J Mol Neurosci (2014) 54:414–429 DOI 10.1007/s12031-014-0381-9
Protein Profiling Reveals Antioxidant and Signaling Activities of NAP (Davunetide) in Rodent Hippocampus Exposed to Hypobaric Hypoxia Niroj Kumar Sethy & Narendra Kumar Sharma & Mainak Das & Kalpana Bhargava
Received: 27 February 2014 / Accepted: 9 July 2014 / Published online: 20 July 2014 # Springer Science+Business Media New York 2014
Niroj Kumar Sethy and Narendra Kumar Sharma contributed to this work equally.
revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.
Electronic supplementary material The online version of this article (doi:10.1007/s12031-014-0381-9) contains supplementary material, which is available to authorized users.
Keywords NAP . Davunetide . Hypoxia . Proteomics . Antioxidant
Abstract NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAPmediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2DGE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAPsupplemented hippocampus after hypoxia. Pathway analysis
N. K. Sethy (*) : N. K. Sharma : K. Bhargava (*) Peptide and Proteomics Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi 110054, India e-mail: [email protected] e-mail: [email protected] K. Bhargava e-mail: [email protected] M. Das Bioscience and Bioengineering (BSBE), Indian Institute of Technology, Kanpur 208016, UP, India Present Address: N. K. Sharma Immunology Laboratory, Division of Infectious Disease, Departmento de medicina-EscolaPaulista de Medicina (EPM), Universidade Federal De Sao paulo (UNIFESP), Sao Paulo 04039032, Brazil
Introduction Cellular hypoxia resulting from asphyxia, impaired cerebral perfusion and exposure to high altitude is the common final pathway for brain function alteration and injury (Wilson et al. 2009). The low barometric pressure of inspired oxygen at high altitudes compromises oxygen supply to brain leading to several neurological clinical syndromes including highaltitude headache (HAH), insomnia, dizziness, sleep disturbance, cognitive dysfunctions, neuroinflammation and highaltitude cerebral edema (HACE) (Hackett et al. 2001; Wilson et al. 2009; Bartsch and Swenson 2013). Clinical evidences suggest that macromorphological damages such as cortical atrophy, cortical and subcortical lesions may occur in individuals with HACE and high-altitude illness (Hackett et al. 1998;
J Mol Neurosci (2014) 54:414–429
Usui et al. 2004; Fayed et al. 2006; Di Paola et al. 2008). Similarly, brain white and grey matter microstructural alterations have also been diagnosed in mountain climbers after one or repeated exposure to extreme altitudes (Zhang et al. 2013a, b). Hence, preserving both the function and structure of brain during physiological or pathological hypoxia is a major neurological challenge. Oxidative stress associated with hypobaric hypoxia is one of the major factors for the observed functional and structural modifications in brain at high altitude (Moller et al. 2001; Zhang et al. 2013a, b). Uncontrolled generation of mitochondrial reactive oxygen species (ROS) and reactive nitrogen species (RNS) during hypoxia induces severe oxidative stress in brain. These reactive species can modify macromolecules like lipids, proteins, carbohydrates and DNA altering cellular function (Bailey et al. 2001; Moller et al. 2001; Magalhaes et al. 2005). Hypoxia exposure also depletes both enzymatic and non-enzymatic antioxidant defence systems in brain making regions like hippocampus and cortex most vulnerable to hypoxic oxidative damage (Maiti et al. 2006; Van Elzen et al. 2008; Sharma et al. 2013). Studying brain regional response to short-term hypobaric hypoxia, we have previously reported that hypoxia-induced deficient energy production and antioxidant enzyme depletion make hippocampus vulnerable to oxidative stress (Sharma et al. 2013). It has been advocated that supplementation of suitable antioxidants can reduce brain oxidative damage during hypoxia (Moller et al. 2001). NAP (davunetide) is an eight amino acid snippet (NAPVSIPQ = Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP) and reportedly possesses antioxidant, neutrotrophic, neuroprotective and cognitive protective activities (Offen et al. 2000; Steingart et al. 2000; Leker et al. 2002; Steingart et al. 2006; Zemlyak et al. 2007, 2009; Blat et al. 2008; Gozes et al. 2011; Gold et al. 2012; Idan-Feldman et al. 2012; Javitt et al. 2012; Jarskog et al. 2013). This peptide drug candidate is safe, well-tolerated, crosses the blood–brain barrier and demonstrates distribution throughout all areas of the brain (Gozes et al. 2000, 2005, 2009; Magen and Gozes 2013; Morimoto et al. 2013). Studies on both cellular and animal models suggest that NAP confers neuroprotection by maintaining microtubule function and inhibition of apoptosis (Divinski et al. 2004, 2006; Fleming et al. 2011; Gold et al. 2012; Oz et al. 2012; Jouroukhin et al. 2013). However, the molecular effects of NAP on brain and associated proteome changes during oxidative stress have been poorly investigated. Studying the effects of NAP during longterm chronic hypobaric hypoxia (7–28 days), we have previously reported that NAP confers neuroprotection by ameliorating oxidative stress (Sharma et al. 2011). However, the effect of NAP on brain hippocampus proteome and associated signaling pathways during hypobaric hypoxia is not yet reported.
