Postnatal calpeptin treatment causes hippocampal neurodevelopmental defects in neonatal rats
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摘要
Our previous studies showed that the early use of calpain inhibitors reduces calpain activity in multiple brain regions, and that postnatal treatment with calpeptin may lead to cerebellar motor dysfunction. However, it remains unclear whether postnatal calpeptin application affects hippocampus-related behaviors. In this study, Sprague-Dawley rats were purchased from the Animal Center of Anhui Medical University of China. For the experiments in the adult stage, rats were intraperitoneally injected with calpeptin, 2 mg/kg, once a day, on postnatal days 7–14. Then on postnatal day 60, the Morris water maze test was used to evaluate spatial learning and memory abilities. The open field test was carried out to assess anxiety-like activities. Phalloidin staining was performed to observe synaptic morphology in the hippocampus. Immunohistochemistry was used to count the number of NeuN-positive cells in the hippocampal CA1 region. DiI was applied to label dendritic spines. Calpeptin administration impaired spatial memory, caused anxiety-like behavior in adulthood, reduced the number and area of apical dendritic spines, and decreased actin polymerization in the hippocampus, but did not affect the number of NeuN-positive cells in the hippocampal CA1 region. For the neonatal experiments, neonatal rats were intraperitoneally injected with calpeptin, 2 mg/kg, on postnatal days 7 and 8. Western blot assay was performed to analyze the protein levels of Akt, Erk, p-Akt, p-Erk1/2, Erk1/2, SCOP, PTEN, mTOR, p-mTOR, CREB and p-CREB in the hippocampus. SCOP expression was increased, and the phosphorylation levels of Akt, mTOR and CREB were reduced in the hippocampus. These findings show that calpeptin administration after birth affects synaptic development in neonatal rats by inhibiting the Akt/mTOR signaling pathway, thereby perturbing hippocampal function. Therefore, calpeptin administration after birth is a risk factor for neurodevelopmental defects.
Our previous studies showed that the early use of calpain inhibitors reduces calpain activity in multiple brain regions, and that postnatal treatment with calpeptin may lead to cerebellar motor dysfunction. However, it remains unclear whether postnatal calpeptin application affects hippocampus-related behaviors. In this study, Sprague-Dawley rats were purchased from the Animal Center of Anhui Medical University of China. For the experiments in the adult stage, rats were intraperitoneally injected with calpeptin, 2 mg/kg, once a day, on postnatal days 7–14. Then on postnatal day 60, the Morris water maze test was used to evaluate spatial learning and memory abilities. The open field test was carried out to assess anxiety-like activities. Phalloidin staining was performed to observe synaptic morphology in the hippocampus. Immunohistochemistry was used to count the number of NeuN-positive cells in the hippocampal CA1 region. DiI was applied to label dendritic spines. Calpeptin administration impaired spatial memory, caused anxiety-like behavior in adulthood, reduced the number and area of apical dendritic spines, and decreased actin polymerization in the hippocampus, but did not affect the number of NeuN-positive cells in the hippocampal CA1 region. For the neonatal experiments, neonatal rats were intraperitoneally injected with calpeptin, 2 mg/kg, on postnatal days 7 and 8. Western blot assay was performed to analyze the protein levels of Akt, Erk, p-Akt, p-Erk1/2, Erk1/2, SCOP, PTEN, mTOR, p-mTOR, CREB and p-CREB in the hippocampus. SCOP expression was increased, and the phosphorylation levels of Akt, mTOR and CREB were reduced in the hippocampus. These findings show that calpeptin administration after birth affects synaptic development in neonatal rats by inhibiting the Akt/mTOR signaling pathway, thereby perturbing hippocampal function. Therefore, calpeptin administration after birth is a risk factor for neurodevelopmental defects.
