metabolite supplementation in IEMs suggests that dysfunction of BCAA metabolism in the fetal and infant brain can be modified with resulting improvement of neurologic outcome. As others have noted for more than a decade [128, 129], there is surprisingly little research on the time and dose-dependent effects of BCAA supplementation and restriction during pregnancy. The safety of BCAA supplementation during pregnancy on neurodevelopmental outcomes has not been fully evaluated, and BCAA use in early pregnancy may be associated with impaired fetal growth [130] and gestational diabetes [131]. Future studies should investigate the effects of maternal diet manipulations on BCAA content in the setting of both typically developing pregnancies and in cases where the fetus experiences insults that may affect brain development and are essential prior to the consideration of altering maternal BCAA dietary content as a therapeutic.

TBI is defined as a forceful mechanical impact to the brain that results in injury and devastating neurological symptomatology. While TBI is restricted to mechanical injury, molecular mechanisms underlying neurological dysfunction are hypothesized to have similar features to developmental brain injuries, such as excitotoxicity, oxidative stress, and neuroinflammation [132]. Classic treatment strategies seek to ameliorate symptoms of concussion, rather than the underlying mechanism(s) of injury. BCAA supplementation has been proposed as a potential treatment in this regard. While the efficacy of BCAA nutritional supplementation in TBI has been previously reviewed [12], here we will briefly summarize major findings over the past 30 years.

BCAA supplementation has been utilized in clinical settings other than TBI. When this approach was used to treat post-exercise fatigue, it became evident that it also enhanced cognitive performance [133]. BCAAs are also significantly reduced following brain injury [134‒136]. Aquilani et al. [137] hypothesized that BCAA supplementation in severe TBI could improve outcomes and demonstrated that this approach improved the disability rating score at 15 days after injury, as compared to the placebo group. These investigators also showed that BCAA supplementation in patients with TBI who were in an unresponsive wakefulness state improved the disability rating score in the experimental group, such that most (>2/3) of treated patients exited a state of unconsciousness, as compared to 0 in the placebo group [138]. These human findings preceded seminal work in mice that demonstrated that BCAA supplementation after TBI improves cognitive and neurological outcomes, including increased net synaptic efficacy in the hippocampus, rescued performance in a fear conditioning paradigm, and an improved sleep-wake cycle [10, 139, 140]. A subsequent study in veterans with severe TBI suggests BCAA supplementation improved sleep [141]. In their comprehensive systematic review, Sharma et al. [12] conclude that BCAA supplementation post-injury is positively associated with improved outcomes in severe TBI, but that there is insufficient evidence to make this claim in mild TBI patients. Recently, Corwin et al. [13] demonstrated that oral BCAA supplementation significantly improved the total symptom score and reduced time to return to physical activity in adolescents and young adults, suggesting that BCAA supplementation could be applicable in supporting recovery from brain injury in younger populations. The authors additionally comment that the treatment was well tolerated and produced no serious adverse events, suggesting safety in adolescents and adults. The foregoing studies imply that BCAA supplementation could be a safe, convenient, and effective therapy for selected forms of developmental brain injury.

BCAA metabolism is critical to many cellular functions, including energy homeostasis, anabolism, and production of glutamate, the primary excitatory neurotransmitter. Dysfunction or disruption of BCAA metabolism may result in impaired or altered neurological functioning. Increasing evidence suggests that BCAA supplementation may attenuate brain injury in diverse clinical settings, including TBI, hypoxia, and inflammatory states. Virtually no research has tested the efficacy of BCAA supplementation in pediatric populations, but recent evidence points to a role for such intervention in adolescents with TBI. Whether modulation of BCAAs is beneficial in developmental brain injury is still unknown. However, emerging evidence suggests BCAA catabolism may increase in the fetal brain in the context of maternal immune activation and intrauterine inflammation. Hypoxic insults to the fetus may increase BCAA content, potentially through inhibition of BCKA oxidation and re-amination to the parent amino acids, or by increased protein breakdown (Fig. 2). While these mechanisms remain to be elucidated, these points of study are undoubtedly crucial prior to preclinical studies involving BCAA supplementation or restriction as treatments for developmental brain injury.

R.C.A.-N. is and advisor for LatusBio and AskBio, on topics unrelated to this work. The University of Pennsylvania and Children’s Hospital of Philadelphia have filed patent applications related to the use of base editing for the treatment of phenylketonuria and urea cycle disorders, unrelated to this work.

R.C.A.-N. is supported by the Chan Zuckerberg Neurodegeneration Challenge Network Collaborative Pairs Grant. A.G.C. is supported by the Robert Wood Johnson Foundation Harold Amos Medical Faculty Development Program and the National Institutes of Health [Grant: 1K08NS119797-01A1 and 1R21HD114071-01A1].

M.M.C., R.C.A.-N., and A.G.C. conceptualized the review. R.C.A.-N. and A.G.C. supervised the study. M.M.C. reviewed the literature. M.M.C., M.Y., R.C.A.-N., and A.G.C. wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.