The triangle of death of neurons: Oxidative damage, mitochondrial dysfunction, and loss of choline-containing biomolecules in brains of mice treated with doxorubicin. Advanced insights into mechanisms of chemotherapy induced cognitive impairment ("chemobrain") involving TNF-α.

Cancer treatments are developing fast and the number of cancer survivors could arise to 20 million in United State by 2025. However, a large fraction of cancer survivors demonstrate cognitive dysfunction and associated decreased quality of life both shortly, and often long-term, after chemotherapy treatment. The etiologies of chemotherapy induced cognitive impairment (CICI) are complicated, made more so by the fact that many anti-cancer drugs cannot cross the blood-brain barrier (BBB). Multiple related factors and confounders lead to difficulties in determining the underlying mechanisms. Chemotherapy induced, oxidative stress-mediated tumor necrosis factor-alpha (TNF-α) elevation was considered as one of the main candidate mechanisms underlying CICI. Doxorubicin (Dox) is a prototypical reactive oxygen species (ROS)-generating chemotherapeutic agent used to treat solid tumors and lymphomas as part of multi-drug chemotherapeutic regimens. We previously reported that peripheral Dox-administration leads to plasma protein damage and elevation of TNF-α in plasma and brain of mice. In the present study, we used TNF-α null (TNFKO) mice to investigate the role of TNF-α in Dox-induced, oxidative stress-mediated alterations in brain. We report that Dox-induced oxidative stress in brain is ameliorated and brain mitochondrial function assessed by the Seahorse-determined oxygen consumption rate (OCR) is preserved in brains of TNFKO mice. Further, we show that Dox-decreased the level of hippocampal choline-containing compounds and brain phospholipases activity are partially protected in TNFKO group in MRS study. Our results provide strong evidence that Dox-targeted mitochondrial damage and levels of brain choline-containing metabolites, as well as phospholipases changes decreased in the CNS are associated with oxidative stress mediated by TNF-α. These results are consistent with the notion that oxidative stress and elevated TNF-α in brain underlie the damage to mitochondria and other pathological changes that lead to CICI. The results are discussed with reference to our identifying a potential therapeutic target to protect against cognitive problems after chemotherapy.

Doxorubicin-induced elevated oxidative stress and neurochemical alterations in brain and cognitive decline: protection by MESNA and insights into mechanisms of chemotherapy-induced cognitive impairment ("chemobrain").

Chemotherapy-induced cognitive impairment (CICI) is now widely recognized as a real and too common complication of cancer chemotherapy experienced by an ever-growing number of cancer survivors. Previously, we reported that doxorubicin (Dox), a prototypical reactive oxygen species (ROS)-producing anti-cancer drug, results in oxidation of plasma proteins, including apolipoprotein A-I (ApoA-I) leading to tumor necrosis factor-alpha (TNF-α)-mediated oxidative stress in plasma and brain. We also reported that co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma. In this study, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. This study also provides further functional and biochemical evidence of the mechanisms of CICI. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits, an effect that was rescued by MESNA. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline-containing compounds assessed by (Cho)/creatine ratios in the hippocampus in mice. To better elucidate a potential mechanism for this MRS observation, we tested the activities of the phospholipase enzymes known to act on phosphatidylcholine (PtdCho), a key component of phospholipid membranes and a source of choline for the neurotransmitter, acetylcholine (ACh). The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox; however, PLD activity was not protected. This study is the first to demonstrate the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine (PCho) levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI.

Superoxide induces protein oxidation in plasma and TNF-α elevation in macrophage culture: Insights into mechanisms of neurotoxicity following doxorubicin chemotherapy.

Chemotherapy-induced cognitive impairment (CICI) is a quality of life-altering consequence of chemotherapy experienced by a large percentage of cancer survivors. Approximately half of FDA-approved anti-cancer drugs are known to produce ROS. Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, generates superoxide (O2(-)•) via redox cycling. Our group previously demonstrated that Dox, which does not cross the BBB, induced oxidative damage to plasma proteins leading to TNF-α elevation in the periphery and, subsequently, in brain following cancer chemotherapy. We hypothesize that such processes play a central role in CICI. The current study tested the notion that O2(-)• is involved and likely responsible for Dox-induced plasma protein oxidation and TNF-α release. Addition of O2(-)• as the potassium salt (KO2) to plasma resulted in significantly increased oxidative damage to proteins, indexed by protein carbonyl (PC) and protein-bound HNE levels. We then adapted this protocol for use in cell culture. Incubation of J774A.1 macrophage culture using this KO2-18crown6 protocol with 1 and 10 µM KO2 resulted in dramatically increased levels of TNF-α produced. These findings, together with our prior results, provide strong evidence that O2(-)• and its resulting reactive species are critically involved in Dox-induced plasma protein oxidation and TNF-α release.

Plasma TNF-α and Soluble TNF Receptor Levels after Doxorubicin with or without Co-Administration of Mesna-A Randomized, Cross-Over Clinical Study.

