In the current study, we correlate biochemical, behavioural and neuropathologic changes in recently developed MSUD mouse models in order to provide mechanistic insight and test therapeutic interventions for MSUD encephalopathy. Examinations of iMSUD mice showed a mouse model that develops neuropathology similar to human MSUD (Crome et al.
; Silberman et al.
), large accumulation of branched-chain amino acids, depletion of other essential large neutral amino acids, and brain specific growth restriction. Both iMSUD and cMSUD mice showed neurotransmitter depletion including dopamine and glutamate, as well as a correlation between the level of depletion with the rise in brain leucine. Placing the iMSUD mice on a high protein diet showed that initiation of behavioural symptoms was concomitant with neurotransmitter depletion and more severe symptoms were associated with energy depletion secondary to Krebs cycle disruption. The use of norleucine revealed a potential acute-treatment for MSUD that effectively delayed encephalopathy in cMSUD pups and iMSUD mice challenged with a high protein.
Current findings indicate a model of brain injury in MSUD that involves two converging mechanisms: (i) amino acid competition at the blood–brain barrier secondary to branched-chain amino acid accumulation; and (ii) disruption of energy metabolism secondary to branched-chain ketoacid accumulation (). On the one hand, accumulation of branched-chain amino acids, especially leucine, likely inhibits transport of other essential large neutral amino acids (threonine, tryptophan and tyrosine) that share the same transporter for brain access. This competition results in the observed depletion of essential large neutral amino acids in the brain with subsequent consequences for both protein and neurotransmitter synthesis similar to previous studies of dietary amino acid manipulation in rats (McKean et al.
; Tews et al.
; Le Masurier et al.
). Impaired protein synthesis secondary to unbalanced essential amino acids results in brain specific growth restriction in iMSUD mice and disruption of dendritic development in cMSUD pups. Additionally, inhibition of neurotransmitter synthesis (dopamine) is associated with early behavioural symptoms such as intermittent dystonia and gait abnormalities within 48 h of protein diet exposure.
Figure 10 Proposed model of brain injury and treatment effect in MSUD. (1) Loss of branched-chain ketoacid dehydrogenase function results in accumulation of branched-chain amino acids (mostly leucine) in blood and tissues including muscle, liver and brain. Rapid (more ...)
The proposed second converging mechanism in MSUD encephalopathy involves energy deprivation secondary to branched-chain ketoacid (αKIC) accumulation. Severe brain damage and death in both cMSUD and iMSUD mice correlated with accumulation of αKIC levels above 100 μmol in the brain. Similarly, enhanced survival of infant cMSUD and adult iMSUD mice with norleucine treatment was associated with reduced levels (<100 μmol) of αKIC. Energy deprivation likely results when αKIC accumulation inhibits Krebs cycle activity as previously demonstrated in vitro
(Patel et al.
; Patel, 1974
; Sgaravatti et al.
). αKIC toxicity is likely responsible for cerebral oedema as ATP depletion leads to Na+
ATPase failure and cell swelling. Increased αKG and αKIC levels found in the brain of iMSUD mice on the high protein diet are consistent with α-ketoglutarate dehyrdrogenase inhibition secondary to αKIC accumulation as previously shown (Patel, 1974
). Additionally, decreases in glutamate and aspartate are consistent with disruption of glutamate cycling secondary to αKIC accumulation as previously postulated (Yudkoff et al.
). These changes are consistent with increased lactate levels and depletion of pyruvate and ATP as shown in . These data underscore the importance of branched-chain ketoacid control and also suggests that branched-chain ketoacids are produced in the brain from available branched-chain amino acids rather than transported in secondary to accumulation in the liver.
Similar biochemical changes were found between iMSUD and cMSUD mice. However, these changes evolved more rapidly over the first few days of life in cMSUD mice, which highlights the delicate nature of MSUD in the newborn requiring prompt treatment. Leucine levels were shown to accumulate exponentially, doubling each day, in the blood and brain of newborn cMSUD mice. These findings suggest enhanced utilization of leucine in the normal immature brain, consistent with previous studies that showed increased transport of branched-chain amino acids across the blood–brain barrier in suckling versus adult rats (Banos et al.