415
In the present study, we aimed to identify the molecular mechanism of NAP-mediated neuroprotection by studying hippocampus proteome after 3, 6 and 12 h of temporal hypobaric hypoxia (25,000 ft, 282 mm Hg). These time points were selected based on the reports that 6 to 12 h of chronic hypobaric hypoxia exposure induces enough oxidative stress to activate apoptotic pathways in brain (Sharma et al. 2011). Using 2D-gel electrophoresis (2D-GE) along with matrixassisted laser desorption ionization time of flight (MALDITOF)/TOF mass spectrometry, we are able to identify 80 differentially expressed proteins in NAP-supplemented hypoxic hippocampus as compared to hypoxic hippocampus. Comparing the protein expression levels at each time point, we have identified higher expression of antioxidant and stress responsive proteins and pathways in NAP-supplemented hippocampus. Further, we have validated the expression levels of Sod1, Eno1, Pebp1, Prdx2 and Prdx5 by Western blotting and Prdx2 expression by immunohistochemistry.
Materials and Methods Animal Experiments and Davunetide Supplementation Animal experiments were performed according to institutional guidelines and were in accordance with the principles and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. Male adult Sprague-Dawley rats (220±10 g) were divided in seven groups (n=18 in each group) (Fig. 1). Group I received vehicle (normal saline) and served as normoxia control (736 mm Hg). Groups II, III and IV served as hypoxia group where as groups V, VI and VII served as NAP (davunetide) supplemented (2 μg/kg of body weight, intranasal) hypoxia groups (Sharma et al. 2011). Animals of hypoxia group (groups II, III and IV) along with NAP-supplemented group (groups V, VI and VII) were exposed to an altitude of 25,000 ft (7,620 m, 282 mm Hg) for 3, 6 and 12 h respectively. Exposure to this altitude reportedly promotes hypoxic brain inflammation and HACE (Patir et al. 2012). After hypoxic exposure, brains from both normoxic as well as hypoxia-exposed animals were removed and dissected hippocampus was snapfrozen in liquid nitrogen and stored at −80 °C. Protein Extraction Total protein from 100-mg wet tissue was extracted in 500 μl lysis buffer containing 40 mM Tris (pH 7.5), 8 M urea, 2.5 M thiourea, 3 % CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF and mammalian protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The homogenate was sonicated, vortexed, and centrifuged at 15,000g at 4 °C for 45 min. Protein
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J Mol Neurosci (2014) 54:414–429
Fig. 1 Representative 2D gel image of rat brain hippocampus proteins during 12 h hypobaric hypoxia with NAP supplementation. The proteins were resolved according to their isoelectric point (pI) followed by molecular weight on 12 % SDSPAGE and silver stained. Differentially expressed protein spots were marked with SwissProt accession number on the representative gel. Molecular weights have been mentioned on the right side of the gel image
concentration of supernatants were determined using the Bradford method and were further stored at −80 °C. Two-dimensional Polyacrylamide Gel Electrophoresis and Image Analysis For each sample, 200 μg of total protein (n=6 for each group run in triplicates) was subjected to isoelectric focusing on Immobiline DryStrip (pH 3–10, 18 cm, GE Healthcare, Sweden) using IPGphor IEF System (Amersham Biotech, Uppsala, Sweden) and subsequently separated on 12 % sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were visualized in SDS-PAGE gels by high sensitivity silver staining as reported earlier (Sharma et al. 2013). The stained gel images were acquired using an InvestigatorTM ProPic II (Genomic Solutions, UK) and analysed using 2D-Progenesis Samespot Software (V 4.0, Nonlinear Dynamics, USA). The relative intensity of individual spots in 2D gels of different time exposures, treatments and control gels were quantified using a grey scale, and the difference between the spot pairs were determined. Differences of matched spots were considered significant whenever a spot group passed statistical analysis (p
Protein Profiling Reveals Antioxidant and Signaling Activities of NAP (Davunetide) in Rodent Hippocampus Exposed to Hypobaric Hypoxia Niroj Kumar Sethy & Narendra Kumar Sharma & Mainak Das & Kalpana Bhargava
Received: 27 February 2014 / Accepted: 9 July 2014 / Published online: 20 July 2014 # Springer Science+Business Media New York 2014
Niroj Kumar Sethy and Narendra Kumar Sharma contributed to this work equally.
revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.