PURPOSE: Chemotherapy-induced cognitive impairment (CICI) is a common sequelae of cancer therapy. Recent preclinical observations have suggested that CICI can be mediated by chemotherapy-induced plasma protein oxidation, which triggers TNF-α mediated CNS damage. This study evaluated sodium-2-mercaptoethane sulfonate (Mesna) co-administration with doxorubicin to reduce doxorubicin-induced plasma protein oxidation and resultant cascade of TNF-α, soluble TNF receptor levels and related cytokines. METHODS: Thirty-two evaluable patients were randomized using a crossover design to receive mesna or saline in either the first or second cycle of doxorubicin in the context of a standard chemotherapy regimen for either non-Hodgkin lymphoma or breast cancer. Mesna (360 mg/m2) or saline administration occurred 15 minutes prior and three hours post doxorubicin. Pre-treatment and post-treatment measurements of oxidative stress, TNF-α and related cytokines were evaluated during the two experimental cycles of chemotherapy. RESULTS: Co-administration of mesna with chemotherapy reduced post-treatment levels of TNF-related cytokines and TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2) (p = 0.05 and p = 0.002, respectively). Patients with the highest pre-treatment levels of each cytokine and its receptors were the most likely to benefit from mesna co-administration. CONCLUSIONS: The extracellular anti-oxidant mesna, when co-administered during a single cycle of doxorubicin, reduced levels of TNF-α and its receptors after that cycle of therapy, demonstrating for the first time a clinical interaction between mesna and doxorubicin, drugs often coincidentally co-administered in multi-agent regimens. These findings support further investigation to determine whether rationally-timed mesna co-administration with redox active chemotherapy may prevent or reduce the cascade of events that lead to CICI. TRIAL REGISTRATION: clinicaltrials.gov NCT01205503.

Dietary vitamin D deficiency in rats from middle to old age leads to elevated tyrosine nitration and proteomics changes in levels of key proteins in brain: implications for low vitamin D-dependent age-related cognitive decline.

In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle age to old age. To address this issue, 27 male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10,000 IU/kg food) VitD beginning at middle age (12 months) and continued for a period of 4-5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low-VitD group showed global elevation of 3-nitrotyrosine compared to control and high-VitD-treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) pathway and NF-κB-mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomics techniques were used to provide insight into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in the low-VitD group compared to the control and high-VitD groups. Three of these proteins, 6-phosphofructokinase, triose phosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (cyclophilin A) has been shown to have multiple roles, including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these results suggest that dietary VitD deficiency contributes to significant nitrosative stress in brain and may promote cognitive decline in middle-aged and elderly adults.

Apolipoprotein A-I: insights from redox proteomics for its role in neurodegeneration.

Proteomics has a wide range of applications, including determination of differences in the proteome in terms of expression and post-translational protein modifications. Redox proteomics allows the identification of specific targets of protein oxidation in a biological sample. Using proteomic techniques, apolipoprotein A-I (ApoA-I) has been found at decreased levels in subjects with a variety of neurodegenerative disorders including in the serum and cerebrospinal fluid (CSF) of Alzheimer disease (AD), Parkinson disease (PD), and Down syndrome (DS) with gout subjects. ApoA-I plays roles in cholesterol transport and regulation of inflammation. Redox proteomics further showed ApoA-I to be highly oxidatively modified and particularly susceptible to modification by 4-hydroxy-2-trans-nonenal (HNE), a lipid peroxidation product. In the current review, we discuss the consequences of oxidation of ApoA-I in terms of neurodegeneration. ROS-associated chemotherapy related ApoA-I oxidation leads to elevation of peripheral levels of tumor necrosis factor-α (TNF-α) that can cross the blood-brain barrier (BBB) causing a signaling cascade that can contribute to neuronal death, likely a contributor to what patients refer to as "chemobrain." Current evidence suggests ApoA-I to be a promising diagnostic marker as well as a potential target for therapeutic strategies in these neurodegenerative disorders.

Cell cycle proteins in brain in mild cognitive impairment: insights into progression to Alzheimer disease.

Recent studies have demonstrated the re-emergence of cell cycle proteins in brain as patients progress from the early stages of mild cognitive impairment (MCI) into Alzheimer's disease (AD). Oxidative stress markers present in AD have also been shown to be present in MCI brain suggesting that these events occur in early stages of the disease. The levels of key cell cycle proteins, such as CDK2, CDK5, cyclin G1, and BRAC1 have all been found to be elevated in MCI brain compared to age-matched control. Further, peptidyl prolyl cis-trans isomerase (Pin1), a protein that plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-ß, and PP2A that are involved in both the phosphorylation state of Tau and in the cell cycle, has been found to be oxidatively modified and downregulated in both AD and MCI brain. Hyperphosphorylation of Tau then results in synapse loss and the characteristic Tau aggregation as neurofibrillary tangles, an AD hallmark. In this review, we summarized the role of cell cycle dysregulation in the progression of disease from MCI to AD. Based on the current literature, it is tempting to speculate that a combination of oxidative stress and cell cycle dysfunction conceivably leads to neurodegeneration.