Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

Reduction of blood Phe levels has been the main treatment goal in PKU patients to minimize toxic Phe effect on brain^16,19,20. The current standard of care to achieve this is the consumption of low Phe diet which is initiated soon after birth to prevent severe brain damage. However, compliance with strict diet steadily decreases with age and among teens and adults, the majority of the patients have higher than recommended Phe levels^8,9. The only other approved therapy for severe PKU patients currently is PEGylated PAL that is a non-mammalian enzyme converting Phe into trans-cinnamic acid^13,14,15. Though efficacious, the therapy requires daily s.c. administrations, long titration period and the patients often develop immune responses both the PEG and PAL²¹. Multiple other therapies are currently in development including rAAV-based PAH gene replacement to provide a normal Phe metabolizing pathway in a more sustained and stable manner¹⁶.

To better understand potential differences between the two strategies for Phe metabolism and to study the effects of elevated Phe on liver and brain, we delivered PAL and PAH genes into livers of PAH^enu2 mice, a model of human PKU. Our data demonstrated that viral mediated gene transfer of either gene resulted in reduced blood Phe levels, though the use of PAL over-corrected when high doses were administered. Hence the use of PAL will require careful optimization to obtain normal blood Phe levels as is currently performed for PEGylated PAL in the clinic¹⁵. This is not surprising as PAL is not subject to allosteric regulation by blood Phe levels. In contrast, multiple studies have demonstrated fine-tuning of PAH activity by Phe binding to N-terminal regulatory domain of PAH^22,23. At lower Phe levels, the enzyme is maintained as a less active dimer form while with increasing Phe levels the enzyme is driven into a more active tetramer form^22,24. As expected, only PAH treatment increased the Tyr levels in the blood.

The mechanism by which high Phe causes neurotoxicity in PKU is unclear. Potential explanations include reduced amino acid transport into brain and subsequent reduction in brain neurotransmitter levels^4,25. High blood Phe competes with the transport of large neutral amino acids (including Tyr and Trp) into brain causing overall lowered levels of these amino acids in brain^4,25. We observed this more for Trp than for Tyr and this may have been due to the use of normal rodent diet. High Phe levels have also been reported to inhibit protein production and enzyme activities, particularly the enzymes involved in neurotransmitter synthesis²⁵. Our brain analyses of animals treated with PAL and PAH vectors showed that both treatments normalized brain Phe levels due to reduction of Phe in the blood. Both treatments also corrected brain Trp levels indicating a better large amino acid transport to brain. The LAT1 transporter has been reported to have high affinity to Phe resulting in lowered amino acid transport of large neutral amino acids in the presence of high Phe²⁶. Only PAH provided elevated Tyr in the brain as expected due to higher Tyr measured in blood. Despite animals being fed the normal diet, the PAL treated mice exhibited low blood Tyr levels which corresponded to lower-than-normal Tyr in the brain (Figs. 1D,E, 2B). This was expected to result in lower dopamine levels since Tyr is a substrate for dopamine synthesis. However, both PAL and PAH treatments normalized brain dopamine levels suggesting that Tyr deficiency is not the main cause for reduced brain dopamine levels in PKU mice. Other products in the dopamine pathway such as L-DOPA and norepinephrine were also consistently increased by both treatments (Fig. 3B,D). While the low Tyr levels in the PAL treatment group did not seem to be limiting for dopamine synthesis the higher Tyr in PAH treatment group may be beneficial during suboptimal Phe reduction. Interestingly, serotonin and its intermediates and breakdown products had a much higher magnitude of correction with PAH and PAL than those in the dopamine pathway. Furthermore, there was a trend of PAL treatment providing higher levels of neurotransmitters which may have been due to slightly lower blood and brain Phe levels (Fig. 3). This suggests that neurotransmitter synthetizing enzymes are highly sensitive to Phe levels in the brain.

Unlike the PAH delivery, the delivery of PAL vector resulted in detectable immune response in liver. An infiltration of inflammatory cells, mostly consisting of B-cells, was observed in all PAL treated animals using histopathology. The nature of these cells as B-cells was also supported by RNA seq analysis of liver that showed upregulation of immunoglobulin genes. The immune response was not unexpected since PAL is a bacterial protein. The material used in the clinic has been PEGylated to reduce the immune response. Despite this, high and sustained levels of antibodies both to PAL and PEG were observed in PKU patients and hence, PKU patients using PEG-PAL (Palynziq) are required to carry EpiPen as a precautionary measure^21,27.

