In this work we present several lines of evidence to suggest that the brain injury of GA-I involves mitochondrial disruption precipitated by glutaric acid production in the neuronal compartment from available lysine. Consistent with initial neuronal injury, expression of Gcdh
and the first enzyme in lysine degradation, lysine-oxoglutarate reductase (29
), are both limited to neurons. These data implicate the neuronal compartment as the predominant location of lysine catabolism and glutaric acid production in the brain. Intracellular glutaric acid accumulation may cause direct mitochondrial toxicity within neurons. Previous studies with isolated mitochondria showed swelling induced with 500–1,000 μM glutaric acid (30
). Higher concentrations of glutaric acid are consistently found in the brains of Gcdh–/–
) and GA-I patients associated with injury (6
). In the current study, mitochondrial swelling and biochemical changes consistent with Krebs cycle disruption were observed when brain glutaric acid levels reached more than 1,000 μM. Glutaric acid is a preferred substrate for the mitochondrial oxodicarboxylate carrier that normally exchanges α-ketoadipate for α-ketoglutarate in a strict counter-exchange mechanism (31
). The level of glutaric acid accumulation, depletion of α-ketoglutarate, and accumulation of acetyl-CoA are all consistent with disruption of Krebs cycle function through loss of cycle intermediates as diagrammed in Figure .
Previous studies have shown a developmental change in BBB transport kinetics for basic and neutral amino acids that switch from low affinity/high capacity in the immature brain to high affinity/low capacity with maturity (15
). Consistent with our current findings, this developmental difference was previously shown to provide a 3- to 4-fold decrease in brain lysine influx between suckling and adult rats (32
). High-capacity amino acid uptake in the immature brain supports the increased metabolic demand and protein turnover during rapid growth and myelination of the brain. However, this enhanced influx of amino acids also provides age-dependent susceptibility to GA-I and other metabolic disorders. The immature brain readily uses alternate energy substrates such as ketone bodies, which may be supplied to the brain as ketogenic amino acids (i.e., lysine and branched-chain amino acids). Ketogenic amino acids have been shown to provide a substantial proportion of ketone bodies used for myelin synthesis (33
). As brain growth and myelination are completed, metabolic demands are reduced and the brain uses glucose more exclusively (15
). Accordingly, transport and catabolic enzyme activity for lysine are reduced in the brain with maturity (32
). Therefore the immature brain is especially susceptible to metabolic disorders of amino acid metabolism such as GA-I and maple syrup urine disease (35
). This susceptibility is typically realized in the context of catabolic stress associated with fasting during a nonspecific illness. Catabolic stress leads to breakdown of muscle protein to free amino acids and enhanced amino acid uptake and turnover in the liver for gluconeogenesis (16
). Hypoglycemia in children with GA-I during metabolic crisis (8
) and in Gcdh–/–
mice on a lysine diet suggests that the process of gluconeogenesis from available amino acids is impaired. Decreased glucose levels place greater dependence on ketone bodies and ketogenic amino acids in the brain. The combination of catabolic stress and enhanced amino acid access to the immature brain provides the opportunity for large accumulations of aberrant metabolites in GA-I and other disorders of amino acid metabolism. These age-dependent susceptibilities, related to GA-I, and the proposed effect of treatments are diagrammed in Figure .
Proposed mechanism of susceptibility and effect of treatment.
Recent evidence shows that glutaric acid has limited BBB permeability, suggesting de novo synthesis rather than diffusion or transport of glutaric acid into the brain (14
). To confirm that glutaric acid is produced in the brain de novo from available lysine, we demonstrated that blocking brain lysine uptake with homoarginine decreased glutaric acid levels, resulting in reduced brain injury and increased survival. These findings are likely to have clinical relevance, since elevated brain glutaric acid levels and brain injury in GA-I patients occur despite the use of low lysine diets (4
). In the present study, controlling brain glutaric acid levels with homoarginine and glucose prevented encephalopathy, underscoring the importance of this strategy for treatment.
