- Email: [email protected]
A Radical Pathway for Minocycline Toxicity: Role of Cytochrome c and Sulfite
results suggest the increase in DJ-1 oxidation in erythrocytes and the brain of patients with PD, particularly at early phases. In this presentation, we will discuss the biochemical changes in peripheral tissues of early-phase PD, which could be used as a marker for the early diagnosis of PD. In addition, we will show that oxDJ-1 interacts with the 20S proteasome, and discuss the relation of DJ1 to the accumulation of modified proteins in PD patients.
and MMP-9, attenuated HT, decreased mortality and improved neurological outcomes. In conclusion, glycyrrhizin could be an adjuvant therapy with t-PA to improve the therapeutic outcome for ischemic stroke.
doi: 10.1016/j.freeradbiomed.2016.10.438
400
398
Ubiquitome Profile in Down Syndrome Brain: Understanding the Mechanisms of Neurodegeneration
Caveolin-1 is Critical for Lymphocyte Trafficking into Central Nervous System during Experimental Autoimmune Encephalomyelitis Jiangang Shen1 and Hao Wu1 School of Chinese Medicine, University of Hong Kong, Hong Kong
1
In the present study, we tested the hypothesis that caveolin-1 plays a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a laboratory murine model of Multiple Sclerosis (MS). After active EAE induction, the increased caveolin1 in serum and CNS tissues was associated with disease incidence and severity in immunized wild-type (WT) mice. After immunization, Cav-1knockout (KO) mice showed remarkable disease resistance with alleviated incidences and clinical symptoms. Cav-1 KO mice had diminished infiltration of encephalitogenic T cells into CNS parenchyma with compromised expressions of adhesion molecules ICAM-1 and VCAM-1 in the white matter of CNS tissues. Consistently, in vitro knockdown of caveolin-1 impaired the upregulation of ICAM-1 in endothelial cells, and ameliorated transendothelial migration of pathogenic TH1 and TH17 cells. Therefore, caveolin-1 serves as an active modulator of lymphocyte trafficking into CNS and could be a therapeutic target for multiple sclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.439 399 Glycyrrhizin Could Inhibit HMGB1-MMP-9 Signaling and Prevent Hemorrhagic Transformation in Ischemic Stroke with Delayed T-PA Treatment Jiangang Shen1 and Hansen Chen1 1 School of Chinese Medicine, University of Hong Kong, Hong Kong Tissue plasminogen activator (t-PA) has restrict therapeutic window for ischemic stroke and hemorrhagic transformation (HT) is the major complications. High mobility group box protein 1 (HMGB1) and MMPs are critical targets for reducing HT. Glycyrrhizin is an antioxidant isolated from Glycyrrhiza glabra. We hypothesize that glycyrrhizin could inhibit HMGB1, MMPs, reduce HT and improve survival rates in ischemic stroke with delayed t-PA tretament. Male SD rats were subjected middle cerebral artery occlusion (MCAO) for 5 hours plus 19 hours of reperfusion. T-PA (10 mg/kg) was intravenously administrated at 4.5 hours after MCAO and continuously injected for 30 min. Glycyrrhizin (30 mg/kg) was administrated at onset of t-PA treatment and saline was used as control. Major discoveries are as below: (1) Delayed t-PA treatment had increased mortality rate, brain swelling, hemorrhagic transformation with the increase of HMGB1 and MMP-9 expression. (2) Glycyrrhizin significantly inhibited HMGB1
doi: 10.1016/j.freeradbiomed.2016.10.440
Antonella Tramutola1, Fabio Di Domenico1, Eugenio Barone1, Elizabeth Head2, D Allan Butterfield2, and Marzia Perluigi1 1 "A.Rossi Fanelli",Sapienza University of Rome, Italy, 2University of Kentucky, Lexington, USA Aims: Among the putative mechanisms proposed to be common factors in Down Syndrome (DS) and Alzheimer disease (AD) neuropathology, deficits in protein quality control has emerged as a unifying mechanism of neurodegeneration. Considering that disturbance of protein degradative systems are present in DS and that oxidized/misfolded proteins require poly-ubiquitination for degradation via the ubiquitin proteasome system (UPS), this study investigated if the dysregulation of protein poly-ubiquitination is associated with AD neurodegeneration in DS. Results: Post-mortem brains from DS cases, prior to and after development of AD neuropathology and age matched controls were analyzed. By selectively isolating poly-ubiquitinated proteins, we were able to identify the specific proteins with an altered pattern of poly-ubiquitination as a function of age. Interestingly, we found that oxidation is coupled with poly-ubiquitination for the majority of proteins, which are mainly involved in protein quality control and energy metabolism. Innovation: This is the first study showing alteration of the polyubiquitination profile as a function of aging in DS brain compared with healthy controls. Understanding the onset of the altered ubiquitome profile in DS brain may contribute to identification of key molecular regulators of age-associated cognitive decline. Conclusions: Disturbance of the poly-ubiquitination machinery may be a key feature of aging and neurodegeneration. In DS, ageassociated deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/poly-ubiquitinated proteins, which are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology.
