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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.

Neira-Peña T, Rojas-Mancilla E, Munoz-Vio V, Perez R, Gutierrez-Hernandez M, Bustamante D, Morales P, Hermoso MA, Gebicke-Haerter P, Herrera-Marschitz M - Neurotox Res (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Millenium Institute BNI-Chile, Santiago, Chile, tanyaneira@gmail.com.

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.

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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)
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Fig5: 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)

Mentions: Figure 4 shows the effect of nicotinamide (CNam, ANam), or saline (CSal, ASal) on pADPr (A, B), PARP-1 levels (C) and PARP-1 activity (D) 2 h after birth. Nicotinamide decreased pADPr levels (B) and PARP-1 activity (D) (ANam versus ASal). No differences were observed in PARP-1 levels (C) between any of the groups.


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.

Neira-Peña T, Rojas-Mancilla E, Munoz-Vio V, Perez R, Gutierrez-Hernandez M, Bustamante D, Morales P, Hermoso MA, Gebicke-Haerter P, Herrera-Marschitz M - Neurotox Res (2015)

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)
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Related In: Results  -  Collection

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Fig5: 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)
Mentions: Figure 4 shows the effect of nicotinamide (CNam, ANam), or saline (CSal, ASal) on pADPr (A, B), PARP-1 levels (C) and PARP-1 activity (D) 2 h after birth. Nicotinamide decreased pADPr levels (B) and PARP-1 activity (D) (ANam versus ASal). No differences were observed in PARP-1 levels (C) between any of the groups.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Millenium Institute BNI-Chile, Santiago, Chile, tanyaneira@gmail.com.

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.

Show MeSH
Related in: MedlinePlus