Syed Z. Imam

Methamphetamine-Induced Dopaminergic Neurotoxicity: Role of Peroxynitrite and Neuroprotective Role of Antioxidants and Peroxynitrite Decomposition Catalysts

Abstract: Oxidative stress, reactive oxygen (ROS), and nitrogen (RNS) species have been known to be involved in a multitude of neurodegenerative disorders such as Parkinsons disease (PD), Alzheimers disease (AD), and amyotrophic lateral sclerosis (ALS). Both ROS and RNS have very short half‐lives, thereby making their identification very difficult as a specific cause of neurodegeneration. Recently, we have developed a high performance liquid chromatography/electrochemical detection (HPLC/EC) method to identify 3‐nitrotyrosine (3‐NT), an in vitro and in vivo biomarker of peroxynitrite production, in cell cultures and brain to evaluate if an agent‐driven neurotoxicity is produced by the generation of peroxynitrite. We show that a single or multiple injections of methamphetamine (METH) produced a significant increase in the formation of 3‐NT in the striatum. This formation of 3‐NT correlated with the striatal dopamine depletion caused by METH administration. We also show that PC12 cells treated with METH has significantly increased formation of 3‐NT and dopamine depletion. Furthermore, we report that pretreatment with antioxidants such as selenium and melatonin can completely protect against the formation of 3‐NT and depletion of striatal dopamine. We also report that pretreatment with peroxynitrite decomposition catalysts such as 5, 10,15,20‐tetrakis(N‐methyl‐4′‐pyridyl)porphyrinato iron III (FeTMPyP) and 5, 10, 15, 20‐tetrakis (2,4,6‐trimethyl‐3,5‐sulfonatophenyl) porphinato iron III (FETPPS) significantly protect against METH‐induced 3‐NT formation and striatal dopamine depletion. We used two different approaches, pharmacological manipulation and transgenic animal models, in order to further investigate the role of peroxynitrite. We show that a selective neuronal nitric oxide synthase (nNOS) inhibitor, 7‐nitroindazole (7‐NI), significantly protect against the formation of 3‐NT as well as striatal dopamine depletion. Similar results were observed with nNOS knockout and copper zinc superoxide dismutase (CuZnSOD)‐overexpressed transgenic mice models. Finally, using the protein data bank crystal structure of tyrosine hydroxylase, we postulate the possible nitration of specific tyrosine moiety in the enzyme that can be responsible for dopaminergic neurotoxicity. Together, these data clearly support the hypothesis that the reactive nitrogen species, peroxynitrite, plays a major role in METH‐induced dopaminergic neurotoxicity and that selective antioxidants and peroxynitrite decomposition catalysts can protect against METH‐induced neurotoxicity. These antioxidants and decomposition catalysts may have therapeutic potential in the treatment of psychostimulant addictions.

Interleukin-18-induced human coronary artery smooth muscle cell migration is dependent on NF-κB- and AP-1-mediated matrix metalloproteinase-9 expression and is inhibited by atorvastatin

The proliferation and migration of arterial smooth muscle cells (SMCs) are key events in the vascular restenosis that frequently follows angioplasty. Furthermore, SMC migration and neointimal hyperplasia are promoted by degradation of the extracellular matrix by matrix metalloproteinases (MMPs). Because we demonstrated previously that the proinflammatory and proatherogenic cytokine interleukin-18 (IL-18) stimulates SMC proliferation (Chandrasekar, B., Mummidi, S., Valente, A. J., Patel, D. N., Bailey, S. R., Freeman, G. L., Hatano, M., Tokuhisa, T., and Jensen, L. E. (2005) J. Biol. Chem. 280, 26263–26277), we investigated whether IL-18 induces SMC migration in an MMP-dependent manner and whether the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin can inhibit this response. IL-18 treatment increased both mRNA and protein expression of MMP9 in human coronary artery SMCs. Gel shift, enzyme-linked immunosorbent, and chromatin immunoprecipitation assays revealed a strong induction of IL-18-mediated AP-1 (c-Fos, c-Jun, and Fra-1) and NF-κB (p50 and p65) activation and stimulation of MMP9 promoter-dependent reporter gene activity in an AP-1- and NF-κB-dependent manner. Ectopic expression of p65, c-Fos, c-Jun, and Fra-1 induced MMP9 promoter activity. Specific antisense or small interfering RNA reagents for these transcription factors reduced IL-18-mediated MMP9 transcription. Furthermore, IL-18 stimulated SMC migration in an MMP9-dependent manner. Atorvastatin effectively suppressed IL-18-mediated AP-1 and NF-κB activation, MMP9 expression, and SMC migration. Together, our results indicate for the first time that the proatherogenic cytokine IL-18 induces human coronary artery SMC migration in an MMP9-dependent manner. Atorvastatin inhibits IL-18-mediated aortic SMC migration and has therapeutic potential for attenuating the progression of atherosclerosis and restenosis.

