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SIGNIFICANCE: Alzheimer disease (AD) is an all-too-common condition in the aging population. However, aging does not automatically equal neurodegeneration and memory decline. RECENT ADVANCES: This review article involves metabolic changes in the AD brain that are related to oxidative stress. Selected pathways are identified as potential targets for intervention in AD. CRITICAL ISSUES: One of the main factors of AD is the oxidative imbalance within the central nervous system, causing a disruption in metabolic processes. Reactive oxygen species (ROS) are a natural consequence of many cellular processes, especially those associated with mitochondria, such as the electron transport chain. Some ROS, when kept under control and maintained at reasonable levels, often play roles in cell signaling. The cellular damage of ROS arises when oxidative imbalance occurs, in which case ROS are not controlled, leading to a myriad of alterations in cellular metabolic processes. These altered pathways include, among others, dysfunctional glycolysis, calcium regulation, lipid metabolism, mitochondrial processes, and mammalian target of rapamycin pathway dysregulation. FUTURE DIRECTIONS: Understanding how ROS can lead to these alterations can, ideally, elucidate therapeutic options for retarding AD progression in the aging population. Antioxid. Redox Signal. 36, 1289–1305.

Proteins identified in both SAL_EVs and DOX_EVs

Purpose: Cardiac injury is a major cause of death in cancer survivors, and biomarkers for it are detectable only after tissue injury has occurred. Extracellular vesicles (EV) remove toxic biomolecules from tissues and can be detected in the blood. Here, we evaluate the potential of using circulating EVs as early diagnostic markers for long-term cardiac injury.Experimental Design: Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we quantified serum EVs, analyzed proteomes, measured oxidized protein levels in serum EVs released after DOX treatment, and investigated the alteration of EV content.Results: Treatment with DOX caused a significant increase in circulating EVs (DOX_EV) compared with saline-treated controls. DOX_EVs exhibited a higher level of 4-hydroxynonenal adducted proteins, a lipid peroxidation product linked to DOX-induced cardiotoxicity. Proteomic profiling of DOX_EVs revealed the distinctive presence of brain/heart, muscle, and liver isoforms of glycogen phosphorylase (GP), and their origins were verified to be heart, skeletal muscle, and liver, respectively. The presence of brain/heart GP (PYGB) in DOX_EVs correlated with a reduction of PYGB in heart, but not brain tissues. Manganese superoxide dismutase (MnSOD) overexpression, as well as pretreatment with cardioprotective agents and MnSOD mimetics, resulted in a reduction of EV-associated PYGB in mice treated with DOX. Kinetic studies indicated that EVs containing PYGB were released prior to the rise of cardiac troponin in the blood after DOX treatment, suggesting that PYGB is an early indicator of cardiac injury.Conclusions: EVs containing PYGB are an early and sensitive biomarker of cardiac injury. Clin Cancer Res; 24(7); 1644–53. ©2017 AACR.See related commentary by Zhu and Gius, p. 1516

Western blots indicating PYGB, PYGM, and PYGL are tissue-specific protein. PYGB is highly expressed in heart and brain. PYGM is highly expressed in skeletal muscle. PYGL is highly expressed in liver. Vinculin and tubulin were used as appropriate positive controls.

Western blots display PYGB, PYGM, and PYGL expression. A. EVs from mice treated with saline or DOX. B. PYGB in EVs and heart tissues from wild-type or MnSOD Tg mice treated with saline or DOX. C. PYGB in EVs and heart tissues from mice treated with MnP or DRZ prior to saline or DOX treatment.

Sucrose gradient isolation process for proteomics analysis. A. Diagram showing the process of EV isolation by sucrose gradient method. B. Western blots of EV markers, CD63 and CD81 of sucrose gradient fraction 1 to 5 (from the top to the bottom). C. EV verification of isolation process by electron microscopy (X49,000).