Electronic supplementary material The online version of this article (doi:10.1007/s12031-014-0381-9) contains supplementary material, which is available to authorized users.
Keywords NAP . Davunetide . Hypoxia . Proteomics . Antioxidant
Abstract NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAPmediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2DGE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAPsupplemented hippocampus after hypoxia. Pathway analysis
N. K. Sethy (*) : N. K. Sharma : K. Bhargava (*) Peptide and Proteomics Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi 110054, India e-mail: [email protected] e-mail: [email protected] K. Bhargava e-mail: [email protected] M. Das Bioscience and Bioengineering (BSBE), Indian Institute of Technology, Kanpur 208016, UP, India Present Address: N. K. Sharma Immunology Laboratory, Division of Infectious Disease, Departmento de medicina-EscolaPaulista de Medicina (EPM), Universidade Federal De Sao paulo (UNIFESP), Sao Paulo 04039032, Brazil
Introduction Cellular hypoxia resulting from asphyxia, impaired cerebral perfusion and exposure to high altitude is the common final pathway for brain function alteration and injury (Wilson et al. 2009). The low barometric pressure of inspired oxygen at high altitudes compromises oxygen supply to brain leading to several neurological clinical syndromes including highaltitude headache (HAH), insomnia, dizziness, sleep disturbance, cognitive dysfunctions, neuroinflammation and highaltitude cerebral edema (HACE) (Hackett et al. 2001; Wilson et al. 2009; Bartsch and Swenson 2013). Clinical evidences suggest that macromorphological damages such as cortical atrophy, cortical and subcortical lesions may occur in individuals with HACE and high-altitude illness (Hackett et al. 1998;
J Mol Neurosci (2014) 54:414–429
Usui et al. 2004; Fayed et al. 2006; Di Paola et al. 2008). Similarly, brain white and grey matter microstructural alterations have also been diagnosed in mountain climbers after one or repeated exposure to extreme altitudes (Zhang et al. 2013a, b). Hence, preserving both the function and structure of brain during physiological or pathological hypoxia is a major neurological challenge. Oxidative stress associated with hypobaric hypoxia is one of the major factors for the observed functional and structural modifications in brain at high altitude (Moller et al. 2001; Zhang et al. 2013a, b). Uncontrolled generation of mitochondrial reactive oxygen species (ROS) and reactive nitrogen species (RNS) during hypoxia induces severe oxidative stress in brain. These reactive species can modify macromolecules like lipids, proteins, carbohydrates and DNA altering cellular function (Bailey et al. 2001; Moller et al. 2001; Magalhaes et al. 2005). Hypoxia exposure also depletes both enzymatic and non-enzymatic antioxidant defence systems in brain making regions like hippocampus and cortex most vulnerable to hypoxic oxidative damage (Maiti et al. 2006; Van Elzen et al. 2008; Sharma et al. 2013). Studying brain regional response to short-term hypobaric hypoxia, we have previously reported that hypoxia-induced deficient energy production and antioxidant enzyme depletion make hippocampus vulnerable to oxidative stress (Sharma et al. 2013). It has been advocated that supplementation of suitable antioxidants can reduce brain oxidative damage during hypoxia (Moller et al. 2001). NAP (davunetide) is an eight amino acid snippet (NAPVSIPQ = Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP) and reportedly possesses antioxidant, neutrotrophic, neuroprotective and cognitive protective activities (Offen et al. 2000; Steingart et al. 2000; Leker et al. 2002; Steingart et al. 2006; Zemlyak et al. 2007, 2009; Blat et al. 2008; Gozes et al. 2011; Gold et al. 2012; Idan-Feldman et al. 2012; Javitt et al. 2012; Jarskog et al. 2013). This peptide drug candidate is safe, well-tolerated, crosses the blood–brain barrier and demonstrates distribution throughout all areas of the brain (Gozes et al. 2000, 2005, 2009; Magen and Gozes 2013; Morimoto et al. 2013). Studies on both cellular and animal models suggest that NAP confers neuroprotection by maintaining microtubule function and inhibition of apoptosis (Divinski et al. 2004, 2006; Fleming et al. 2011; Gold et al. 2012; Oz et al. 2012; Jouroukhin et al. 2013). However, the molecular effects of NAP on brain and associated proteome changes during oxidative stress have been poorly investigated. Studying the effects of NAP during longterm chronic hypobaric hypoxia (7–28 days), we have previously reported that NAP confers neuroprotection by ameliorating oxidative stress (Sharma et al. 2011). However, the effect of NAP on brain hippocampus proteome and associated signaling pathways during hypobaric hypoxia is not yet reported.
