INTRODUCTION

Phenylketonuria (PKU), a rare inherited disorder due to mutation of the phenylalanine hydroxylase (PAH) gene, causes phenylalanine build-up in the body. Untreated, PKU can lead to intellectual disability, seizures, behavioral problems, and mental disorders. However, with early diagnosis (all infants are screened at birth), PKU can be managed by the combination of a low-Phe diet and medication. A growing body of research indicates that the white-matter integrity of the brain is compromised in individuals with PKU. Using diffusion tensor imaging (DTI), our team has shown that microstructural white-matter compromise is widespread and is present even in continuously-treated individuals who have no visible of signs structural anomaly1-4. In spite of clear evidence of compromised white-matter in individuals with PKU, however, the underlying neuropathological mechanisms remain unknown. Neuropathological research is rare in individuals with PKU due to the limited availability of brain tissue, but such research is feasible in the C57Bl6/J-Pahenu2 (Pahenu2) mouse model of PKU. Herein, we report the results of the first study to examine microstructural white-matter compromise in Pahenu2 (PKU) mice using DTI.

METHODS

Beginning at weaning, cohorts of Pahenu2 mice (n=10) were fed either no-Phe chow, supplemented with Phe added to the drinking water at 100 mg/L (“low-Phe” diet), or normal chow. C57Bl6 (n=5) mice were fed on normal chow. Blood Phe levels were monitored biweekly in mice on the low-Phe diet. DTI data were collected on an Agilent/Varian 4.7-T small-animal MR scanner using an actively decoupled transmit (volume) and receive (surface) coil pair with a multi-echo, spin-echo sequence employing 26 b-values5. ADC and FA maps were calculated by standard methods using MatLab (MathWorks®). The corpus callosum (CC) and internal capsule (IC) were manually segmented using ITK-SNAP (http://www.itksnap.org).

RESULTS

DTI parametric maps were segmented for CC and IC volumes and diffusion parameters, including ADC, Daxial, Dradial and FA (Fig. 1). At postnatal month two, the volume of the CC in C57Bl6 control mice (6.7 ± 0.62 µL) is larger than that of both low-Phe-diet Pahenu2 mice (5.2 ± 0.63 µL; p = 0.04) and Pahenu2 mice fed a normal diet (5.4 ± 1.4 µL; p = 0.07). IC volumes are comparable in all cohorts (Fig. 2A). FA values for both CC and IC are higher in control mice than in Pahenu2 mice (p < 0.01, Fig. 2B). For Pahenu2 mice, the DTI parameters of CC and IC at postnatal months two and four were analyzed with ANOVA (Type III Wald test, Figure 3). In the low-Phe group, Daxial increased from two to four months for both CC (0.95 ± 0.04 à 0.99 ± 0.03 µm2/ms; p< 0.001) and IC (0.99 ± 0.03 à 1.02 ± 0.04 µm2/ms; p<0.001), while in the normal chow group, Daxial decreased for both CC (1.01 ± 0.04 à 0.97 ± 0.05; p<0.001) and IC (1.05 ± 0.05 à 1.03 ± 0.03 µm2/ms; p<0.001). In the low-Phe group, FA increased for CC (0.44 ± 0.01 à 0.46 ± 0.01) and IC (0.57 ± 0.02 à0.59 ± 0.02; p<0.001). By contrast, for Pahenu2 mice fed normal chow, FA is the same for two- and four-month-old animals.

DISCUSSION

At two months of age, both the volume and FA of the CC are greater in C57Bl6 controls than in Pahenu2 mice. FA of the IC is also greater. Compared with two-month-old mice, Daxial and FA of CC and IC of four-month-old Pahenu2 on the low-Phe diet increased significantly, while Daxial and FA decreased significantly in Pahenu2 mice fed normal chow. Increased Daxial is associated with normal axonal development. Thus, our findings suggest that compromised axonal development in Pahenu2 mice fed a normal diet can be rescued by a low-Phe diet. In total, our data demonstrate that controlling Phe levels in Pahenu2 mice is critical for proper white-matter development, an observation that is consistent with clinical studies of PKU patients.

CONCLUSION

DTI experiments were used to probe white-matter development, and its dependence on diet, in Pahenu2 mice. Differences in white-matter integrity for Pahenu2 mice fed normal and low-Phe diets are consistent with those observed clinically. The Pahenu2 mouse can serve as a model for studying PKU-related metabolism, treatment response, and animal behavior.

Acknowledgements

Research was supported, in part, by the Washington University Division of Biology and Biomedical (DBBS) and Mallinckrodt Institute of Radiology.

References

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