Western blots confirming the existence of PYGB, PYGM, and PYGL in SAL_EVs and DOX_EVs after treatment with proteinase K. Cell lysate was used as a positive control for PYGB, PYGM and PYGL detection. IgG and albumin were measured as a reference for proteinase K efficiency to digest extravesicular proteins.

Western blots of EV markers (CD63 and Alix) and endoplasmic reticulum marker (Calnexin) in SAL_EVs and DOX_EVs. The cell lysate was used as a positive control.

Chemotherapy-induced cognitive impairment (CICI) has been observed in a large fraction of cancer survivors. Although many of the chemotherapeutic drugs do not cross the blood-brain barrier, following treatment, the structure and function of the brain are altered and cognitive dysfunction occurs in a significant number of cancer survivors. The means by which CICI occurs is becoming better understood, but there still remain unsolved questions of the mechanisms involved. The hypotheses to explain CICI are numerous. More than 50% of FDA-approved cancer chemotherapy agents are associated with reactive oxygen species (ROS) that lead to oxidative stress and activate a myriad of pathways as well as inhibit pathways necessary for proper brain function. Oxidative stress triggers the activation of different proteins, one in particular is tumor necrosis factor alpha (TNFα). Following treatment with various chemotherapy agents, this pro-inflammatory cytokine binds to its receptors at the blood-brain barrier and translocates to the parenchyma via receptor-mediated endocytosis. Once in brain, TNFα initiates pathways that may eventually lead to neuronal death and ultimately cognitive impairment. TNFα activation of the c-jun N-terminal kinases (JNK) and Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathways may contribute to both memory decline and loss of higher executive functions reported in patients after chemotherapy treatment. Chemotherapy also affects the brain's antioxidant capacity, allowing for accumulation of ROS. This review expands on these topics to provide insights into the possible mechanisms by which the intersection of oxidative stress and TNFΑ are involved in chemotherapy-induced cognitive impairment.

The cells possess several mechanisms to counteract the over-production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Moreover, an important sensor involved in the anti-oxidant response is KEAP1-NRF2-ARE signaling complex. Under oxidative stress (OS), the transcription factor NRF2 can dissociate from the KEAP1-complex in the cytosol and translocate into the nucleus to promote the transcriptional activation of anti-oxidant genes, such as heme oxygenase 1 and NADPH quinone oxidoreductase. Within this context, the activation of NRF2 response is further regulated by BACH1, a transcription repressor, that compete with the KEAP1-NRF2-ARE complex. In this work, we focused on the role of BACH1/NRF2 ratio in the regulation of the anti-oxidant response, proposing their antithetical relation as a valuable target for a therapeutic strategy to test drugs able to exert neuroprotective effects, notably in aging and neurodegenerative diseases. Among these, Down syndrome (DS) is a complex genetic disorder characterized by BACH1 gene triplication that likely results in the impairment of NRF2 causing increased OS. Our results revealed that BACH1 overexpression alters the BACH1/NRF2 ratio in the nucleus and disturbs the induction of antioxidant response genes ultimately resulting in the accumulation of oxidative damage both in Ts2Cje mice (a mouse model of DS) and human DS lymphoblastoid cell lines (LCLs). Based on this evidence, we tested Caffeic Acid Phenethyl Ester (CAPE) and the synthetic analogue VP961, which have been proven to modulate NRF2 activity. We showed that CAPE and VP961 administration to DS LCLs was able to promote NRF2 nuclear translocation, which resulted in the amelioration of antioxidant response. Overall, our study supports the hypothesis that BACH1 triplication in DS subjects is implicated in the alteration of redox homeostasis and therapeutic strategies to overcome this effect are under investigation in our laboratory.

Amnestic mild cognitive impairment (MCI), arguably the earliest clinical stage of Alzheimer disease (AD), is characterized by normal activities of daily living but with memory issues but no dementia. Oxidative stress, with consequent damaged key proteins and lipids, are prominent even in this early state of AD. This review article outlines oxidative stress in MCI and how this can account for neuronal loss and potential therapeutic strategies to slow progression to AD.

