Oligodendrocyte development and myelinogenesis are not impaired by high concentrations of phenylalanine or its metabolites

Abstract

Phenylketonuria (PKU) is a metabolic genetic disease characterized by deficient phenylalanine hydroxylase (PAH) enzymatic activity. Brain hypomyelination has been reported in untreated patients, but its mechanism remains unclear. We therefore investigated the influence of phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) on oligodendrocytes. We first showed in a mouse model of PKU that the number of oligodendrocytes is not different in corpus callosum sections from adult mutants or from control brains. Then, using enriched oligodendroglial cultures, we detected no cytotoxic effect of high concentrations of Phe, PP, or PA. Finally, we analyzed the impact of Phe, PP, and PA on the myelination process in myelinating cocultures using both an in vitro index of myelination, based on activation of the myelin basic protein (MBP) promoter, and the direct quantification of myelin sheaths by both optical measurement and a bioinformatics method. None of these parameters was affected by the increased levels of Phe or its derivatives. Taken together, our data demonstrate that high levels of Phe, such as in PKU, are unlikely to directly induce brain hypomyelination, suggesting involvement of alternative mechanisms in this myelination defect.

Fig. 1

a Oligodendrocyte proliferation rate in the subventricular zone (SVZ) from P8 BTBR Pah^enu2/J heterozygous (Pah+/−) and double-mutant (Pah−/−) mice. Results are expressed in percentages of oligodendrocyte transcription factor 2 (Olig-2)-positive cells having incorporated 5-bromo-2-deoxyuridine (BrdU) after 24-h exposure versus the total number of Olig-2 cells present in the SVZ. b Immunohistochemistry of P8 BTBR Pah^enu2/J brain slice along the SVZ at ×40 magnification. Red labelling is Olig-2 and green is BrdU. Arrows indicate double-positive cells

Fig. 2

a Oligodendroglial density in the corpus callosum of 2-month-old BTBR Pah^enu2/J wild-type (Pah+/+) and double mutant (Pah−/−) mice. Data represent the density of mature oligodendrocytes, expressing both oligodendrocyte transcription factor 2 (Olig-2) and CC1. b Immunolabeling of a brain section across the corpus callosum from BTBR Pah^enu2/J with anti-Olig-2 (green) and anti-CC1 (red) antibodies. Most cells are coexpressing the two markers and thus correspond to mature oligodendrocytes

Fig. 3

a Viability test using methylthiazolyldiphenyl-tetrazolium bromide (MTT) after a 6 days exposure to phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) at pathologically relevant (Patho) and suprapathological (High) concentrations of an enriched population of oligodendrocytes isolated from postnatal-day 1 (P1) rat brains. Pathological/high concentrations are 1.6/5 mM, 2.2/22 µM, 7.4/74 µM for Phe, PP, and PA, respectively. b Anti-oligodendrocyte marker 4 (anti-O4) immunolabeling of rat preoligodendrocyte population 48 h after purification

Fig. 4

a β-Galactosidase activity assay from murine myelinating cocultures. The degree of maturation of oligodendrocytes in the cultures was assessed by quantifying the activation of the myelin basic protein (MBP) promoter through luminometric β-galactosidase activity assay. Results are expressed as relative counts per second (CPS) compared with control condition. b Optical counting of proteolipid protein (PLP)-positive internodes (i.e., myelin sheaths) in murine myelinating cocultures. Results are expressed as ratios of the count from cultures exposed to phenylalanine (Phe) (5 mM), phenylpyruvate (PP) (22 µM), or phenylacetate (PA) (74 µM) to the score from the corresponding control samples. P>0.05 versus control group

Fig. 5

Bioinformatics analysis: a Counting of myelin sheaths from the whole surface of murine myelinating coculture samples. b Evaluation of the mean length of myelin sheaths from the same samples. Results are expressed as ratios of the score from cultures exposed to phenylalanine (Phe) (5 mM), phenylpyruvate (PP) (22 µM), or phenylacetate (PA) (74 µM) to the score from the corresponding control samples. P>.05 versus control group