Limits...
Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury.

Xie C, Ginet V, Sun Y, Koike M, Zhou K, Li T, Li H, Li Q, Wang X, Uchiyama Y, Truttmann AC, Kroemer G, Puyal J, Blomgren K, Zhu C - Autophagy (2016)

Bottom Line: There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI).Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death.Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells.

View Article: PubMed Central - PubMed

Affiliation: a Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg , Gothenburg , Sweden.

ABSTRACT
Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.

No MeSH data available.


Related in: MedlinePlus

Neuronal Atg7 deficiency prevents neuronal basal autophagy and does not alter expression of mitochondria and cell death-related proteins. (A) Representative immunoblotting of ATG7, SQSTM1, and LC3BB in the atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl (Atg7flox/+; Nes-Cre) mice showing that ATG7 was strongly decreased in KO mice leading to an accumulation of SQSTM1 and absence of LC3BB-II. (B) Representative immunostaining of SQSTM1 in the cortex of Ctrl and atg7 KO mice in the non-HI control condition, confirming the accumulation of SQSTM1 in KO compared to the faint and diffuse expression in Ctrl. (C) Punctate and strong SQSTM1 staining was observed in the cytoplasm of neuronal cells (RBFOX3) in the cortex of atg7 KO mice, but not in the Ctrl as shown by RBFOX3 and SQSTM1. (D) Representative immunoblots from cortical tissue homogenates of P9 Ctrl and atg7 KO mice. Quantification did not show any significant differences between the 2 types of mice for cell death-related proteins AIFM1, CYCS and CASP3, mitochondria-related proteins (SOD2, HSP70, CAT and mitochondrial respiratory chain complexes (COXV, COXIII, COXIV, COXII, COXI). (n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835980&req=5

f0002: Neuronal Atg7 deficiency prevents neuronal basal autophagy and does not alter expression of mitochondria and cell death-related proteins. (A) Representative immunoblotting of ATG7, SQSTM1, and LC3BB in the atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl (Atg7flox/+; Nes-Cre) mice showing that ATG7 was strongly decreased in KO mice leading to an accumulation of SQSTM1 and absence of LC3BB-II. (B) Representative immunostaining of SQSTM1 in the cortex of Ctrl and atg7 KO mice in the non-HI control condition, confirming the accumulation of SQSTM1 in KO compared to the faint and diffuse expression in Ctrl. (C) Punctate and strong SQSTM1 staining was observed in the cytoplasm of neuronal cells (RBFOX3) in the cortex of atg7 KO mice, but not in the Ctrl as shown by RBFOX3 and SQSTM1. (D) Representative immunoblots from cortical tissue homogenates of P9 Ctrl and atg7 KO mice. Quantification did not show any significant differences between the 2 types of mice for cell death-related proteins AIFM1, CYCS and CASP3, mitochondria-related proteins (SOD2, HSP70, CAT and mitochondrial respiratory chain complexes (COXV, COXIII, COXIV, COXII, COXI). (n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.

Mentions: In order to investigate the specific role of neuronal enhanced autophagy after HI, we characterized mice bearing a neuron-specific knockout of Atg7 resulting from the inactivation of floxed Atg7 by Cre recombinase expressed under the Nes/nestin promoter (Nes-Cre; Atg7flox/flox). The Nes promoter is commonly used when targeting brain neurons.15 These mice display a nearly complete deficiency of neuronal ATG7 protein, as well as defective autophagy, as shown by the absence of LC3B-II (Fig. 2A) and pronounced neuronal SQSTM1 accumulation (Fig. 2A to C) as early as on postnatal d 9 (P9). Punctate SQSTM1-positive staining was observed in neurons from the cortex, hippocampus, and striatum of atg7 KO mice but not in Ctrl mice, which presented a diffuse labeling (Fig. 2B).Figure 2.


Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury.

Xie C, Ginet V, Sun Y, Koike M, Zhou K, Li T, Li H, Li Q, Wang X, Uchiyama Y, Truttmann AC, Kroemer G, Puyal J, Blomgren K, Zhu C - Autophagy (2016)

Neuronal Atg7 deficiency prevents neuronal basal autophagy and does not alter expression of mitochondria and cell death-related proteins. (A) Representative immunoblotting of ATG7, SQSTM1, and LC3BB in the atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl (Atg7flox/+; Nes-Cre) mice showing that ATG7 was strongly decreased in KO mice leading to an accumulation of SQSTM1 and absence of LC3BB-II. (B) Representative immunostaining of SQSTM1 in the cortex of Ctrl and atg7 KO mice in the non-HI control condition, confirming the accumulation of SQSTM1 in KO compared to the faint and diffuse expression in Ctrl. (C) Punctate and strong SQSTM1 staining was observed in the cytoplasm of neuronal cells (RBFOX3) in the cortex of atg7 KO mice, but not in the Ctrl as shown by RBFOX3 and SQSTM1. (D) Representative immunoblots from cortical tissue homogenates of P9 Ctrl and atg7 KO mice. Quantification did not show any significant differences between the 2 types of mice for cell death-related proteins AIFM1, CYCS and CASP3, mitochondria-related proteins (SOD2, HSP70, CAT and mitochondrial respiratory chain complexes (COXV, COXIII, COXIV, COXII, COXI). (n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835980&req=5

f0002: Neuronal Atg7 deficiency prevents neuronal basal autophagy and does not alter expression of mitochondria and cell death-related proteins. (A) Representative immunoblotting of ATG7, SQSTM1, and LC3BB in the atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl (Atg7flox/+; Nes-Cre) mice showing that ATG7 was strongly decreased in KO mice leading to an accumulation of SQSTM1 and absence of LC3BB-II. (B) Representative immunostaining of SQSTM1 in the cortex of Ctrl and atg7 KO mice in the non-HI control condition, confirming the accumulation of SQSTM1 in KO compared to the faint and diffuse expression in Ctrl. (C) Punctate and strong SQSTM1 staining was observed in the cytoplasm of neuronal cells (RBFOX3) in the cortex of atg7 KO mice, but not in the Ctrl as shown by RBFOX3 and SQSTM1. (D) Representative immunoblots from cortical tissue homogenates of P9 Ctrl and atg7 KO mice. Quantification did not show any significant differences between the 2 types of mice for cell death-related proteins AIFM1, CYCS and CASP3, mitochondria-related proteins (SOD2, HSP70, CAT and mitochondrial respiratory chain complexes (COXV, COXIII, COXIV, COXII, COXI). (n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
Mentions: In order to investigate the specific role of neuronal enhanced autophagy after HI, we characterized mice bearing a neuron-specific knockout of Atg7 resulting from the inactivation of floxed Atg7 by Cre recombinase expressed under the Nes/nestin promoter (Nes-Cre; Atg7flox/flox). The Nes promoter is commonly used when targeting brain neurons.15 These mice display a nearly complete deficiency of neuronal ATG7 protein, as well as defective autophagy, as shown by the absence of LC3B-II (Fig. 2A) and pronounced neuronal SQSTM1 accumulation (Fig. 2A to C) as early as on postnatal d 9 (P9). Punctate SQSTM1-positive staining was observed in neurons from the cortex, hippocampus, and striatum of atg7 KO mice but not in Ctrl mice, which presented a diffuse labeling (Fig. 2B).Figure 2.

Bottom Line: There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI).Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death.Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells.

View Article: PubMed Central - PubMed

Affiliation: a Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg , Gothenburg , Sweden.

ABSTRACT
Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.

No MeSH data available.


Related in: MedlinePlus