Parkinson disease (PD) is the second most common neurodegenerative disease associated with aging. Dopaminergic neuronal degeneration and α-synuclein aggregation are commonly found in PD brain. Oxidative damage and inflammation often are considered as etiological factors of PD, although the detailed mechanisms still remain unknown. Gender and aging are two important risk factors to PD, and gene mutations and certain environmental factors have been implicated in this disease. The current study employed PTEN-induced putative kinase -1 (PINK1) knockout (KO) rats, since mutations in PINK-1 lead to familial PD. We evaluated the oxidative damage in the brain of PINK1 KO rats, and we used MRI and MRS to measure the ventricle sizes and neurochemical metabolite profiles in these rats as a function of age and gender. Distinct gender- and age-related alterations were found. The results are discussed with respect to the suitabililty of this unique rat as a faithful model of known characteristics of PD.

Alzheimer disease (AD) is the major locus of dementia worldwide. In the USA there are nearly 6 million persons with this disorder, and estimates of 13–20 million AD cases in the next three decades. The molecular bases for AD remain unknown, though processes involving amyloid beta-peptide as small oligomeric forms are gaining attention as known agents to both lead to oxidative stress and synaptic dysfunction associated with cognitive dysfunction in AD and its earlier forms, including amnestic mild cognitive impairment (MCI) and possibly preclinical Alzheimer disease (PCAD). Altered brain protein phosphorylation is a hallmark of AD, and phosphoproteomics offers an opportunity to identify these altered phosphoproteins in order to gain more insights into molecular mechanisms of neuronal dysfunction and death that lead to cognitive loss. This paper reviews what, to this author, are believed to be the known phosphoproteomics studies related to in vitro and in vivo models of AD as well as phosphoproteomics studies of brains from subjects with AD, and in at least one case in MCI and PCAD as well. The results of this review are discussed with relevance to new insights into AD brain protein dysregulation in critical neuronal functions and to potential therapeutic targets to slow, or in favorable cases, halt progression of this dementing disorder that affects millions of persons and their families worldwide.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which these metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we found that S100 calcium-binding protein A4 (S100A4), a major metastasis-promoting protein, confers metabolic plasticity to drive tumor invasion and metastasis of non-small cell lung cancer cells. Investigating how S100A4 regulates metabolism, we found that S100A4 depletion decreases oxygen consumption rates, mitochondrial activity, and ATP production and also shifts cell metabolism to higher glycolytic activity. We further identified that the 49-kDa mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types as determined by database-driven analysis of genomes in clinical samples using cBioPortal for Cancer Genomics. Importantly, we noted that S100A4 or NDUFS2 silencing inhibits mitochondrial complex I activity, reduces cellular ATP level, decreases invasive capacity in three-dimensional growth, and dramatically decreases metastasis rates as well as tumor growth in vivo. Finally, we provide evidence that cells depleted in S100A4 or NDUFS2 shift their metabolism toward glycolysis by up-regulating hexokinase expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Our findings uncover a novel S100A4 function and highlight its importance in controlling NDUFS2 expression to regulate the plasticity of mitochondrial metabolism and thereby promote the invasive and metastatic capacity in lung cancer.

Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids’ production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.

