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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 decreased CASP3 activation after hypoxia-ischemia. (A) Representative active CASP3/caspase-3 staining 24 h after HI and the corresponding quantifications of the number of CASP3-positive cells (B) in the cortex (Cx) (81,630 ± 7,671/mm3 vs. 51,210 ± 11,970/mm3; *, P < 0.05), (C) in the striatum (Str) (132,800 ± 5,368/mm3 vs. 47,350 ± 13,110/mm3; ***, P < 0.001), (D) in the CA1 (41,810 cells ± 3,243 cells/mm3 vs. 22,860 cells ± 4,685 cells/mm3; **, P < 0.01) and (E) dentate gyrus (DG) (214,000 cells ± 14,700 cells/mm3 vs. 144,000 cells ± 23,940 cells/mm3; *, P < 0.05, n = 11/group). (F) CASP3 enzymatic activity 24 h after HI was 50% lower in the ipsilateral (IL) hemisphere of atg7 KO mice than in Ctrl (n = 10 for Ctrl, n = 6 for atg7 KO). There was no difference in the nonischemic control brains (Cont) (n = 13 for Ctrl and n = 6 for atg7 KO) or in the contralateral (CL) hemispheres (n = 10 for Ctrl, n = 6 for atg7 KO). (G) Representative SPTAN1/α-fodrin (240 kDa) western blots from control (Cont) and ipsilateral (IL) hemispheres 24 h after HI and (H) the quantification of the caspase-dependent 120 kDa breakdown products confirmed that CASP3 activation was more pronounced in the Ctrl mice. (*, P < 0.05, n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
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f0005: Neuronal Atg7 deficiency decreased CASP3 activation after hypoxia-ischemia. (A) Representative active CASP3/caspase-3 staining 24 h after HI and the corresponding quantifications of the number of CASP3-positive cells (B) in the cortex (Cx) (81,630 ± 7,671/mm3 vs. 51,210 ± 11,970/mm3; *, P < 0.05), (C) in the striatum (Str) (132,800 ± 5,368/mm3 vs. 47,350 ± 13,110/mm3; ***, P < 0.001), (D) in the CA1 (41,810 cells ± 3,243 cells/mm3 vs. 22,860 cells ± 4,685 cells/mm3; **, P < 0.01) and (E) dentate gyrus (DG) (214,000 cells ± 14,700 cells/mm3 vs. 144,000 cells ± 23,940 cells/mm3; *, P < 0.05, n = 11/group). (F) CASP3 enzymatic activity 24 h after HI was 50% lower in the ipsilateral (IL) hemisphere of atg7 KO mice than in Ctrl (n = 10 for Ctrl, n = 6 for atg7 KO). There was no difference in the nonischemic control brains (Cont) (n = 13 for Ctrl and n = 6 for atg7 KO) or in the contralateral (CL) hemispheres (n = 10 for Ctrl, n = 6 for atg7 KO). (G) Representative SPTAN1/α-fodrin (240 kDa) western blots from control (Cont) and ipsilateral (IL) hemispheres 24 h after HI and (H) the quantification of the caspase-dependent 120 kDa breakdown products confirmed that CASP3 activation was more pronounced in the Ctrl mice. (*, P < 0.05, n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.

Mentions: Apoptotic cell death pathways, including caspase-dependent and –independent pathways, were investigated by quantifying the activation of CASP3 (Fig. 5A to E) and the mitochondrial-nuclear translocation of AIFM (Fig. 6A to E). Immunohistochemical detection of active cleaved CASP3 (but not its inactive precursor, pro-CASP3) revealed a similar pattern as that shown by Fluoro-Jade staining (Fig. 4). Moreover, CASP3 enzymatic activity was 50% lower in the ipsilateral hemispheres of atg7 KO compared to the Ctrl mice 24 h after HI (Fig. 5F). CASP3 data were confirmed by immunoblotting against SPTAN1/α-fodrin, revealing a decrease in the production of the caspase-dependent 120-kDa breakdown product (Fig. 5G, E). Caspase-independent apoptotic cell death—as indicated by AIFM1 nuclear translocation, as shown previously26—was also reduced in atg7 KO compared to Ctrl mice in all brain regions (Fig. 6).Figure 5.


