Maternal low-protein diet decreases brain-derived neurotrophic factor expression in the brains of the neonatal rat offspring

Abstract

Prenatal exposure to a maternal low-protein (LP) diet has been known to cause cognitive impairment, learning and memory deficits. However, the underlying mechanisms have not been identified. Herein, we demonstrate that a maternal LP diet causes, in the brains of the neonatal rat offspring, an attenuation in the basal expression of the brain-derived neurotrophic factor (BDNF), a neurotrophin indispensable for learning and memory. Female rats were fed either a 20% normal protein (NP) diet or an 8% LP 3 weeks before breeding and during the gestation period. Maternal LP diet caused a significant reduction in the Bdnf expression in the brains of the neonatal rats. We further found that the maternal LP diet reduced the activation of the cAMP/protein kinase A/cAMP response element binding protein (CREB) signaling pathway. This reduction was associated with a significant decrease in CREB binding to the Bdnf promoters. We also show that prenatal exposure to the maternal LP diet results in an inactive or repressed exon I and exon IV promoter of the Bdnf gene in the brain, as evidenced by fluxes in signatory hallmarks in the enrichment of acetylated and trimethylated histones in the nucleosomes that envelop the exon I and exon IV promoters, causing the Bdnf gene to be refractory to transactivation. Our study is the first to determine the impact of a maternal LP diet on the basal expression of BDNF in the brains of the neonatal rats exposed prenatally to an LP diet.

Keywords: Brain-derived neurotrophic factor; Cyclic adenosine monophosphate; Histone acetylation; Histone methylation; Maternal low-protein diet; cAMP response element binding protein.

Figure 1. Effects of maternal LP diet on Bdnf expression in the brains of the neonatal offspring rats

(A, B) Representative western blots (A) and densitometric analysis (B) showing the effects of prenatal exposure to the maternal LP diet on the pro-BDNF and mature BDNF protein levels in the whole-brain homogenates of the neonatal offspring. (C) ELISA immunoassay showing the effects of a maternal LP diet on total BDNF protein levels (free and Trk-bound pro-BDNF as well as mature BDNF) in whole brain homogenates of the neonatal offspring. (D) Real-time RT-PCR analysis demonstrating the effects of prenatal exposure to a maternal LP diet on the abundance of the BDNF mRNA transcripts containing exons I, III, IV, and IX. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. **p<0.01, ***p<0.001 versus male neonatal offspring exposed prenatally to a maternal normal protein (NP) diet; ^††p<0.01, ^†††p<0.001 versus female neonatal offspring exposed prenatally to a maternal NP diet.

Figure 2. Effects of maternal LP diet on cAMP levels and PKA activity in the brains of the neonatal offspring

(A) ELISA immunoassay demonstrating the effects of a maternal LP diet on cAMP levels in the whole cell, cytosolic, and membranes fractions from the whole brain homogenates of the neonatal offspring. (B) ELISA immunoassay showing the effects of prenatal exposure to a LP diet on PKA activity in the cytosolic, nuclear, and membranes fractions from the whole brain homogenates of the neonatal offspring. (C) Representative western blots showing the effects of maternal LP diet on the phosphorylation of the PKA catalytic subunits, PKA-Cα and PKA-Cβ, as well as the subsequent nuclear translocation of PKA-Cα and PKA-Cβ, in the brains of the neonatal offspring. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. *p<0.05, **p<0.01, ***p<0.001 versus male neonatal offspring exposed prenatally to a maternal NP diet; ^†p<0.05, ^††p<0.01 versus female neonatal offspring exposed prenatally to a maternal NP diet.

Figure 3. Effects of maternal LP diet on CREB transcriptional activity and the subsequent binding of CREB to the exon I promoter and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring

(A, B) Representative western blots (A) and densitometric analysis (B) showing the effects of prenatal exposure to a maternal LP diet on the activation phosphorylation at the Ser¹³³ residue of CREB, in the whole brains of the neonatal offspring. (C) ELISA immunoassay showing the effects of a maternal LP diet on CREB transcriptional activity in the brains of the neonatal offspring. (D) ChIP-qPCR assay showing the effects of prenatal exposure to a LP diet on the binding of CREB to the CRE sites in the exon I promoter and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring. (E, F) Representative western blots (E) and densitometric analysis (F) of the reverse cross-linked CREB-ChIP samples showing the extent of association of the known CREB coactivators, p300, CBP, and CRTC2, with the chromatin-bound CREB, in the brains of the neonatal offspring exposed prenatally to a maternal LP diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. ***p<0.001 versus male neonatal offspring exposed prenatally to a maternal NP diet; ^††p<0.01, ^†††p<0.001 versus female neonatal offspring exposed prenatally to a maternal NP diet.

Figure 4. Effects of maternal LP diet on the acetylation of the known lysine residues of Histone H3 and Histone H4 in the exon I and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring

(A–D) ChIP-qPCR analysis shows that a maternal LP diet results in a non-permissive chromatin in the exon I and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring. The ChIP-qPCR data show a reduced enrichment of Histone H3 and Histone H4 acetylation in the nucleosomes that envelop the exon I promoter and exon IV promoter of the Bdnf gene, in the brains of the neonatal offspring exposed prenatally to a maternal LP diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. ***p<0.001 versus male neonatal offspring exposed prenatally to a maternal NP diet; ^†††p<0.001 versus female neonatal offspring exposed prenatally to a maternal NP diet.

Figure 5. Maternal LP diet alters Histone H3 methylation in the exon I and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring

(A–D) ChIP-qPCR analysis shows that a maternal LP diet results in a non-permissive chromatin in the exon I and exon IV promoter of the Bdnf gene in the brains of the neonatal offspring. (A, B) The ChIP-qPCR data show an augmentation in the enrichment of H3K9me3 and H3K27me3, a signature of an inactive or repressed promoter, in the nucleosomes that envelop the exon I and exon IV promoter of the Bdnf gene, in the brains of the neonatal offspring exposed prenatally to a maternal LP diet. (C, D) The ChIP-qPCR data also show a reduction in the enrichment of H3K4me3 and H3K36me3, a signature of an active promoter, in the nucleosomes that envelop the exon I promoter and exon IV promoter of the Bdnf gene, in the brains of the neonatal offspring exposed prenatally to a maternal LP diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. ***p<0.001 versus male neonatal offspring exposed prenatally to a maternal NP diet; ^†††p<0.001 versus female neonatal offspring exposed prenatally to a maternal NP diet.