Chemotherapy-induced cognitive impairment (CICI) has been observed in a large fraction of cancer survivors. Although many of the chemotherapeutic drugs do not cross the blood-brain barrier, following treatment, the structure and function of the brain are altered and cognitive dysfunction occurs in a significant number of cancer survivors. The means by which CICI occurs is becoming better understood, but there still remain unsolved questions of the mechanisms involved. The hypotheses to explain CICI are numerous. More than 50% of FDA-approved cancer chemotherapy agents are associated with reactive oxygen species (ROS) that lead to oxidative stress and activate a myriad of pathways as well as inhibit pathways necessary for proper brain function. Oxidative stress triggers the activation of different proteins, one in particular is tumor necrosis factor alpha (TNFα). Following treatment with various chemotherapy agents, this pro-inflammatory cytokine binds to its receptors at the blood-brain barrier and translocates to the parenchyma via receptor-mediated endocytosis. Once in brain, TNFα initiates pathways that may eventually lead to neuronal death and ultimately cognitive impairment. TNFα activation of the c-jun N-terminal kinases (JNK) and Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathways may contribute to both memory decline and loss of higher executive functions reported in patients after chemotherapy treatment. Chemotherapy also affects the brain's antioxidant capacity, allowing for accumulation of ROS. This review expands on these topics to provide insights into the possible mechanisms by which the intersection of oxidative stress and TNFΑ are involved in chemotherapy-induced cognitive impairment.

Age-related brain iron accumulation is linked with oxidative stress, neurodegeneration and cognitive decline. Certain nutrients can reduce brain iron concentration in animal models, however, this association is not well established in humans. Moreover, it remains unknown if nutrition can moderate the effects of age on brain iron concentration and/or cognition. Here, we explored these issues in a sample of seventy-three healthy older adults (61–86 years old), while controlling for several factors such as age, gender, years of education, physical fitness and alcohol-intake. Quantitative susceptibility mapping was used for assessment of brain iron concentration and participants performed an N-Back paradigm to evaluate working memory performance. Nutritional-intake was assessed via a validated questionnaire. Nutrients were grouped into nutrition factors based on previous literature and factor analysis. One factor, comprised of vitamin E, lysine, DHA omega-3 and LA omega-6 PUFA, representing food groups such as nuts, healthy oils and fish, moderated the effects of age on both brain iron concentration and working memory performance, suggesting that these nutrients may slow the rate of brain iron accumulation and working memory declines in aging.

Parkinson disease (PD) is the second most common age-related neurodegenerative disease in the world, and PD significantly impacts the quality of life, especially as in general people are living longer. Because of the numerous and complex features of sporadic PD that progressively develops, it is difficult to build an ideal animal model for PD research. Genetically modified PD rodent animal models are considered as a major tool with which to study the mechanisms and potential therapeutic targets for PD. Up to now, none of the rodent animal models displays all PD characteristics. The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded SAGE Laboratories to generate a PTEN-induced putative kinase-1 (PINK1) knockout (KO) rat model for familial PD using zinc finger nuclease (ZFN) technology. In the current paper, we review all papers from PubMed that report studies with PINK1 KO rats, presenting the research results, and discussing the fidelity of this rat model to PD according to its phenotypes studied by several laboratories. This review will serve as a critical reference for future studies with this rodent model, providing a better understanding of PD etiology, pathology, and potential treatment strategies.

This article aims to alert the medical community and public health authorities to accumulating evidence on health benefits from sun exposure, which suggests that insufficient sun exposure is a significant public health problem. Studies in the past decade indicate that insufficient sun exposure may be responsible for 340,000 deaths in the United States and 480,000 deaths in Europe per year, and an increased incidence of breast cancer, colorectal cancer, hypertension, cardiovascular disease, metabolic syndrome, multiple sclerosis, Alzheimer’s disease, autism, asthma, type 1 diabetes and myopia. Vitamin D has long been considered the principal mediator of beneficial effects of sun exposure. However, oral vitamin D supplementation has not been convincingly shown to prevent the above conditions; thus, serum 25(OH)D as an indicator of vitamin D status may be a proxy for and not a mediator of beneficial effects of sun exposure. New candidate mechanisms include the release of nitric oxide from the skin and direct effects of ultraviolet radiation (UVR) on peripheral blood cells. Collectively, this evidence indicates it would be wise for people living outside the tropics to ensure they expose their skin sufficiently to the sun. To minimize the harms of excessive sun exposure, great care must be taken to avoid sunburn, and sun exposure during high ambient UVR seasons should be obtained incrementally at not more than 5–30 min a day (depending on skin type and UV index), in season-appropriate clothing and with eyes closed or protected by sunglasses that filter UVR.