Our previous studies showed that the early use of calpain inhibitors reduces calpain activity in multiple brain regions, and that postnatal treatment with calpeptin may lead to cerebellar motor dysfunction. However, it remains unclear whether postnatal calpeptin application affects hippocampus-related behaviors. In this study, Sprague-Dawley rats were purchased from the Animal Center of Anhui Medical University of China. For the experiments in the adult stage, rats were intraperitoneally injected with calpeptin, 2 mg/kg, once a day, on postnatal days 7–14. Then on postnatal day 60, the Morris water maze test was used to evaluate spatial learning and memory abilities. The open field test was carried out to assess anxiety-like activities. Phalloidin staining was performed to observe synaptic morphology in the hippocampus. Immunohistochemistry was used to count the number of NeuN-positive cells in the hippocampal CA1 region. DiI was applied to label dendritic spines. Calpeptin administration impaired spatial memory, caused anxiety-like behavior in adulthood, reduced the number and area of apical dendritic spines, and decreased actin polymerization in the hippocampus, but did not affect the number of NeuN-positive cells in the hippocampal CA1 region. For the neonatal experiments, neonatal rats were intraperitoneally injected with calpeptin, 2 mg/kg, on postnatal days 7 and 8. Western blot assay was performed to analyze the protein levels of Akt, Erk, p-Akt, p-Erk1/2, Erk1/2, SCOP, PTEN, mTOR, p-mTOR, CREB and p-CREB in the hippocampus. SCOP expression was increased, and the phosphorylation levels of Akt, mTOR and CREB were reduced in the hippocampus. These findings show that calpeptin administration after birth affects synaptic development in neonatal rats by inhibiting the Akt/mTOR signaling pathway, thereby perturbing hippocampal function. Therefore, calpeptin administration after birth is a risk factor for neurodevelopmental defects.
引文
Amini M,Ma CL,Farazifard R,Zhu G,Zhang Y,Vanderluit J,Zoltewicz JS,Hage F,Savitt JM,Lagace DC,Slack RS,Beique JC,Baudry M,Greer PA,Bergeron R,Park DS(2013)Conditional disruption of calpain in the CNS alters dendrite morphology,impairs LTP,and promotes neuronal survival following injury.J Neurosci 33:5773-5784.
Andreae LC,Burrone J(2014)The role of neuronal activity and transmitter release on synapse formation.Curr Opin Neurobiol 27:47-52.
Baudry M,Bi X(2016)Calpain-1 and Calpain-2:The Yin and Yang of synaptic plasticity and neurodegeneration.Trends Neurosci 39:235-245.
Baudry M,Kramar E,Xu X,Zadran H,Moreno S,Lynch G,Gall C,Bi X(2012)Ampakines promote spine actin polymerization,long-term potentiation,and learning in a mouse model of Angelman syndrome.Neurobiol Dis 47:210-215.
Briz V,Baudry M(2016)Calpains:master regulators of synaptic plasticity.Neuroscientist doi:10.1177/1073858416649178.
Briz V,Hsu YT,Li Y,Lee E,Bi X,Baudry M(2013)Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis.J Neurosci 33:4317-4328.
Cheng SY,Wang SC,Lei M,Wang Z,Xiong K(2018)Regulatory role of calpain in neuronal death.Neural Regen Res 13:556-562.
Cuesto G,Enriquez-Barreto L,Carames C,Cantarero M,Gasull X,Sandi C,Ferrús A,Acebesá,Morales M(2011)Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons.J Neurosci 31:2721-2733.
D’Orsi B,Bonner H,Tuffy LP,Dussmann H,Woods I,Courtney MJ,Ward MW,Prehn JH(2012)Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.J Neurosci 32:1847-1858.
Fan Y,Tang X,Vitriol E,Chen G,Zheng JQ(2011)Actin capping protein is required for dendritic spine development and synapse formation.JNeurosci 31:10228-10233.
Furukawa M,Tsukahara T,Tomita K,Iwai H,Sonomura T,Miyawaki S,Sato T(2017)Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.Biochem Biophys Res Commun 493:1243-1249.