Though neurotoxicity is the hallmark of PKU pathology, we attempted to understand the effect of hyperphenylalanemia in the liver, the major site for PAH expression. Both the transcriptome and proteomics data revealed changes in large number of genes (Figs. 5A, 6A). Most of these changes were reversed post Phe normalization by PAH and PAL treatments suggesting that these changes were caused by elevated Phe levels and hence were disease specific changes (Figs. 5A,B, 6A). Both analyses demonstrated the cholesterol synthesis pathway was upregulated in PKU with elevated expression levels of many enzymes involved in this complex multistep pathway (Fig. 5C,D, Supplementary Fig. S4D). These included SREB2, the master-regulator of the pathway while the SREB1, controlling the fatty acid synthesis was reduced. This upregulation could be in response to the low total cholesterol levels in sera of PAH^enu2 mice and PAH-KO mice observed by us and others^28,29. In PKU patients, serum cholesterol, HDL, LDL are lower than in healthy controls³⁰. Similarly, long-chain unsaturated fatty acids levels such as docosahexaenoic acid (DHA) and arachidonic acid (AA) have been reported to be lower in PKU patients³⁰. The lowered cholesterol has been proposed to be caused by the inhibition of Phe or its metabolites on HMG-CoA reductase and mevalonate 5-pyrophosphate decarboxylase^28,31.

The highest degree of over expression in the PKU naïve mice livers was from CYP4A family (Cyp4a10, Cyp4a14 and Cyp17a1) of heme-containing monooxygenases that are largely involved in oxidation of lipids (Figs. 5E, 6C). Of these, Cyp4a10 and 14 are involved in microsomal oxidation of medium to long chain fatty acids specifically with a hydroxylated terminal ώ-carbon³². Upregulation of these Cyp proteins has not been previously reported in the liver though Cyp4a14 was elevated in the brains of PAHenu2 mice³³. In the liver, Cyp4a10 and Cyp4a14 expression is known to be induced by a nuclear receptor peroxisome proliferator-activated receptor (PPARα), a ligand activated transcription factor that regulates lipid and lipoprotein metabolism. PPARα is mainly activated by elevated free fatty acids and upon activation it enhances hepatic lipid metabolism by upregulation of fatty acid translocase (FAT) /CD36 that functions to mediate the uptake of long chain fatty acids into the cell³². Fatty acids can subsequently be removed via increased peroxisomal and mitochondrial fatty acid beta-oxidation. Our data showed that the expression of both PPARα and FAT/CD36 were slightly increased in PAH^enu2 mice suggestive of potential biological impact of Cyp4a10/Cyp4a14 upregulation (Fig. 5F). Substrates of Cyp4a10 (Cyp4a11 in humans), long chain poly unsaturated fatty acid (LC-PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to be low in PKU patients suggesting possible downstream consequences of increased Cyp4a10 expression³⁴. In-fact lower levels of LC-PUFA are also observed in children on unrestricted diet suggesting that these changes are specific to the disease and not merely a consequence of PKU diet³⁵. Besides potential microsomal omega oxidation via increased Cyp4a14, enzymes involved beta oxidation, Acox1 and Acot4, were also increased by proteomics. Elevation in Cyp4a14 has also been reported in models of nonalcoholic fatty liver disease and shown to contribute to hepatic steatosis and nonalcoholic steatohepatitis by increased oxidative stress due to lipid accumulation³². While we observed an increase in expression of various glutathione transferases (Fig. 6B, Supplementary Fig. S4C), enzymes that protect cell from reactive species, it should be noted that liver pathology indicative of massive oxidative stress or consistent elevation of liver enzymes was not observed in untreated PAH^enu2 mice. Furthermore, neither liver pathology nor oxidative stress and elevated liver enzymes have been reported in PKU patients. However, PKU mice exhibit poor growth which can be corrected with Phe reduction suggesting Phe effect on energy metabolism and lipid dysfunction³⁶. Taken together, our data highlights a novel observation of family of Cyp4a protein upregulation that may increase uptake of fatty acids and their oxidation. Whether this may contribute to lowered sera cholesterol, HDL and LDL observed in PKU patients is currently unclear.

It should be noted that the changes in cholesterol and lipid levels in our PAH^enu2 mouse study were obtained with animals fed with normal rodent diet during their entire life span. However, understanding the causative nature of changes in lipid and cholesterol levels in PKU patients is clearly more complex as the disease pathology, compliance with Phe-restricted diet, fluctuations in Phe levels and other underlying genetic factors all likely impact lipid levels^30,37,38,39. It has been generally thought that the consumption of Phe-restricted diet consisting little animal derived products contributes to lower levels of lipids and cholesterol. However, even non-compliant PKU patients tend to have lower HDL levels⁴⁰ suggesting that lipid alterations in PKU patients are influenced by disease pathology in addition to diet. Interestingly, compliant PKU patients also tend to have high rate of being overweight thought to be at partially caused by the use of protein substitutes and commercial low-Phe products with high carbohydrate content^37,38. However, the effect of Phe on lipid metabolism with increase fatty acid uptake and metabolism may also play a role. Lastly, the impact of lipid alterations and increased bodyweights in PKU patients on various co-morbidities such as cardiovascular disease and atherosclerosis are important topics of discussion for the care of PKU patients and should be aided with better understanding the underlying disease.

In summary, our data demonstrated that Phe reduction could effectively be obtained with PAH or PAL expression and resulted in improved brain health in mice. Obtaining normal brain Phe levels appeared to be especially critical for restoring neurotransmitter synthesis while amino acid transport was less critical. Our data also highlighted novel changes in lipid metabolism pathways in the PKU liver indicating elevated Phe has profound effects in other organs beyond the known neurotoxicity.