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. OBJECTIVE: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. METHODS: C57BL/6 and BALB/c female mice were i.p. injected with 10mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1β, and caspase-1 determination. RESULTS: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. CONCLUSIONS: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.

Cranial radiation is important for treating both primary brain tumors and brain metastases. A potential delayed side effect of cranial radiation is neurocognitive function decline. Early detection of CNS injury might prevent further neuronal damage. Extracellular vesicles (EVs) have emerged as a potential diagnostic tool because of their unique membranous characteristics and cargos. We investigated whether EVs can be an early indicator of CNS injury by giving C57BJ/6 mice 10 Gy cranial IR. EVs were isolated from sera to quantify: 1) number of EVs using nanoparticle tracking analysis (NTA); 2) Glial fibrillary acidic protein (GFAP), an astrocyte marker; and 3) protein-bound 4-hydroxy-2-nonenal (HNE) adducts, an oxidative damage marker. Brain tissues were prepared for immunohistochemistry staining and protein immunoblotting. The results demonstrate: 1) increased GFAP levels (p 0.05) in EVs, but not brain tissue, in the IR group; and 2) increased HNE-bound protein adduction levels (p 0.05). The results support using EVs as an early indicator of cancer therapy-induced neuronal injury.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory, reasoning and other cognitive functions. Pathologically, patients with AD are characterized by deposition of senile plaques (SPs), formed by β-amyloid (Aβ), and neurofibrillary tangles (NTFs) that consist of aggregated hyperphosphorylated tau protein. The accumulation of insoluble protein aggregates in AD brain can be associated with an impairment of degradative systems. This current study investigated if the disturbance of protein polyubiquitination is associated with AD neurodegeneration. By using a novel proteomic approach, we found that 13 brain proteins are increasingly polyubiquitinated in AD human brain compared to age-matched controls. Moreover, the majority of the identified proteins were previously found to be oxidized in our prior proteomics, and these proteins are mainly involved in protein quality control and glucose metabolism. This is the first study showing alteration of the poly-ubiquitin profile in AD brain compared with healthy controls. Understanding the onset of the altered ubiquitin profile in AD brain may contribute to identification of key molecular regulators of cognitive decline. In AD, deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins that are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology and cognitive decline.

Oxidative stress, an overproduction of free radicals or a diminution of free radical scavenging ability relative to those of cognitively aged-matched controls, is widely recognized as a critical component of the pathogenesis and progression of Alzheimer's disease (AD). This recognition arose in significant part from the work in the author's laboratory, complemented by research from others' laboratories. The Butterfield laboratory discovered the oxidative stress associated with oligomeric amyloid-β peptide manifested primarily as elevated oxidative modification of proteins and peroxidation of lipids. Such oxidative damage caused neuronal death, which undoubtedly underlies the progressive loss of cognition in AD. Identification of specific oxidatively modified brain proteins in subjects with AD or amnestic mild cognitive impairment was achieved by the methods of redox proteomics, pioneered in the author's laboratory. The importance and significance of the research emanating from the Butterfield laboratory rest on the paradigm shift of thinking regarding the roles of oxidative stress and resulting damage to key proteins and biochemical pathways in the pathogenesis and progression of AD. Predictions of future research directions also are presented. Given the enormous financial and personal burden placed upon citizens (and governments) of the US from AD, and the surety that the number of AD patients will greatly increase over the next 20-30 years, greater understanding of the molecular basis of pathogenesis and progression of AD is essential. Our laboratory is privileged to have contributed to better understanding of AD and provided rationales to identify effective therapeutic targets for this devastating dementing disorder.