Gatto CL,Broadie K(2010)Genetic controls balancing excitatory and inhibitory synaptogenesis in neurodevelopmental disorder models.Front Synaptic Neurosci 2:4.
Han L,Tian X(2005)Dynamic-average characteristics of late wave of auditory evoked potentials for synaptic malfunction rat model.Conf Proc IEEE Eng Med Biol Soc 4:3664-3667.
Hojo Y,Munetomo A,Mukai H,Ikeda M,Sato R,Hatanaka Y,Murakami G,Komatsuzaki Y,Kimoto T,Kawato S(2015)Estradiol rapidly modulates spinogenesis in hippocampal dentate gyrus:Involvement of kinase networks.Horm Behav 74:149-156.
Huang F,Chotiner JK,Steward O(2007)Actin polymerization and ERKphosphorylation are required for Arc/Arg3.1 mRNA targeting to activated synaptic sites on dendrites.J Neurosci 27:9054-9067.
Kaech S,Fischer M,Doll T,Matus A(1997)Isoform specificity in the relationship of actin to dendritic spines.J Neurosci 17:9565-9572.
Komatsuzaki Y,Hatanaka Y,Murakami G,Mukai H,Hojo Y,Saito M,Kimoto T,Kawato S(2012)Corticosterone induces rapid spinogenesis via synaptic glucocorticoid receptors and kinase networks in hippocampus.PLoS One 7:e34124.
Kuchibhotla KV,Goldman ST,Lattarulo CR,Wu HY,Hyman BT,Bacskai BJ(2008)Abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks.Neuron 59:214-225.
Li J,Yang S,Zhu G(2017a)Postnatal calpain inhibition elicits cerebellar cell death and motor dysfunction.Oncotarget 8:87997-88007.
Li J,Chen H,Wu S,Cheng Y,Li Q,Wang J,Zhu G(2017b)MPP(+)inhibits mGluR1/5-mediated long-term depression in mouse hippocampus by calpain activation.Eur J Pharmacol 795:22-27.
Li JP,Lu W,Yang L,Xie MX,He X,Pan AH(2017c)Neurons in the hippocampus of chemobrain versus non-chemotherapy brain.Zhongguo Zuzhi Gongcheng Yanjiu 21:4523-4528.
Li K,Chen Y,Jiang R,Chen D,Wang H,Xiong W,Li D,Liu Z,Li X,Li J,Yuan K(2017d)Protective effects of astragaloside IV against ovalbumin-induced allergic rhinitis are mediated by T-box protein expressed in T cells/GATA-3 and forkhead box protein 3/retinoic acid-related orphan nuclear receptor gammat.Mol Med Rep 16:1207-1215.
Li XY,Zhan XR,Liu XM,Wang XC(2011)CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet beta-cells mediated by hepatocyte growth factor.Biochem Biophys Res Commun 404:711-716.
Ma M(2013)Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon.Neurobiol Dis 60:61-79.
Prangsaengtong O,Senda K,Doki Y,Park JY,Jo M,Sakurai H,Shibahara N,Saiki I,Koizumi K(2012)Calpain 1 and-2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation.Hum Cell 25:36-44.
Samantaray S,Knaryan VH,Shields DC,Cox AA,Haque A,Banik NL(2015)Inhibition of calpain activation protects MPTP-induced nigral and spinal cord neurodegeneration,reduces inflammation,and improves gait dynamics in mice.Mol Neurobiol 52:1054-1066.
Shimizu K,Mackenzie SM,Storm DR(2010)SCOP/PHLPP and its functional role in the brain.Mol Biosyst 6:38-43.
Shimizu K,Phan T,Mansuy IM,Storm DR(2007)Proteolytic degradation of SCOP in the hippocampus contributes to activation of MAPkinase and memory.Cell 128:1219-1229.
Sindi IA,Dodd PR(2015)New insights into Alzheimer’s disease pathogenesis:the involvement of neuroligins in synaptic malfunction.Neurodegener Dis Manag 5:137-145.
