Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity

Minocycline is neuroprotective in animal models of a number of acute CNS injuries and neurodegenerative diseases. While anti-inflammatory and anti-apoptotic effects of minocycline have been characterized, the molecular basis for the neuroprotective effects of minocycline remains unclear. We report here that minocycline and a number of antioxidant compounds protect mixed neuronal cultures in an oxidative stress assay. To evaluate the role of minocycline's direct antioxidant properties in neuroprotection, we determined potencies for minocycline, other tetracycline antibiotics, and reference antioxidant compounds using a panel of in vitro radical scavenging assays. Data from in vitro rat brain homogenate lipid peroxidation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays show that minocycline, in contrast to tetracycline, is an effective antioxidant with radical scavenging potency similar to vitamin E. Our findings suggest that the direct antioxidant activity of minocycline may contribute to its neuroprotective effects in some cell-based assays and animal models of neuronal injury.     

Oxidative and nitrative alpha‐synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies

Alpha‐synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single‐point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinson's disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over‐expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN‐related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post‐mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYN's degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging.     

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Glucose-modulated tyrosine nitration in beta cells: Targets and consequences

Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified function in protein folding, energy metabolism, antioxidant capacity, and membrane permeability. Nitration of heat shock protein 60 in vitro was found to decrease its ATP hydrolysis and interaction with proinsulin, suggesting a mechanism by which protein nitration could diminish insulin secretion. This was supported by our finding of a decrease in stimulated insulin secretion following glycolytic stress in cultured cells. Our results reveal that protein tyrosine nitration may be a previously unrecognized factor in beta-cell dysfunction and the pathogenesis of diabetes.     

Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate.     

Protein nitration increased by simulated weightlessness and decreased by melatonin and quercetin in PC12 cells.

A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.     

Serum Paraoxonase and Arylesterase Activities and Oxidative Stress Levels in Patients with SSRI Intoxication

Oxidative stress is a critical route of damage in various psychological stress-induced disorders, such as depression. Paraoxonase-1 (PON1) plays an important role as an endogenous free-radical scavenging molecule. The aim of this study was to evaluate the influence of serum PON1 activity and oxidative stress in patients with selective serotonin reuptake inhibitor (SSRI) intoxication. A total of 11 patients with SSRI intoxication and 20 healthy controls were enrolled. The serum total antioxidant capacity (TAC) and malondialdehyde (MDA) levels, as well as the paraoxonase and arylesterase activities, were measured spectrophotometrically. The serum TAC levels and the paraoxonase and arylesterase activities were significantly lower (for all, p < 0.001), whereas the serum MDA levels were significantly higher in the patients with SSRI intoxication than in the controls (p < 0.001). These results indicated that decreased PON1 activity and increased oxidative stress represent alternative mechanisms in SSRI toxicity. More studies are needed to elucidate the role of PON1 activity in the etiology of SSRI intoxication.     

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ₁₀), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H₂O₂)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ₁₀ can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ₁₀ not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ₁₀ prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ₁₀ preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ₁₀ may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.     

Oxidative inactivation of paraoxonase1, an antioxidant protein and its effect on antioxidant action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu₂₊ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe₂₊ (2.0 μM), the inclusion of Cu₂₊ (0.1-1.0 μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5 mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu₂₊ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu₂₊-catalyzed oxidative inactivation. In addition, Cu₂₊ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu₂₊-induced oxidation of LDL. Noteworthy, ascorbate/Cu₂₊-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu₂₊-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia

Glutamate excitotoxicity to a large extent is mediated through activation of the N-methyl-D-aspartate (NMDA)-gated ion channels in several neurodegenerative diseases and ischemic stroke. Minocycline, a tetracycline derivative with antiinflammatory effects, inhibits IL-1beta-converting enzyme and inducible nitric oxide synthase up-regulation in animal models of ischemic stroke and Huntington's disease and is therapeutic in these disease animal models. Here we report that nanomolar concentrations of minocycline protect neurons in mixed spinal cord cultures against NMDA excitotoxicity. NMDA treatment alone induced microglial proliferation, which preceded neuronal death, and administration of extra microglial cells on top of these cultures enhanced the NMDA neurotoxicity. Minocycline inhibited all these responses to NMDA. Minocycline also prevented the NMDA-induced proliferation of microglial cells and the increased release of IL-1beta and nitric oxide in pure microglia cultures. Finally, minocycline inhibited the NMDA-induced activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells, and a specific p38 MAPK inhibitor, but not a p44/42 MAPK inhibitor, reduced the NMDA toxicity. Together, these results suggest that microglial activation contributes to NMDA excitotoxicity and that minocycline, a tetracycline derivative, represents a potential therapeutic agent for brain diseases.     