415
In the present study, we aimed to identify the molecular mechanism of NAP-mediated neuroprotection by studying hippocampus proteome after 3, 6 and 12 h of temporal hypobaric hypoxia (25,000 ft, 282 mm Hg). These time points were selected based on the reports that 6 to 12 h of chronic hypobaric hypoxia exposure induces enough oxidative stress to activate apoptotic pathways in brain (Sharma et al. 2011). Using 2D-gel electrophoresis (2D-GE) along with matrixassisted laser desorption ionization time of flight (MALDITOF)/TOF mass spectrometry, we are able to identify 80 differentially expressed proteins in NAP-supplemented hypoxic hippocampus as compared to hypoxic hippocampus. Comparing the protein expression levels at each time point, we have identified higher expression of antioxidant and stress responsive proteins and pathways in NAP-supplemented hippocampus. Further, we have validated the expression levels of Sod1, Eno1, Pebp1, Prdx2 and Prdx5 by Western blotting and Prdx2 expression by immunohistochemistry.
Materials and Methods Animal Experiments and Davunetide Supplementation Animal experiments were performed according to institutional guidelines and were in accordance with the principles and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. Male adult Sprague-Dawley rats (220±10 g) were divided in seven groups (n=18 in each group) (Fig. 1). Group I received vehicle (normal saline) and served as normoxia control (736 mm Hg). Groups II, III and IV served as hypoxia group where as groups V, VI and VII served as NAP (davunetide) supplemented (2 μg/kg of body weight, intranasal) hypoxia groups (Sharma et al. 2011). Animals of hypoxia group (groups II, III and IV) along with NAP-supplemented group (groups V, VI and VII) were exposed to an altitude of 25,000 ft (7,620 m, 282 mm Hg) for 3, 6 and 12 h respectively. Exposure to this altitude reportedly promotes hypoxic brain inflammation and HACE (Patir et al. 2012). After hypoxic exposure, brains from both normoxic as well as hypoxia-exposed animals were removed and dissected hippocampus was snapfrozen in liquid nitrogen and stored at −80 °C. Protein Extraction Total protein from 100-mg wet tissue was extracted in 500 μl lysis buffer containing 40 mM Tris (pH 7.5), 8 M urea, 2.5 M thiourea, 3 % CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF and mammalian protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The homogenate was sonicated, vortexed, and centrifuged at 15,000g at 4 °C for 45 min. Protein
416
J Mol Neurosci (2014) 54:414–429
Fig. 1 Representative 2D gel image of rat brain hippocampus proteins during 12 h hypobaric hypoxia with NAP supplementation. The proteins were resolved according to their isoelectric point (pI) followed by molecular weight on 12 % SDSPAGE and silver stained. Differentially expressed protein spots were marked with SwissProt accession number on the representative gel. Molecular weights have been mentioned on the right side of the gel image
concentration of supernatants were determined using the Bradford method and were further stored at −80 °C. Two-dimensional Polyacrylamide Gel Electrophoresis and Image Analysis For each sample, 200 μg of total protein (n=6 for each group run in triplicates) was subjected to isoelectric focusing on Immobiline DryStrip (pH 3–10, 18 cm, GE Healthcare, Sweden) using IPGphor IEF System (Amersham Biotech, Uppsala, Sweden) and subsequently separated on 12 % sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were visualized in SDS-PAGE gels by high sensitivity silver staining as reported earlier (Sharma et al. 2013). The stained gel images were acquired using an InvestigatorTM ProPic II (Genomic Solutions, UK) and analysed using 2D-Progenesis Samespot Software (V 4.0, Nonlinear Dynamics, USA). The relative intensity of individual spots in 2D gels of different time exposures, treatments and control gels were quantified using a grey scale, and the difference between the spot pairs were determined. Differences of matched spots were considered significant whenever a spot group passed statistical analysis (p