Skilbeck KJ,Johnston GAR,Hinton T(2018)Long-lasting effects of early-life intervention in mice on adulthood behaviour,GABAA receptor subunit expression and synaptic clustering.Pharmacol Res 128:179-189.
Smith IJ,Dodd SL(2007)Calpain activation causes a proteasome-dependent increase in protein degradation and inhibits the Akt signalling pathway in rat diaphragm muscle.Exp Physiol 92:561-573.
Stalker TJ,Skvarka CB,Scalia R(2003)A novel role for calpains in the endothelial dysfunction of hyperglycemia.FASEB J 17:1511-1513.
Tashiro A,Makino H,Gage FH(2007)Experience-specific functional modification of the dentate gyrus through adult neurogenesis:a critical period during an immature stage.J Neurosci 27:3252-3259.
Tatti R,Haley MS,Swanson OK,Tselha T,Maffei A(2017)Neurophysiology and regulation of the balance between excitation and inhibition in neocortical circuits.Biol Psychiatry 81:821-831.
Vetulani J(2013)Early maternal separation:a rodent model of depression and a prevailing human condition.Pharmacol Rep 65:1451-1461.
Wang Y,Hersheson J,Lopez D,Hammer M,Liu Y,Lee KH,Pinto V,Seinfeld J,Wiethoff S,Sun J,Amouri R,Hentati F,Baudry N,Tran J,Singleton AB,Coutelier M,Brice A,Stevanin G,Durr A,Bi X,et al.(2016)Defects in the CAPN1 gene result in alterations in cerebellar development and cerebellar ataxia in mice and humans.Cell Rep 16:79-91.
Wang YL,Li F,Chen X(2015)Pten inhibitor-bpV ameliorates early postnatal propofol exposure-induced memory deficit and impairment of hippocampal LTP.Neurochem Res 40:1593-1599.
Xu F(2011)Interaction effects between AMP-activated protein kinase and mammalian target of rapamycin signaling pathways.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 15:3775-3777.
Xu W,Cao J,Zhou Y,Wang L,Zhu G(2018)GPR30 activation improves memory and facilitates DHPG-induced LTD in the hippocampal CA3of middle-aged mice.Neurobiol Learn Mem 149:10-19.
Zhu G,Wang Y,Li J,Wang J(2015a)Chronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged mice.Neuroscience 292:81-89.
Zhu G,Yang S,Xie Z,Wan X(2018)Synaptic modification by L-theanine,a natural constituent in green tea,rescues the impairment of hippocampal long-term potentiation and memory in AD mice.Neuropharmacology 138:331-340.
Zhu G,Liu Y,Wang Y,Bi X,Baudry M(2015b)Different patterns of electrical activity lead to long-term potentiation by activating different intracellular pathways.J Neurosci 35:621-633.
Zhu G,Briz V,Seinfeld J,Liu Y,Bi X,Baudry M(2017)Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction.Sci Rep 7:42788.
Andreae LC,Burrone J(2014)The role of neuronal activity and transmitter release on synapse formation.Curr Opin Neurobiol 27:47-52.
Baudry M,Bi X(2016)Calpain-1 and Calpain-2:The Yin and Yang of synaptic plasticity and neurodegeneration.Trends Neurosci 39:235-245.
Baudry M,Kramar E,Xu X,Zadran H,Moreno S,Lynch G,Gall C,Bi X(2012)Ampakines promote spine actin polymerization,long-term potentiation,and learning in a mouse model of Angelman syndrome.Neurobiol Dis 47:210-215.
Briz V,Baudry M(2016)Calpains:master regulators of synaptic plasticity.Neuroscientist doi:10.1177/1073858416649178.
Briz V,Hsu YT,Li Y,Lee E,Bi X,Baudry M(2013)Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis.J Neurosci 33:4317-4328.