Currently, there is no reliable marker that can be used noninvasively for predicting the risk of brain injury in human GA-I. Brain glutaric acid levels were previously shown to correlate with injury in this mouse model (12
) but currently cannot be measured noninvasively. Glutamate and GABA depletion monitored by 1
H NMR spectroscopy correlates with increased brain glutaric acid levels and may translate for use in human GA-I to detect risk of brain injury. Glutamate depletion in this mouse model is consistent with reduced brain glucose utilization, as previously shown with immature rats (36
). Reduced brain glucose utilization was previously shown in human GA-I using 18
fluoro-2-deoxyglucose uptake studies (37
), indicating that glutamate levels may also be compromised in human GA-I with encephalopathy. Glutamate is the precursor for GABA (38
), and reduced GABA levels in this mouse model and human GA-I (7
) correlate with glutamate depletion and further indicate that glutamate levels may be compromised in human GA-I. GABA levels may be initially reduced along with glutamate as brain glucose utilization is compromised during encephalopathy (37
). Glutaric acid accumulation suppresses GABA production (21
), and the return of glutamate levels with restored brain glucose utilization may set up unopposed excitatory neurotransmission resulting in seizures and excitotoxic lesions found in this model and in human GA-I (9
). This possibility may explain the differences between glucose and homoarginine treatments and emphasizes the importance of controlling brain glutaric acid accumulation for neuroprotection.
Establishment of this mouse model provides the opportunity to test the effects of current and novel treatment strategies. Intravenous glucose administration is standard treatment for GA-I encephalopathy, but there are currently no data on the effect of this treatment in the brain. The lysine diet induces a catabolic state in Gcdh–/–
mice similar to that seen in children with GA-I during crisis, including hypoglycemia and ketosis (Figure C). Catabolic stress leads to increased amino acid accumulation and breakdown in the liver (16
). For lysine, increased catabolism was shown to be regulated by mitochondrial influx (39
), and glucose availability reduces this effect (39
). We propose that a similar mechanism regulates lysine utilization in the immature brain. In the context of catabolic stress with reduced serum glucose levels, the immature brain depends on alternate energy substrates such as ketone bodies and ketogenic amino acids. Glucose supplementation provides adequate preferred substrate and reduces the demand for alternate substrates. This reduced demand correlates with reduced brain lysine accumulation in adult brains that utilize glucose more exclusively (15
). Here we demonstrate that dietary glucose therapy reduced lysine catabolism (glutaric acid formation) more than lysine accumulation in the brain. Serum glutaric acid levels were also lower with glucose treatment (data not shown), consistent with the influence of glucose to reduce amino acid turnover in the liver (41
Combined homoarginine and glucose treatment may provide the best protection by reducing the substrate and the drive for glutaric acid production. However, the lack of cumulative effect on glutaric acid levels suggests that each treatment affects a different part of the same pathway. Glutaric acid is produced from lysine breakdown in the context of GCDH deficiency. The rate-limiting step of lysine breakdown at the mitochondrial level was shown to be lysine influx, which was increased by a high-protein diet and reduced by glucose availability (39
). Our current data support a model in which lysine access is limited by homoarginine at the BBB and by glucose at the mitochondrial level. Both glucose and homoarginine reduced lysine breakdown (glutaric acid formation) similarly, but brain lysine accumulation remained higher with glucose treatment compared with homoarginine. Therefore, both treatments reduce brain glutaric acid production by limiting lysine access, although to different compartments.
Similar to Huntington disease, the expression of the affected gene in GA-I is not specific to the striatum but results in selective striatal degeneration. Medium spiny neurons are primarily affected in both disorders (4
). Although the clinical presentation of GA-I is commonly associated with acute onset, the resulting neuropathology in this mouse model is strikingly similar to that of Huntington disease, including the involvement of cortical pyramidal neurons (12
). Corticostriatal circuitry may play a role in the pathophysiology of both disorders (43
). Further study of this mouse model may reveal factors underlying striatal-specific susceptibility that can be used to develop protective strategies.
The current findings provide insight into the age-dependent mechanism, treatment, and monitoring of GA-I and offer new strategies for the prediction and prevention of brain injury. Enhanced amino acid accumulation in the immature brain may contribute to the age-dependent susceptibility in other neurometabolic disorders (44
). Competitive transport inhibition, shown in this study using homoarginine, provides an attractive approach to developing potential treatments for these disorders.