doi: 10.1016/j.freeradbiomed.2016.10.441 401 A Radical Pathway for Minocycline Toxicity: Role of Cytochrome c and Sulfite Murugesan Velayutham1, Shanmugam Nagarajan1, and Arturo J Cardounel1 1 Vascular Medicine Institute, University of Pittsburgh, USA Minocycline (MC) is a second-generation semi-synthetic analogue of tetracycline. MC has been used as an antibiotic and for the treatment of various diseases such as acne vulgaris, sexually transmitted diseases, and rheumatoid arthritis. MC is currently being explored in various clinical trials for the treatment of cardiovascular diseases such as stroke, vascular aneurisms, and coronary by-pass surgery. However, the clinical use of MC has
SfRBM / SFRRI 2016
S167
been associated with potential side effects. It has been demonstrated that MC interacts with cytochrome c (Cyt-c) and inhibits its peroxidase activity. However, molecular mechanism involved in the interaction between Cyt-c and MC is not studied. In this study, UV-visible spectroscopic measurements were carried out to understand the reaction between ferric cytochrome c (Fe3+Cyt-c) and MC. MC reduces Fe3+Cyt-c to ferrous cytochrome c (Fe2+Cyt-c). The reduction of Fe3+Cyt-c increases with increasing concentration of MC. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with Fe3+Cyt-c, MC, and sulfite. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3– ) was obtained. The formation of DMPO-SO3– was very low in the absence of MC. Interestingly, the addition of MC enhanced the DMPO-SO3– adducts. Furthermore, the formation of DMPO-SO3– adducts increased with increasing concentration of MC. From these results, we propose a mechanism that the Fe3+Cyt-c oxidizes MC to MC radical, which subsequently oxidizes sulfite to sulfite radical. Our results suggest that Fe3+Cyt-c could have a novel role in the deleterious effects of MC in biological systems by increasing the production of sulfite radical.
doi: 10.1016/j.freeradbiomed.2016.10.442 402 Thiol Oxidation Mediated Redox Signaling Triggers Specific Dopaminergic Neurodegeneration in Parkinson′s Disease Aditi Verma1, Ajit Ray1, Smitha Karunakaran1, Lalitha Durgadoss1, and Vijayalakshmi Ravindranath1 1 Indian Institute of Science, Bangalore, India Oxidative stress has been implicated in pathogenesis of Parkinson’s disease (PD), but it is yet unclear as to how the generalized ROS generation leads to degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Activation of redox driven signaling pathways may contribute to the specific cell death seen in PD. Using the MPTP mouse model of PD, we have established that stress-regulated MAPK signaling, specifically p38 MAPK-dependent death signaling, is selectively activated in dopaminergic neurons, while JNK is activated in microglia. Further, we found ASK1 as an upstream MAP3K responsible for p38 activation. ASK1, in turn, was selectively activated by TNF upregulation acting through thiol oxidation of Trx1, a negative regulator of ASK1 activity. Further, we also observed inhibition of Akt-mediated cell survival pathway through cysteine oxidation in Akt. As protein thiol oxidation emerged as a major contributor towards selective cell death signaling, we developed a novel diamide-induced mouse model of PD to examine whether thiol oxidation alone, caused by diamide, could trigger PD-relevant phenotype. Stereotaxic injection of diamide into mouse brain led to extensive dopaminergic neurodegeneration, coupled with biochemical and behavioral deficits as observed in PD, and activation of ASK1-mediated cell death signaling. αSynuclein aggregation, not often seen in other models of PD, was also reproduced in this model. Current studies are underway to investigate whether Grx1 inhibition in vivo (leading to increased glutathionylation and protein thiol oxidation), results in PD pathology. Overall, these studies demonstrate the critical role of thiol oxidation in triggering neurodegeneration in PD.