Cheng SY,Wang SC,Lei M,Wang Z,Xiong K(2018)Regulatory role of calpain in neuronal death.Neural Regen Res 13:556-562.
Cuesto G,Enriquez-Barreto L,Carames C,Cantarero M,Gasull X,Sandi C,Ferrús A,Acebesá,Morales M(2011)Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons.J Neurosci 31:2721-2733.
D’Orsi B,Bonner H,Tuffy LP,Dussmann H,Woods I,Courtney MJ,Ward MW,Prehn JH(2012)Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.J Neurosci 32:1847-1858.
Fan Y,Tang X,Vitriol E,Chen G,Zheng JQ(2011)Actin capping protein is required for dendritic spine development and synapse formation.JNeurosci 31:10228-10233.
Furukawa M,Tsukahara T,Tomita K,Iwai H,Sonomura T,Miyawaki S,Sato T(2017)Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.Biochem Biophys Res Commun 493:1243-1249.
Gatto CL,Broadie K(2010)Genetic controls balancing excitatory and inhibitory synaptogenesis in neurodevelopmental disorder models.Front Synaptic Neurosci 2:4.
Han L,Tian X(2005)Dynamic-average characteristics of late wave of auditory evoked potentials for synaptic malfunction rat model.Conf Proc IEEE Eng Med Biol Soc 4:3664-3667.
Hojo Y,Munetomo A,Mukai H,Ikeda M,Sato R,Hatanaka Y,Murakami G,Komatsuzaki Y,Kimoto T,Kawato S(2015)Estradiol rapidly modulates spinogenesis in hippocampal dentate gyrus:Involvement of kinase networks.Horm Behav 74:149-156.
Huang F,Chotiner JK,Steward O(2007)Actin polymerization and ERKphosphorylation are required for Arc/Arg3.1 mRNA targeting to activated synaptic sites on dendrites.J Neurosci 27:9054-9067.
Kaech S,Fischer M,Doll T,Matus A(1997)Isoform specificity in the relationship of actin to dendritic spines.J Neurosci 17:9565-9572.
Komatsuzaki Y,Hatanaka Y,Murakami G,Mukai H,Hojo Y,Saito M,Kimoto T,Kawato S(2012)Corticosterone induces rapid spinogenesis via synaptic glucocorticoid receptors and kinase networks in hippocampus.PLoS One 7:e34124.
Kuchibhotla KV,Goldman ST,Lattarulo CR,Wu HY,Hyman BT,Bacskai BJ(2008)Abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks.Neuron 59:214-225.
Li J,Yang S,Zhu G(2017a)Postnatal calpain inhibition elicits cerebellar cell death and motor dysfunction.Oncotarget 8:87997-88007.
Li J,Chen H,Wu S,Cheng Y,Li Q,Wang J,Zhu G(2017b)MPP(+)inhibits mGluR1/5-mediated long-term depression in mouse hippocampus by calpain activation.Eur J Pharmacol 795:22-27.
Li JP,Lu W,Yang L,Xie MX,He X,Pan AH(2017c)Neurons in the hippocampus of chemobrain versus non-chemotherapy brain.Zhongguo Zuzhi Gongcheng Yanjiu 21:4523-4528.
Li K,Chen Y,Jiang R,Chen D,Wang H,Xiong W,Li D,Liu Z,Li X,Li J,Yuan K(2017d)Protective effects of astragaloside IV against ovalbumin-induced allergic rhinitis are mediated by T-box protein expressed in T cells/GATA-3 and forkhead box protein 3/retinoic acid-related orphan nuclear receptor gammat.Mol Med Rep 16:1207-1215.
Li XY,Zhan XR,Liu XM,Wang XC(2011)CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet beta-cells mediated by hepatocyte growth factor.Biochem Biophys Res Commun 404:711-716.
Ma M(2013)Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon.Neurobiol Dis 60:61-79.