Minocycline decreases in vitro microglial motility, beta1-integrin, and Kv1.3 channel expression

Minocycline is a semisynthetic, tetracycline derivative that exerts anti-inflammatory and neuroprotective effects unrelated to its anti-microbial action. We have previously shown that minocycline prevented peripheral nerve injury-induced mechanical allodynia. Minocycline's mechanisms of action as a neuroprotective and anti-allodynic agent are unknown. In response to injury, microglia become activated, proliferate, and migrate. Resting microglia express voltage-dependent inward K⁺ currents and blocking Kv1.3 channels has been shown to inhibit microglial-mediated neuronal death. We investigated the effect of minocycline on the expression of Kv channels, cell motility, and β-integrin expression using primary rat cortical microglia, transwell assays, and by flow cytometry. Minocycline significantly reduced microglial migration to cellular debris, astrocyte-conditioned medium, ADP, and algesic mediators and significantly reduced the expression of CD29 (β₁-integrin) but not CD18 (β₂-integrin). Minocycline reduced the effect of extracellular potassium and later decreased microglial Kv1.3 expression. In summary, we uncovered a novel effect of minocycline that demonstrates this agent decreases microglial β₁-integrin expression, which leads to inhibition of motility. We propose an in vivo model whereby reduced microglial trafficking to injured neurons following nerve injury decreases the release of proinflammatory mediators into the synaptic milieu, preventing neuronal sensitization, the pathological correlate to chronic pain.     

Antioxidant Activity of Sulfur and Selenium: A Review of Reactive Oxygen Species Scavenging,Glutathione Peroxidase,and Metal-Binding Antioxidant Mechanisms

It is well known that oxidation caused by reactive oxygen species (ROS) is a major cause of cellular damage and death and has been implicated in cancer, neurodegenerative, and cardiovascular diseases. Small-molecule antioxidants containing sulfur and selenium can ameliorate oxidative damage, and cells employ multiple antioxidant mechanisms to prevent this cellular damage. However, current research has focused mainly on clinical, epidemiological, and in vivo studies with little emphasis on the antioxidant mechanisms responsible for observed sulfur and selenium antioxidant activities. In addition, the antioxidant properties of sulfur compounds are commonly compared to selenium antioxidant properties; however, sulfur and selenium antioxidant activities can be quite distinct, with each utilizing different antioxidant mechanisms to prevent oxidative cellular damage. In the present review, we discuss the antioxidant activities of sulfur and selenium compounds, focusing on several antioxidant mechanisms, including ROS scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms. Findings of several recent clinical, epidemiological, and in vivo studies highlight the need for future studies that specifically focus on the chemical mechanisms of sulfur and selenium antioxidant behavior.     

Antioxidants from defatted Indian Mustard (Brassica Juncea) protect biomolecules against in vitro oxidation

Indian mustard seeds were defatted by distillation with hexane and the residue extracted with methanol was analyzed for potential antioxidants; ascorbate, riboflavin, and polyphenols. Gallic acid (129.796 μg), caffeic acid (753.455 μg), quercetin (478.352 μg) and kaempferol (48.060 μg)/g dry seeds were identified by HPLC analysis of the extract. DPPH free radical scavenging activity and protection of lipids, proteins and DNA against metal induced oxidation was examined. Defatted mustard seed remnant had excellent free radical scavenging activity and protects biomolecules with IC₅₀ value 2.0–2.25 mg dry seed weight. Significant content of polyphenols in methanol extract of defatted seeds accounts for high antioxidant potential. We are the first to report the detailed analysis of antioxidant composition and protection of biomolecules against oxidative damage by methanol extract of mustard seed remnant after oil extraction.     

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1)

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu²⁺ system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe²⁺, Cu²⁺ (0.05–1.0 µM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02–0.1 mM). Moreover, Cu²⁺ alone inhibited the lactonase activity at concentrations as low as 1 µM. The ascorbate/Cu²⁺-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu²⁺-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu²⁺-catalyzed oxidation. Separately, ascorbate/Cu²⁺-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu²⁺-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu²⁺ among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.     

Glucose-mediated tyrosine nitration in adipocytes: Targets and consequences

Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes.     

DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson''s disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP⁺ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.     

Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.     

Hydrogen peroxide attenuates insulin-like growth factor-1 neuroprotective effect, prevented by minocycline

Oxidative stress-induced neuronal death due to hydrogen peroxide overload plays a critical role in the pathogenesis of numerous neurological diseases. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation in the central nervous system. We now report that sublethal doses of hydrogen peroxide attenuated IGF-1 neuroprotective activity on cultured cerebellar granule neurons under potassium and serum deprivation. Interestingly, this attenuation can be prevented by minocycline, an antibiotic that has been shown to have neuroprotective activity in animal models of neuronal injury. Furthermore, hydrogen peroxide also blocked IGF-1's neuroprotection for cortical neurons deprived of neurotrophic factors (B27), which was prevented by minocycline. Our data suggest that inhibition of IGF-1 signaling by hydrogen peroxide may constitute an additional pathway contributing to its neurotoxicity. More importantly, combining minocycline and IGF-1 could be an effective treatment in neurological diseases associated with both oxidative stress and deficiency of IGF-1.