Alzheimer's disease (AD) is the most common form of dementia in the elderly followed by vascular dementia. In addition to clinically diagnosed dementia, cognitive dysfunction has been reported in diabetic patients. Recent studies are now beginning to recognize type 2 diabetes mellitus (T2DM), characterized by chronic hyperglycemia and insulin resistance, as a risk factor for AD and other cognitive disorders. While studies on insulin action have remained traditionally in the domain of peripheral tissues, the detrimental effects of insulin resistance in the central nervous system on cognitive dysfunction are increasingly being reported in recent clinical and preclinical studies. Brain functions require continuous supply of glucose and oxygen and a tight regulation of metabolic processes. Loss of this metabolic regulation has been proposed to be a contributor to memory dysfunction associated with neurodegeneration. Within the above scenario, this review will focus on the interplay among oxidative stress (OS), insulin resistance and AMPK dysfunctions in the brain by highlighting how these neurotoxic events contribute to neurodegeneration. We provide an overview on the detrimental effects of OS on proteins regulating insulin signaling and how these alterations impact cell metabolic dysfunctions through AMPK dysregulation. Such processes, we assert, are critically involved in the molecular pathways that underlie AD.

Doxorubicin (DOX) is a potent chemotherapeutic agent known to cause acute and long-term cognitive impairments in cancer patients. Cognitive function is presumed to be primarily mediated by neuronal circuitry in the frontal cortex (FC) and hippocampus, where glutamate is the primary excitatory neurotransmitter. Mice treated with DOX (25 mg/kg i.p.) were subjected to in vivo recordings under urethane anesthesia at 24h post-DOX injection or 5 consecutive days of cognitive testing (Morris Water Maze; MWM). Using novel glutamate-selective microelectrode arrays, amperometric recordings measured parameters of extracellular glutamate clearance and potassium-evoked release of glutamate within the medial FC and dentate gyrus (DG) of the hippocampus. By 24h post-DOX injection, glutamate uptake was 45% slower in the FC in comparison to saline-treated mice. In the DG, glutamate took 48% longer to clear than saline-treated mice. Glutamate overflow in the FC was similar between treatment groups, however, it was significantly increased in the DG of DOX treated mice. MWM data indicated that a single dose of DOX impaired swim speed without impacting total length traveled. These data indicate that systemic DOX treatment changes glutamate neurotransmission in key nuclei associated with cognitive function within 24h, without a lasting impact on spatial learning and memory. Understanding the functional effects of DOX on glutamate neurotransmission may help us understand and prevent some of the debilitating side effects of chemotherapeutic treatment in cancer survivors.

One of the major complaints patients who survive cancer often make is chemotherapy induced cognitive impairment (CICI), which survivors often call "chemo brain." CICI is a side effect of chemotherapy due to the cytotoxicity and neurotoxicity of anti-cancer drugs causing structural and functional changes in brain, even when drugs that do not cross the blood brain barrier (BBB) are used. Diminished cognitive functions including diminution of learning and memory, concentration and attention, processing speed and executive functions that reduce quality of life and ability to work are common signs and symptoms of CICI. There still is not a clarified and complete mechanism for CICI, but researchers have pointed to several biochemical candidates. Chemotherapy-induced, cytokine-mediated involvement in CICI will be mainly discussed in this review paper with emphasis on different types of cytokines, correlated with BBB and epigenetic changes. Mechanisms of ROS-generating, anti-cancer drugs and their relation to cytokine-mediated CICI will be emphasized.