doi: 10.1016/j.freeradbiomed.2016.10.443
403 Clearing Amyloid-β Through PPARγ/ApoE Activation by Genistein is an Experimental Treatment of Alzheimer's Disease Jose Vina1, Bonet-Costa Vicent1, MariCarmen Blanco-Gandía1, Vicente Herranz-Pérez1, Cristina Mas-Bargues1, Marta Ingles1, Patricia GarciaTarraga1, Marta Rodriguez-Arias1, Jose Minarro1, Consuelo Borras1, and Jose- Manuel Garcia- Verdugo1 1 University of Valencia, Spain Amyloid-β (Aβ) clearance from brain, which is decreased in Alzheimer’s disease, is facilitated by apolipoprotein E. Apo E is upregulated by activation of the retinoid X receptor moiety of the RXR/PPARγ dimeric receptor. Genistein, a non-toxic, well tested and inexpensive drug has a multifaceted protective effect: antioxidant (because it stimulates the expression of antioxidant genes), anit-inflammatory and stimulator of activates the PPARγ receptor, which results in increased expression of ApoE. Treatment of an Alzheimer’s mouse model with genistein results in a remarkable and rapid improvement in various parameters of cognition, such as hippocampal learning, recognition memory, implicit memory and odor discrimination. This is associated with a lowering of Aβ levels in brain, in the number and the area of amyloid plaques (confirmed in vivo by positron emission tomography) as well as in microglial reactivity. Finally, incubation of primary astrocytes with genistein results in a PPARγ-mediated increased release of ApoE. Our results strongly suggest that controlled clinical trials should be performed to test the effect of genistein as treatment of human Alzheimer’s disease.
doi: 10.1016/j.freeradbiomed.2016.10.444 404 Nitrosylation of Vesicular Transporters in Brain of Amyloid Precursor Protein/presenilin 1 Double Transgenic Mice Ying Wang1 and Jun-Feng Wang1 University of Manitoba, Winnipeg, Canada
1
Nitric oxide can attack thiols of protein cysteine residues and induce cysteine S-nitrosylation. Cholinergic and glutamatergic systems are dysregulated in Alzheimer’s disease (AD). Vesicular acetylcholine transporter (VAChT) and vesicular glutamate transporter 1 (VGLUT1) are important in packaging acetylcholine and glutamate into vesicles that is an important step for neurotransmission. Previously we found that VAChT and VGLUT1 can be nitrosylated and this nitrosylation inhibits vesicular uptake of acetylcholine and glutamate. To understand the role of VAChT and VGLUT1 nitrosylation in the pathophysiological development of AD, we analyzed nitrosylation to VAChT and VGLUT1 in brain of amyloid precursor protein (APP) and presenilin 1 (PS1) transgenic mice, an animal model for AD. We found that 9 and 12 month old APP/PS1 mice showed memory deficit. We further found that total protein nitrosylation was increased in frontal cortex and hippocampus of 9 and 12 month old APP/PS1 mice when compared to wild type mice. Although nitrosylation of VAChT and VGLUT1 was not changed in hippocampus, nitrosylation of VAChT and VGLUT1 was increased in frontal cortex of 9 and 12 month old APP/PS1 mice. These findings suggest that nitrosylation of VAChT and VGLUT1 may be associated with dysfunctional acetylcholinergic and glutamatergic neurotransmission in AD.
