Perinatal asphyxia leads to PARP-1 overactivity, p65 translocation, IL-1β and TNF-α overexpression, and apoptotic-like cell death in mesencephalon of neonatal rats: prevention by systemic neonatal nicotinamide administration

Abstract

Perinatal asphyxia (PA) is a leading cause of neuronal damage in newborns, resulting in long-term neurological and cognitive deficits, in part due to impairment of mesostriatal and mesolimbic neurocircuitries. The insult can be as severe as to menace the integrity of the genome, triggering the overactivation of sentinel proteins, including poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 overactivation implies increased energy demands, worsening the metabolic failure and depleting further NAD(+) availability. Using a global PA rat model, we report here evidence that hypoxia increases PARP-1 activity, triggering a signalling cascade leading to nuclear translocation of the NF-κB subunit p65, modulating the expression of IL-1β and TNF-α, pro-inflammatory molecules, increasing apoptotic-like cell death in mesencephalon of neonate rats, monitored with Western blots, qPCR, TUNEL and ELISA. PARP-1 activity increased immediately after PA, reaching a maximum 1-8 h after the insult, while activation of the NF-κB signalling pathway was observed 8 h after the insult, with a >twofold increase of p65 nuclear translocation. IL-1β and TNF-α mRNA levels were increased 24 h after the insult, together with a >twofold increase in apoptotic-like cell death. A single dose of the PARP-1 inhibitor nicotinamide (0.8 mmol/kg, i.p.), 1 h post delivery, prevented the effect of PA on PARP-1 activity, p65 translocation, pro-inflammatory cytokine expression and apoptotic-like cell death. The present study demonstrates that PA leads to PARP-1 overactivation, increasing the expression of pro-inflammatory cytokines and cell death in mesencephalon, effects prevented by systemic neonatal nicotinamide administration, supporting the idea that PARP-1 inhibition represents a therapeutic target against the effects of PA.

Fig. 1

Effect of perinatal asphyxia on pADPr, PARP-1 levels and PARP-1 activity in mesencephalon of rat neonates, 0–24 h after delivery. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were euthanized immediately after delivery (0 h), or following re-oxygenation (1–24 h), brain tissue sampled and treated for Western blots. a Representative immunoblots for pADPr (170–70 kDa), PARP-1 (113 kDa) and β-actin (42 kDa) levels following hypoxia and/or re-oxygenation. b pADPr levels (expressed as arbitrary units, A.U.) (CS open columns, AS grey columns). c PARP-1 levels, normalized to β-actin, A.U.). d PARP-1 activity (pADPr/PARP-1 ratio) (CS open circles, AS filled triangles). Pair-wise comparisons were analysed with Student t test (*p < 0.05, **p < 0.005; n = 5–7, for each condition and experiment)

Fig. 2

Effect of perinatal asphyxia on IκBα, nuclear translocation of p65, IL-1β and TNF-α mRNA levels in mesencephalon of rat neonates, 1–24 h after birth. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were euthanized 1–24 h after delivery, and brain tissue sampled and treated for Western blots or RT-qPCR. a Representative immunoblots for IκBα (35–41 kDa) and β-actin (42 kDa) levels. b IκBα levels (normalized to β-actin; A.U. arbitrary units) (CS open columns, AS grey columns). c Representative immunoblots for p65 (65 kDa), α-tubulin (45 kDa) and histone H4 (15–11 kDa) levels, measured in cytoplasmic (c) and nuclear (n) protein extracts. d Nuclear p65 levels normalized to total p65. e, f IL-1β (E) and TNF-α f mRNA levels measured by RT-qPCR with specific primers; analysed in triplicates with MxPro software and normalized to GAPDH mRNA levels. Pair-wise comparisons analysed with Student t test (*p < 0.05, ***p < 0.0005; n = 5–7, for each condition and experiment)

Fig. 3

Effect of perinatal asphyxia on apoptotic-like cell death in mesencephalon of rat neonates, 24 h after delivery: prevention by systemic nicotinamide treatment. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were anaesthetized and transcardially perfused with buffered saline and a formalin solution 24 h after delivery. DNA fragmentation was evaluated in coronal brain sections (20-μm thick) with the TUNEL assay, counterstained with methyl green. The number of TUNEL-positive cells per mm³ was determined in substantia nigra (SN), and ventral tegmental area (VTA). a Representative microphotographs, from SN of control and asphyxia-exposed animals; TUNEL-positive cells are brown (arrows) (bar 10 μm). Inset shows a section from Foster atlas (1998), indicating the sampling area (white rectangles). b TUNEL-positive cells were manually counted on the stage of a Nikon TS100 microscope (magnification ×100), expressed as number of TUNEL-positive cells/mm³ in CS and AS animals, or in parallel animal series following a single dose of nicotinamide (0.8 mmol/kg, i.p.) (CNam, ANam) or saline (0.1 ml, i.p.) (CSal, ASal) 1 h after delivery; also fixed at 24 h (CSal open columns; CNam dashed columns, ASal grey columns, ANam doubled dashed columns). Pair-wise comparisons analysed with Student t test (*p < 0.05; n = 4–5, for each condition)

Fig. 4

Effect of nicotinamide or saline on PARP-1 activity in mesencephalon of asphyxia-exposed and control rats. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were treated 1 h after birth with a single dose of Nam (0.8 mmol/kg, i.p.) (CNam, ANam) or saline (0.1 ml, i.p.) (CSal, ASal), and euthanized 2 h after delivery. Brain tissue sampled and treated for Western blots. pADPr and PARP-1 protein levels were measured in total protein extracts. PARP-1 activity was estimated as the pADPr/PARP-1 ratio. a Representative immunoblots for pADPr, PARP-1 and β-actin levels; b pADPr levels (expressed as arbitrary units, A.U.); c PARP-1 levels (normalized to β-actin, A.U.); d PARP-1 activity (pADPr/PARP-1 ratio) (CSal open columns, CNam dashed columns, ASal grey columns, ANam double dashed columns). Pair-wise comparisons analysed with Student t test (*p < 0.05, **p < 0.005, ***p < 0.0005; n = 4–5, for each condition and experiment)

Fig. 5

Effect of nicotinamide or saline on p65 translocation; on p65, IL-1β and TNF-α mRNA levels, and on ELISA TNF-α protein levels in mesencephalon of asphyxia-exposed and control rats. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were treated with a single dose of nicotinamide (0.8 mmol/kg, i.p.) (CNam, ANam) or saline (0.1 ml, i.p.) (CSal, ASal), 1 h after delivery, euthanized at 8 or 24 h after birth. Selected brain tissue was treated for Western blots, RT-qPCR or ELISA. a Representative immunoblots for p65, α-tubulin and histone H4 levels, measured in cytoplasmic (c) and nuclear (n) protein extracts. b Nuclear p65 levels normalized to total p65. c De novo synthesis of p65, d IL-1β and e TNF-α, measured by RT-qPCR 8 h and/or 24 h after delivery (data analysed in triplicates with MxPro software, normalized to GAPDH mRNA levels). f TNF-α protein levels measured in total protein extracts with Quantikine^® ELISA Rat (normalized by mg of protein for each sample, in duplicate) (CSal open columns, CNam dashed columns, ASal grey columns, ANam doubled dashed columns). Pair-wise comparisons analysed with Student t test (*p < 0.05, **p < 0.005)