A Specific Nutrient Combination Attenuates the Reduced Expression of PSD-95 in the Proximal Dendrites of Hippocampal Cell Body Layers in a Mouse Model of Phenylketonuria

Abstract

The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice.

Keywords: Hippocampus; PSD-95; nutrient combination; phenylketonuria; synaptic proteins.

Figure 1

PSD-95 expression in the C57BL/6 PKU mouse. (A) An overview picture of the PSD-95 immunostaining; The following 10 areas of interest were measured: (B) CA3: stratum lucidum (SL), CA3 pyramidal cell layer (CA3 pyr); (C) DG: outer and middle molecular layer (OML/MML), the inner molecular layer (IML), inner (DG-IB) and outer blade (DG-OB) of the dentate gyrus, hilus; (D) CA1: stratum oriens (SO), CA1 pyramidal cell layer (CA1 pyr) and the stratum radiatum (SR); (E) a detailed picture of the granular layers present in the DG; (F) a detailed picture of the CA1 pyramidal cell layer. Arrows indicate clear staining within the granular layer. The size bar indicates 100 µm. The detail pictures within (E) and (F) are digitally enlarged.

Figure 1

PSD-95 expression in the C57BL/6 PKU mouse. (A) An overview picture of the PSD-95 immunostaining; The following 10 areas of interest were measured: (B) CA3: stratum lucidum (SL), CA3 pyramidal cell layer (CA3 pyr); (C) DG: outer and middle molecular layer (OML/MML), the inner molecular layer (IML), inner (DG-IB) and outer blade (DG-OB) of the dentate gyrus, hilus; (D) CA1: stratum oriens (SO), CA1 pyramidal cell layer (CA1 pyr) and the stratum radiatum (SR); (E) a detailed picture of the granular layers present in the DG; (F) a detailed picture of the CA1 pyramidal cell layer. Arrows indicate clear staining within the granular layer. The size bar indicates 100 µm. The detail pictures within (E) and (F) are digitally enlarged.

Figure 2

PSD-95 expression is reduced specifically in the hippocampus of the PKU mouse model. (A) No significant differences are found between the three groups in striatum (Kruskal-Wallis: p = 0.830); (B) No significant differences are found between the three groups in the prefrontal cortex (Kruskal-Wallis: p = 0.930); (C) Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus (Kruskal-Wallis: DG-IB p = 0.009, DG-OB p = 0.008), with a similar trend in the CA1 and CA3 pyramidal cell layer (Kruskal-Wallis: CA1 pyr p = 0.053, CA3 pyr p = 0.064). A significant difference was found between the WT group and both PKU groups for the DG-IB and DG-OB (WT compared to PKU without SNC: Mann-Whitney U test DG-IB; p = 0.005, DG-OB; p = 0.005. WT compared to PKU with SNC: Mann-Whitney U test DG-IB; p = 0.031, DG-OB; p = 0.026) (arrow bars depict SEM).