doi: 10.1016/j.freeradbiomed.2016.10.445 S168
SfRBM / SFRRI 2016
and MMP-9, attenuated HT, decreased mortality and improved neurological outcomes. In conclusion, glycyrrhizin could be an adjuvant therapy with t-PA to improve the therapeutic outcome for ischemic stroke.
doi: 10.1016/j.freeradbiomed.2016.10.438
400
398
Ubiquitome Profile in Down Syndrome Brain: Understanding the Mechanisms of Neurodegeneration
Caveolin-1 is Critical for Lymphocyte Trafficking into Central Nervous System during Experimental Autoimmune Encephalomyelitis Jiangang Shen1 and Hao Wu1 School of Chinese Medicine, University of Hong Kong, Hong Kong
1
In the present study, we tested the hypothesis that caveolin-1 plays a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a laboratory murine model of Multiple Sclerosis (MS). After active EAE induction, the increased caveolin1 in serum and CNS tissues was associated with disease incidence and severity in immunized wild-type (WT) mice. After immunization, Cav-1knockout (KO) mice showed remarkable disease resistance with alleviated incidences and clinical symptoms. Cav-1 KO mice had diminished infiltration of encephalitogenic T cells into CNS parenchyma with compromised expressions of adhesion molecules ICAM-1 and VCAM-1 in the white matter of CNS tissues. Consistently, in vitro knockdown of caveolin-1 impaired the upregulation of ICAM-1 in endothelial cells, and ameliorated transendothelial migration of pathogenic TH1 and TH17 cells. Therefore, caveolin-1 serves as an active modulator of lymphocyte trafficking into CNS and could be a therapeutic target for multiple sclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.439 399 Glycyrrhizin Could Inhibit HMGB1-MMP-9 Signaling and Prevent Hemorrhagic Transformation in Ischemic Stroke with Delayed T-PA Treatment Jiangang Shen1 and Hansen Chen1 1 School of Chinese Medicine, University of Hong Kong, Hong Kong Tissue plasminogen activator (t-PA) has restrict therapeutic window for ischemic stroke and hemorrhagic transformation (HT) is the major complications. High mobility group box protein 1 (HMGB1) and MMPs are critical targets for reducing HT. Glycyrrhizin is an antioxidant isolated from Glycyrrhiza glabra. We hypothesize that glycyrrhizin could inhibit HMGB1, MMPs, reduce HT and improve survival rates in ischemic stroke with delayed t-PA tretament. Male SD rats were subjected middle cerebral artery occlusion (MCAO) for 5 hours plus 19 hours of reperfusion. T-PA (10 mg/kg) was intravenously administrated at 4.5 hours after MCAO and continuously injected for 30 min. Glycyrrhizin (30 mg/kg) was administrated at onset of t-PA treatment and saline was used as control. Major discoveries are as below: (1) Delayed t-PA treatment had increased mortality rate, brain swelling, hemorrhagic transformation with the increase of HMGB1 and MMP-9 expression. (2) Glycyrrhizin significantly inhibited HMGB1
doi: 10.1016/j.freeradbiomed.2016.10.440
Antonella Tramutola1, Fabio Di Domenico1, Eugenio Barone1, Elizabeth Head2, D Allan Butterfield2, and Marzia Perluigi1 1 "A.Rossi Fanelli",Sapienza University of Rome, Italy, 2University of Kentucky, Lexington, USA Aims: Among the putative mechanisms proposed to be common factors in Down Syndrome (DS) and Alzheimer disease (AD) neuropathology, deficits in protein quality control has emerged as a unifying mechanism of neurodegeneration. Considering that disturbance of protein degradative systems are present in DS and that oxidized/misfolded proteins require poly-ubiquitination for degradation via the ubiquitin proteasome system (UPS), this study investigated if the dysregulation of protein poly-ubiquitination is associated with AD neurodegeneration in DS. Results: Post-mortem brains from DS cases, prior to and after development of AD neuropathology and age matched controls were analyzed. By selectively isolating poly-ubiquitinated proteins, we were able to identify the specific proteins with an altered pattern of poly-ubiquitination as a function of age. Interestingly, we found that oxidation is coupled with poly-ubiquitination for the majority of proteins, which are mainly involved in protein quality control and energy metabolism. Innovation: This is the first study showing alteration of the polyubiquitination profile as a function of aging in DS brain compared with healthy controls. Understanding the onset of the altered ubiquitome profile in DS brain may contribute to identification of key molecular regulators of age-associated cognitive decline. Conclusions: Disturbance of the poly-ubiquitination machinery may be a key feature of aging and neurodegeneration. In DS, ageassociated deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/poly-ubiquitinated proteins, which are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology.