Prangsaengtong O,Senda K,Doki Y,Park JY,Jo M,Sakurai H,Shibahara N,Saiki I,Koizumi K(2012)Calpain 1 and-2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation.Hum Cell 25:36-44.
Samantaray S,Knaryan VH,Shields DC,Cox AA,Haque A,Banik NL(2015)Inhibition of calpain activation protects MPTP-induced nigral and spinal cord neurodegeneration,reduces inflammation,and improves gait dynamics in mice.Mol Neurobiol 52:1054-1066.
Shimizu K,Mackenzie SM,Storm DR(2010)SCOP/PHLPP and its functional role in the brain.Mol Biosyst 6:38-43.
Shimizu K,Phan T,Mansuy IM,Storm DR(2007)Proteolytic degradation of SCOP in the hippocampus contributes to activation of MAPkinase and memory.Cell 128:1219-1229.
Sindi IA,Dodd PR(2015)New insights into Alzheimer’s disease pathogenesis:the involvement of neuroligins in synaptic malfunction.Neurodegener Dis Manag 5:137-145.
Skilbeck KJ,Johnston GAR,Hinton T(2018)Long-lasting effects of early-life intervention in mice on adulthood behaviour,GABAA receptor subunit expression and synaptic clustering.Pharmacol Res 128:179-189.
Smith IJ,Dodd SL(2007)Calpain activation causes a proteasome-dependent increase in protein degradation and inhibits the Akt signalling pathway in rat diaphragm muscle.Exp Physiol 92:561-573.
Stalker TJ,Skvarka CB,Scalia R(2003)A novel role for calpains in the endothelial dysfunction of hyperglycemia.FASEB J 17:1511-1513.
Tashiro A,Makino H,Gage FH(2007)Experience-specific functional modification of the dentate gyrus through adult neurogenesis:a critical period during an immature stage.J Neurosci 27:3252-3259.
Tatti R,Haley MS,Swanson OK,Tselha T,Maffei A(2017)Neurophysiology and regulation of the balance between excitation and inhibition in neocortical circuits.Biol Psychiatry 81:821-831.
Vetulani J(2013)Early maternal separation:a rodent model of depression and a prevailing human condition.Pharmacol Rep 65:1451-1461.
Wang Y,Hersheson J,Lopez D,Hammer M,Liu Y,Lee KH,Pinto V,Seinfeld J,Wiethoff S,Sun J,Amouri R,Hentati F,Baudry N,Tran J,Singleton AB,Coutelier M,Brice A,Stevanin G,Durr A,Bi X,et al.(2016)Defects in the CAPN1 gene result in alterations in cerebellar development and cerebellar ataxia in mice and humans.Cell Rep 16:79-91.
Wang YL,Li F,Chen X(2015)Pten inhibitor-bpV ameliorates early postnatal propofol exposure-induced memory deficit and impairment of hippocampal LTP.Neurochem Res 40:1593-1599.
Xu F(2011)Interaction effects between AMP-activated protein kinase and mammalian target of rapamycin signaling pathways.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 15:3775-3777.
Xu W,Cao J,Zhou Y,Wang L,Zhu G(2018)GPR30 activation improves memory and facilitates DHPG-induced LTD in the hippocampal CA3of middle-aged mice.Neurobiol Learn Mem 149:10-19.
Zhu G,Wang Y,Li J,Wang J(2015a)Chronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged mice.Neuroscience 292:81-89.
Zhu G,Yang S,Xie Z,Wan X(2018)Synaptic modification by L-theanine,a natural constituent in green tea,rescues the impairment of hippocampal long-term potentiation and memory in AD mice.Neuropharmacology 138:331-340.
Zhu G,Liu Y,Wang Y,Bi X,Baudry M(2015b)Different patterns of electrical activity lead to long-term potentiation by activating different intracellular pathways.J Neurosci 35:621-633.
Zhu G,Briz V,Seinfeld J,Liu Y,Bi X,Baudry M(2017)Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction.Sci Rep 7:42788.