Mitochondrial and nuclear DNA-repair capacity of various brain regions in mouse is altered in an age-dependent manner.

Aging is associated with increased susceptibility to neuronal loss and disruption of cerebral function either as a component of senescence, or as a consequence of neurodegenerative disease or stroke. Here we report differential changes in the repair of oxidative DNA damage in various brain regions during aging. We evaluated mitochondrial and nuclear incision activities of oxoguanine DNA glycosylase (OGG1), uracil DNA glycosylase (UDG) and the endonuclease III homologue (NTH1) in the caudate nucleus (CN), frontal cortex (FC), hippocampus (Hip), cerebellum (CE) and brain stem (BS) of 6- and 18-month-old male C57Bl/6 mice. We observed a significant age-dependent decrease in incision activities of all three glycosylases in the mitochondria of all brain regions, whereas variable patterns of changes were seen in nuclei. No age- or region-specific changes were observed in the mitochondrial repair synthesis incorporation of uracil-initiated base-excision repair (BER). We did not observe any age or region dependent differences in levels of BER proteins among the five brain regions. In summary, our data suggest that a decreased efficiency of mitochondrial BER-glycosylases and increased oxidative damage to mitochondrial DNA might contribute to the normal aging process. These data provide a novel characterization of oxidative DNA damage processing in different brain regions implicated in various neurodegenerative disorders, and suggest that this process is regulated in an age-dependent manner. Manipulation of DNA repair mechanisms may provide a strategy to prevent neuronal loss during age-dependent neurodegenerative disorders.

Selenium, an antioxidant, attenuates methamphetamine-induced dopaminergic toxicity and peroxynitrite generation.

Methamphetamine (METH) has been known to produce neurotoxicity via generation of reactive oxygen and nitrogen species. Selenium, an antioxidant, was reported to protect against METH-induced dopaminergic neurotoxicity in mouse caudate nucleus. In the present study, the in vitro and in vivo efficacy of the supplementation of selenium was studied in METH-induced generation of peroxynitrite. PC12 cell cultures were exposed to 200 microM METH either with or without 10 microM and 20 microM selenium (30 min prior to METH exposure). After 24 h, METH exposure resulted in the significant depletion of dopamine, and its metabolites DOPAC and HVA, as well as the significant formation of 3-nitrotyrosine (3-NT), a marker of peroxynitrite generation, in PC12 cell cultures. Selenium supplementation attenuated the depletion of dopamine and its metabolites, DOPAC and HVA and the formation of 3-NT in PC12 cells. For in vivo studies, adult male mice were supplemented with selenium in drinking water, 1 week before and 1 week after the multiple injections of METH (4x10 mg/kg, i.p. at 2-h interval) or an equivalent volume of saline. The supplementation of Se attenuated the formation of 3-NT in the striatum resulting from METH treatment. These data suggest that METH-induced neurotoxicity is mediated by the production of peroxynitrite, and selenium plays a protective role in METH-induced neurotoxicity.