doi: 10.1016/j.freeradbiomed.2016.10.441 401 A Radical Pathway for Minocycline Toxicity: Role of Cytochrome c and Sulfite Murugesan Velayutham1, Shanmugam Nagarajan1, and Arturo J Cardounel1 1 Vascular Medicine Institute, University of Pittsburgh, USA Minocycline (MC) is a second-generation semi-synthetic analogue of tetracycline. MC has been used as an antibiotic and for the treatment of various diseases such as acne vulgaris, sexually transmitted diseases, and rheumatoid arthritis. MC is currently being explored in various clinical trials for the treatment of cardiovascular diseases such as stroke, vascular aneurisms, and coronary by-pass surgery. However, the clinical use of MC has
SfRBM / SFRRI 2016
S167
been associated with potential side effects. It has been demonstrated that MC interacts with cytochrome c (Cyt-c) and inhibits its peroxidase activity. However, molecular mechanism involved in the interaction between Cyt-c and MC is not studied. In this study, UV-visible spectroscopic measurements were carried out to understand the reaction between ferric cytochrome c (Fe3+Cyt-c) and MC. MC reduces Fe3+Cyt-c to ferrous cytochrome c (Fe2+Cyt-c). The reduction of Fe3+Cyt-c increases with increasing concentration of MC. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with Fe3+Cyt-c, MC, and sulfite. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3– ) was obtained. The formation of DMPO-SO3– was very low in the absence of MC. Interestingly, the addition of MC enhanced the DMPO-SO3– adducts. Furthermore, the formation of DMPO-SO3– adducts increased with increasing concentration of MC. From these results, we propose a mechanism that the Fe3+Cyt-c oxidizes MC to MC radical, which subsequently oxidizes sulfite to sulfite radical. Our results suggest that Fe3+Cyt-c could have a novel role in the deleterious effects of MC in biological systems by increasing the production of sulfite radical.
doi: 10.1016/j.freeradbiomed.2016.10.442 402 Thiol Oxidation Mediated Redox Signaling Triggers Specific Dopaminergic Neurodegeneration in Parkinson′s Disease Aditi Verma1, Ajit Ray1, Smitha Karunakaran1, Lalitha Durgadoss1, and Vijayalakshmi Ravindranath1 1 Indian Institute of Science, Bangalore, India Oxidative stress has been implicated in pathogenesis of Parkinson’s disease (PD), but it is yet unclear as to how the generalized ROS generation leads to degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Activation of redox driven signaling pathways may contribute to the specific cell death seen in PD. Using the MPTP mouse model of PD, we have established that stress-regulated MAPK signaling, specifically p38 MAPK-dependent death signaling, is selectively activated in dopaminergic neurons, while JNK is activated in microglia. Further, we found ASK1 as an upstream MAP3K responsible for p38 activation. ASK1, in turn, was selectively activated by TNF upregulation acting through thiol oxidation of Trx1, a negative regulator of ASK1 activity. Further, we also observed inhibition of Akt-mediated cell survival pathway through cysteine oxidation in Akt. As protein thiol oxidation emerged as a major contributor towards selective cell death signaling, we developed a novel diamide-induced mouse model of PD to examine whether thiol oxidation alone, caused by diamide, could trigger PD-relevant phenotype. Stereotaxic injection of diamide into mouse brain led to extensive dopaminergic neurodegeneration, coupled with biochemical and behavioral deficits as observed in PD, and activation of ASK1-mediated cell death signaling. αSynuclein aggregation, not often seen in other models of PD, was also reproduced in this model. Current studies are underway to investigate whether Grx1 inhibition in vivo (leading to increased glutathionylation and protein thiol oxidation), results in PD pathology. Overall, these studies demonstrate the critical role of thiol oxidation in triggering neurodegeneration in PD.
