Proteomic analysis of hypoxia/ischemia-induced alteration of cortical development and dopamine neurotransmission in neonatal rat

Abstract

Perinatal hypoxia/ischemia (HI) is a common cause of neurological deficits in children. Our goal was to elucidate the underlying mechanisms that contribute to the neurological sequelae of HI-induced brain injury. HI was induced by permanent ligation of the left carotid artery followed by 90 min of hypoxia (7.8% O2) in female P7 rats. A two-dimensional differential proteome analysis was used to assess changes in protein expression in cortex 2 h after HI. In total, 17 proteins reflecting a 2-fold or higher perturbation of expression after HI as compared to sham-treated pups were identified by mass spectrometry. Of the altered proteins, 14-3-3epsilon and TUC-2, both playing an important role in the development of the central nervous system, decrease after HI, consistent with an early disturbance of cortical development. Also affected, DARPP-32 and alpha-synuclein, two proteins important for dopamine neurotransmission, increased more than 2-fold 2 h after HI injury. The differential expression of these proteins was validated by individual Western blot assays. The expression of several metabolic enzymes and translational factors was also perturbed early after HI brain injury. These findings provide initial insights into the mechanisms underlying neurodegenerative events after HI and may allow for the rational design of therapeutic strategies that enhance neuronal adaptation and compensation after HI.

Figure 1

Sypro-ruby stained two-dimensional gels. The cytoplasmic extracts from sham (A) or HI injured (B) P7 rat cortexes were separated by two-dimensional electrophoresis. Differential expression of spots was evaluated with Progenesis. These changed spots were identified by MALDI-TOF-TOF-MS. Identified protein spots are listed in Table 1. Magnified image shows changes in spot intensity of TUC-2 (arrow).

Figure 2

14-3-3ε decreased after HI. (A) Enlarged images of 2D protein profile representing decreases in protein expression of 14-3-3ε after HI. (B) Immunolabeling (×60) of sham-treated P7 rat cortex shows distribution of 14-3-3ε (Red) and co-localization of 14-3-3ε with nuclear DAPI (blue) stain. (C) Representative western blot assays and graphic representation of densitometry analyses of 14-3-3ε protein expression in cytoplasmic extracts of P7 rat cortices. Lane 1–3: Sham-treated; lane 4–6: HI-treated. β-actin was used as loading control. Values are mean ± SEM. N=7 in each group. *: p<0.05 compared to sham-treated group.

Figure 3

TUC-2 decreased after HI. (A) Enlarged images of 2D protein profile representing decreases in protein expression of TUC-2 after HI. (B) Immunolabeling (×60) of sham-treated P7 rat cortex shows distribution of TUC-2 (Red) and co-localization of TUC-2 with nuclear DAPI (blue) stain. (C) Representative western blot assays and graphic representation of densitometry analyses of TUC-2 protein expression in cytoplasmic extracts of P7 rat cortices. Lane 1–3: Sham-treated; lane 4–6: HI-treated. β-actin was used as loading control. Values are mean ± SEM. N=7 in each group. *: p<0.05 compared to sham-treated group.

Figure 4

Alpha Synuclein increased after HI. (A) Enlarged images of 2D protein profile representing increases in alpha Synuclein after HI. (B) Representative western blot assays and graphic representation of densitometry analyses of alpha Synuclein protein expression in cytoplasmic extracts of P7 rat cortices. Lane 1–3: Sham-treated; lane 4–6: HI-treated. β-actin was used as loading control. Values are mean ± SEM. N=7 in each group. *: p<0.05 compared to sham-treated group.

Figure 5

DARPP-32 increased after HI. (A) Enlarged images of 2D protein profile representing decreases in protein expression of DARPP-32 after HI. (B) Representative western blot assays and graphic representation of densitometry analyses of DARPP-32 protein expression in cytoplasmic extracts of P7 rat cortices. Lane 1–3: Sham-treated; lane 4–6: HI-treated. β-actin was used as loading control. Values are mean ± SEM. N=7 in each group. *: p<0.05 compared to sham-treated group.