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 decreased CASP3 activation after hypoxia-ischemia. (A) Representative active CASP3/caspase-3 staining 24 h after HI and the corresponding quantifications of the number of CASP3-positive cells (B) in the cortex (Cx) (81,630 ± 7,671/mm3 vs. 51,210 ± 11,970/mm3; *, P < 0.05), (C) in the striatum (Str) (132,800 ± 5,368/mm3 vs. 47,350 ± 13,110/mm3; ***, P < 0.001), (D) in the CA1 (41,810 cells ± 3,243 cells/mm3 vs. 22,860 cells ± 4,685 cells/mm3; **, P < 0.01) and (E) dentate gyrus (DG) (214,000 cells ± 14,700 cells/mm3 vs. 144,000 cells ± 23,940 cells/mm3; *, P < 0.05, n = 11/group). (F) CASP3 enzymatic activity 24 h after HI was 50% lower in the ipsilateral (IL) hemisphere of atg7 KO mice than in Ctrl (n = 10 for Ctrl, n = 6 for atg7 KO). There was no difference in the nonischemic control brains (Cont) (n = 13 for Ctrl and n = 6 for atg7 KO) or in the contralateral (CL) hemispheres (n = 10 for Ctrl, n = 6 for atg7 KO). (G) Representative SPTAN1/α-fodrin (240 kDa) western blots from control (Cont) and ipsilateral (IL) hemispheres 24 h after HI and (H) the quantification of the caspase-dependent 120 kDa breakdown products confirmed that CASP3 activation was more pronounced in the Ctrl mice. (*, P < 0.05, n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
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Related In: Results  -  Collection

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

f0005: Neuronal Atg7 deficiency decreased CASP3 activation after hypoxia-ischemia. (A) Representative active CASP3/caspase-3 staining 24 h after HI and the corresponding quantifications of the number of CASP3-positive cells (B) in the cortex (Cx) (81,630 ± 7,671/mm3 vs. 51,210 ± 11,970/mm3; *, P < 0.05), (C) in the striatum (Str) (132,800 ± 5,368/mm3 vs. 47,350 ± 13,110/mm3; ***, P < 0.001), (D) in the CA1 (41,810 cells ± 3,243 cells/mm3 vs. 22,860 cells ± 4,685 cells/mm3; **, P < 0.01) and (E) dentate gyrus (DG) (214,000 cells ± 14,700 cells/mm3 vs. 144,000 cells ± 23,940 cells/mm3; *, P < 0.05, n = 11/group). (F) CASP3 enzymatic activity 24 h after HI was 50% lower in the ipsilateral (IL) hemisphere of atg7 KO mice than in Ctrl (n = 10 for Ctrl, n = 6 for atg7 KO). There was no difference in the nonischemic control brains (Cont) (n = 13 for Ctrl and n = 6 for atg7 KO) or in the contralateral (CL) hemispheres (n = 10 for Ctrl, n = 6 for atg7 KO). (G) Representative SPTAN1/α-fodrin (240 kDa) western blots from control (Cont) and ipsilateral (IL) hemispheres 24 h after HI and (H) the quantification of the caspase-dependent 120 kDa breakdown products confirmed that CASP3 activation was more pronounced in the Ctrl mice. (*, P < 0.05, n = 6/group). KO: atg7 KO (Atg7flox/flox; Nes-Cre) and Ctrl: Atg7flox/+; Nes-Cre.
Mentions: Apoptotic cell death pathways, including caspase-dependent and –independent pathways, were investigated by quantifying the activation of CASP3 (Fig. 5A to E) and the mitochondrial-nuclear translocation of AIFM (Fig. 6A to E). Immunohistochemical detection of active cleaved CASP3 (but not its inactive precursor, pro-CASP3) revealed a similar pattern as that shown by Fluoro-Jade staining (Fig. 4). Moreover, CASP3 enzymatic activity was 50% lower in the ipsilateral hemispheres of atg7 KO compared to the Ctrl mice 24 h after HI (Fig. 5F). CASP3 data were confirmed by immunoblotting against SPTAN1/α-fodrin, revealing a decrease in the production of the caspase-dependent 120-kDa breakdown product (Fig. 5G, E). Caspase-independent apoptotic cell death—as indicated by AIFM1 nuclear translocation, as shown previously26—was also reduced in atg7 KO compared to Ctrl mice in all brain regions (Fig. 6).Figure 5.

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