doi: 10.1016/j.freeradbiomed.2016.10.443
403 Clearing Amyloid-β Through PPARγ/ApoE Activation by Genistein is an Experimental Treatment of Alzheimer's Disease Jose Vina1, Bonet-Costa Vicent1, MariCarmen Blanco-Gandía1, Vicente Herranz-Pérez1, Cristina Mas-Bargues1, Marta Ingles1, Patricia GarciaTarraga1, Marta Rodriguez-Arias1, Jose Minarro1, Consuelo Borras1, and Jose- Manuel Garcia- Verdugo1 1 University of Valencia, Spain Amyloid-β (Aβ) clearance from brain, which is decreased in Alzheimer’s disease, is facilitated by apolipoprotein E. Apo E is upregulated by activation of the retinoid X receptor moiety of the RXR/PPARγ dimeric receptor. Genistein, a non-toxic, well tested and inexpensive drug has a multifaceted protective effect: antioxidant (because it stimulates the expression of antioxidant genes), anit-inflammatory and stimulator of activates the PPARγ receptor, which results in increased expression of ApoE. Treatment of an Alzheimer’s mouse model with genistein results in a remarkable and rapid improvement in various parameters of cognition, such as hippocampal learning, recognition memory, implicit memory and odor discrimination. This is associated with a lowering of Aβ levels in brain, in the number and the area of amyloid plaques (confirmed in vivo by positron emission tomography) as well as in microglial reactivity. Finally, incubation of primary astrocytes with genistein results in a PPARγ-mediated increased release of ApoE. Our results strongly suggest that controlled clinical trials should be performed to test the effect of genistein as treatment of human Alzheimer’s disease.
doi: 10.1016/j.freeradbiomed.2016.10.444 404 Nitrosylation of Vesicular Transporters in Brain of Amyloid Precursor Protein/presenilin 1 Double Transgenic Mice Ying Wang1 and Jun-Feng Wang1 University of Manitoba, Winnipeg, Canada
1
Nitric oxide can attack thiols of protein cysteine residues and induce cysteine S-nitrosylation. Cholinergic and glutamatergic systems are dysregulated in Alzheimer’s disease (AD). Vesicular acetylcholine transporter (VAChT) and vesicular glutamate transporter 1 (VGLUT1) are important in packaging acetylcholine and glutamate into vesicles that is an important step for neurotransmission. Previously we found that VAChT and VGLUT1 can be nitrosylated and this nitrosylation inhibits vesicular uptake of acetylcholine and glutamate. To understand the role of VAChT and VGLUT1 nitrosylation in the pathophysiological development of AD, we analyzed nitrosylation to VAChT and VGLUT1 in brain of amyloid precursor protein (APP) and presenilin 1 (PS1) transgenic mice, an animal model for AD. We found that 9 and 12 month old APP/PS1 mice showed memory deficit. We further found that total protein nitrosylation was increased in frontal cortex and hippocampus of 9 and 12 month old APP/PS1 mice when compared to wild type mice. Although nitrosylation of VAChT and VGLUT1 was not changed in hippocampus, nitrosylation of VAChT and VGLUT1 was increased in frontal cortex of 9 and 12 month old APP/PS1 mice. These findings suggest that nitrosylation of VAChT and VGLUT1 may be associated with dysfunctional acetylcholinergic and glutamatergic neurotransmission in AD.
doi: 10.1016/j.freeradbiomed.2016.10.445 S168
SfRBM / SFRRI 2016