Role of nitric oxide in rotenone-induced nigro-striatal injury

Rotenone, a widely used pesticide, causes a syndrome in rats that mimics, both behaviorally and pathologically, the symptoms of Parkinsons disease. The present study evaluated the role of nitric oxide in rotenone‐induced nigro‐striatal injury. After administration of rotenone in rats for 40 days, there was a moderate but significant injury of the nigro‐striatal pathway indicated by a 47% decrease in striatal dopamine levels and a 28% loss of substantia nigra tyrosine hydroxylase‐immunopositive neurons. Furthermore, a significant (37%) increase in the number of cells positive for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‐d) in the striatum was observed, accompanied by a 83% increase in nitric oxide synthase (NOS) activity and a significant increase in the production of 3‐nitrotyrosine (3‐NT). There was a significant increase (45%) in the optical density of NADPH‐d staining and an increase (72%) in NOS activity in the substantia nigra. Moreover, administration of the neuronal NOS inhibitor 7‐nitroindazole significantly attenuated the increased NOS activity and 3‐NT production, and provided significant protection against rotenone‐induced nigro‐striatal injury. Our data suggest that chronic rotenone administration can lead to significant injury to the nigro‐striatal system, mediated by increased generation of nitric oxide.

Peroxynitrite plays a role in methamphetamine-induced dopaminergic neurotoxicity: evidence from mice lacking neuronal nitric oxide synthase gene or overexpressing copper-zinc superoxide dismutase

The use of methamphetamine (METH) leads to neurotoxic effects in mammals. These neurotoxic effects appear to be related to the production of free radicals. To assess the role of peroxynitrite in METH‐induced dopaminergic, we investigated the production of 3‐nitrotyrosine (3‐NT) in the mouse striatum. The levels of 3‐NT increased in the striatum of wild‐type mice treated with multiple doses of METH (4 × 10 mg/kg, 2 h interval) as compared with the controls. However, no significant production of 3‐NT was observed either in the striata of neuronal nitric oxide synthase knockout mice (nNOS –/–) or copper–zinc superoxide dismutase overexpressed transgenic mice (SOD‐Tg) treated with similar doses of METH. The dopaminergic damage induced by METH treatment was also attenuated in nNOS–/– or SOD‐Tg mice. These data further confirm that METH causes its neurotoxic effects via the production of peroxynitrite.

Novel Regulation of Parkin Function Through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinsons disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.

Selenium, an antioxidant, protects against methamphetamine-induced dopaminergic neurotoxicity

Dopaminergic changes were studied in the caudate nucleus of adult female mice after pre- and post-treatment with an antioxidant, selenium, 72 h after the multiple injections of methamphetamine (METH, 4x10 mg/kg, i.p. at 2-h interval) or an equivalent volume of saline. Selenium treatment prevented the depletion of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in caudate nucleus resulting from the METH treatment. These data suggest that METH-induced neurotoxicity is mediated by free radical and selenium plays a protective role against METH-induced dopaminergic neurotoxicity.

Aging increases the susceptiblity to methamphetamine-induced dopaminergic neurotoxicity in rats : correlation with peroxynitrite production and hyperthermia

Methamphetamine (METH) produces dopaminergic neurotoxicity by the production of reactive oxygen (ROS) and nitrogen (RNS) species. The role of free radicals has also been implicated in the process of aging. The present study was designed to evaluate whether METH‐induced dopaminergic neurotoxicity and hyperthermia is a result of peroxynitrite production and if these effects correlate with age. One‐, six‐ and 12‐month‐old male rats (n = 8) were administered a single dose of METH (0, 5, 10, 20, and 40 mg/kg, intraperitoneally). The formation of 3‐nitrotyrosine (3‐NT) as a marker of peroxynitrite production as well as dopamine and its metabolites DOPAC and HVA were measured in the striatum 4‐h after METH‐administration. Rectal temperature was monitored every 30 min after METH administration until 4 h. At 40 mg/kg METH, a 100% mortality in 12‐month‐old animals was observed, whereas no deaths occurred in 1‐ or 6‐month‐old rats. An age‐dependent increase in hyperthermia was observed after METH‐administration. A similar pattern of dose‐dependent increase in the formation of 3‐NT and in the depletion of dopamine and its metabolites with age was observed in the striatum. Furthermore, no effect was observed at 5 mg/kg METH in 1‐month‐old animals, whereas the effect was significant in 6‐ and 12‐month‐old animals. These data suggest that aging increases the susceptibility of the animals toward METH‐induced peroxynitrite generation and striatal dopaminergic neurotoxicity.

Macrophage-mediated GDNF Delivery Protects Against Dopaminergic Neurodegeneration: A Therapeutic Strategy for Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinsons disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH+ terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinsons disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH(+) terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.