). The immature brain readily uses alternate energy substrates such as ketone bodies (Cremer, 1982
), which may be supplied as ketogenic amino acids (i.e. leucine and lysine). Additionally, ketogenic amino acids have been shown to provide a substantial proportion of ketone bodies used for myelin synthesis (Dhopeshwarkar and Subramanian, 1979
). The pathway for ketone body production from leucine is blocked in MSUD and may account, in part, for disrupted myelination (Crome et al.
; Silberman et al.
). Accordingly, we observed an increase in brain lysine levels, which may represent compensation for ketogenic substrates similar to previously observed accumulation of brain leucine when lysine breakdown is disrupted (Zinnanti et al.
Neuropathological changes identified in iMSUD mice are consistent with previous reports of MSUD patients with inadequate dietary control or repeated catabolic crises (Morton et al.
; Simon et al.
). Similar to human MSUD, the iMSUD mice were responsive to a low branched-chain amino acid diet. Neurochemical analysis of these mice are consistent with MR spectroscopy findings in human MSUD that suggested dietary intervention could reduce brain branched-chain amino acid accumulation to near normal levels (Jan et al.
). However, adult iMSUD mice maintained on the low branched-chain amino acid diet continue to have increased T2
signals on MRI, which is similar to previous reports in adolescents and adults with MSUD and suggests lingering myelin disruption (Schonberger et al.
). Further study of this model may provide insight into the cause of these residual changes and suggest ways to prevent them.
Challenging the iMSUD mice with a high protein diet induced a catabolic crisis, which allowed the correlation of early neurochemical changes with appearance of symptoms. Using this strategy, we showed that whole brain dopamine, glutamate and GABA depletion are concomitant with onset of initial behaviour deficits. These findings correlate with neuropathological and behavioural changes during encephalopathy in human MSUD, that indicate susceptibility of motor control centers (Crome et al.
; Silberman et al.
; Hauber, 1998
; Morton et al.
). Furthermore, this model shows the first direct experimental evidence that dopamine levels are depleted in the brain during MSUD encephalopathy, which has been previously suggested based on clinical observations and reduced blood tyrosine levels in human MSUD (Morton et al.
). This finding also suggests that other catecholamines and serotonin are likely compromised secondary to tyrosine and tryptophan depletion. Therefore, tyrosine and tryptophan supplementation is expected to benefit long-term outcomes in MSUD patients and possibly lessen or prevent learning disabilities.
In the current study, whole brain homogenates were used for biochemical analysis to correlate and compare changes in the cellular amino acid pool directly with effects on Krebs cycle function and neurotransmitter synthesis. Although, associations can be made from these studies about whole brain metabolism, these findings also provide targets for future studies in CSF, which can then be compared with human MSUD for possible translational value.
Despite careful management of MSUD patients, non-specific illness can trigger catabolic crisis leading to life threatening cerebral oedema, which can be extremely difficult to manage clinically (Morton et al.
). Norleucine may provide an acute strategy to control αKIC levels and prevent cerebral oedema during catabolic crisis. In the current study, norleucine attenuated brain leucine accumulation, but had a proportionally greater affect to control αKIC levels. These data suggest that norleucine may inhibit branched-chain transaminase in addition to competing with leucine for brain access. In astrocytes, branched-chain transaminase functions in the glutamate cycle by transferring amines from branched-chain amino acids to αKG to produce glutamate and the associated branched-chain ketoacid (Yudkoff et al.
) (). The opposite reaction takes place in neurons to complete the glutamate cycle. In the case of MSUD, αKIC accumulation in neurons may drive formation of leucine and αKG over glutamate. A high αKG/glutamate ratio can disrupt mitochondrial glutamate, aspartate, malate and αKG transport resulting in Krebs cycle failure and impaired glutamate cycling (Yudkoff et al.
). This hypothesis is consistent with our current findings including increased αKG, decreased glutamate and aspartate with lactate accumulation and ATP depletion with αKIC above 100 μM. Additionally, control of αKIC levels with norleucine treatment prevented these changes and strongly supports the hypothesis that αKIC disrupts Krebs cycle function and glutamate cycling (Yudkoff et al.
The current findings provide insight into the mechanism of brain injury and potential treatment strategy for MSUD. Both iMSUD and cMSUD mouse models were shown to be invaluable for the study of MSUD and may also be used to study cerebral glutamate and dopamine metabolism. Further studies in these mouse models can help define the role of tyrosine and tryptophan supplementation in MSUD. Use of atypical amino acids to limit toxic accumulation may provide effective treatments for other neurometabolic disorders.