Brains from persons with Alzheimer disease (AD) and its earlier stage, amnestic mild cognitive impairment (MCI), exhibit high levels of oxidative damage, including that to phospholipids. One type of oxidative damage is lipid peroxidation, the most important index of which is protein-bound 4-hydroxy-2-trans-nonenal (HNE). This highly reactive alkenal changes the conformations and lowers the activities of brain proteins to which HNE is covalently bound. Evidence exists that suggests that lipid peroxidation is the first type of oxidative damage associated with amyloid β-peptide (Aβ), a 38-42 amino acid peptide that is highly neurotoxic and critical to the pathophysiology of AD. The Butterfield laboratory is one of, if not the, first research group to show that Aβ42 oligomers led to lipid peroxidation and to demonstrate this modification in brains of subjects with AD and MCI. The Mattson laboratory, particularly when Dr. Mattson was a faculty member at the University of Kentucky, also showed evidence for lipid peroxidation associated with Aβ peptides, mostly in in vitro systems. Consequently, there is synergy between our two laboratories. Since this special tribute issue of Aging Research Reviews is dedicated to the career of Dr. Mattson, a review of some aspects of this synergy of lipid peroxidation and its relevance to AD, as well as the role of lipid peroxidation in the progression of this dementing disorder seems germane. Accordingly, this review outlines some of the individual and/or complementary research on lipid peroxidation related to AD published from our two laboratories either separately or jointly.

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

e24051 Background: Chemotherapy-induced cognitive impairment (CICI) is a common side effect of cancer therapy, affecting up to 75% of cancer patients. Oxidative stress is thought to play a key role in CICI and patients who have been treated with chemotherapy have demonstrated biochemical brain changes on magnetic resonance spectroscopy (MRS). Methods: Following institutional review board approval, chemotherapy-naïve gynecologic cancer patients scheduled to receive intravenous platinum/taxane-based chemotherapy were identified. The primary objective of this study was identification of chemotherapy-induced cognitive function changes using the Montreal Cognitive Assessment (MoCA) tool. This exploratory analysis evaluates correlations between changes in MoCA scores, oxidative analytes TNF-a, protein carbonyls (PC), and 4-hydroxynonenal (HNE) protein adducts, and MRS metabolic signals. Testing was performed at baseline and three weeks post-treatment. Simple linear regression was performed to evaluate the correlation between changes in MoCA scores and oxidative metabolites. Results: Fourteen patients were enrolled. Six patients had complete pre/post oxidative stress data, and four patients had complete pre/post MRS data. Five of six had decreased PC values (mean net change -15.17%, SD 0.15), half had increased HNE levels (mean net change +16.38%, SD 0.38), and four of six had decreased TNF-a values (mean net change -15.10%, SD 0.27) (Table). MoCA scores were positively correlated with serum PC levels ( r = 0.86, p = 0.03) and not significantly correlated with TNF-a or HNE levels. Of those with complete pre/post MRS data, all patients had a decrease in glycerophosphocholine + phosphocholine/creatinine ratio signals. MoCA raw score prior to and after completion of chemotherapy. A higher score indicates improved test performance. Positive percentage of oxidative stress markers (PC, HNE, TNF-a) indicates increased post-therapy levels compared to pre-therapy. Conclusions: In this study, we evaluated candidate oxidative stress serum markers and changes in MRS metabolite signals for CICI. Serum PC levels correlate with cognitive screening scores. For patients undergoing brain imaging, MRS evaluation for individual choline components, glycerophosphocholine and phosphocholine, may serve as an additional marker of oxidative damage. This pilot study highlights the potential value of serum PC levels and dedicated brain imaging for signs of oxidative stress, including MRS, to guide future CICI identification studies. Clinical trial information: NCT03324945. [Table: see text]

A diet consisting of a high intake of saturated fat and refined sugars is characteristic of a Western-diet and has been shown to have a substantial negative effect on human health. Expression proteomics were used to investigate changes to the parietal lobe proteome of rhesus monkeys consuming either a high fat and sugar (HFS) diet, a HFS diet supplemented with resveratrol (HFS+RSV), or a healthy control diet for 2 years. Here we discuss the modifications in the levels of 12 specific proteins involved in various cellular systems including metabolism, neurotransmission, structural integrity, and general cellular signaling following a nutritional intervention. Our results contribute to a better understanding of the mechanisms by which resveratrol functions through the up- or down-regulation of proteins in different cellular sub-systems to affect the overall health of the brain.

Oxidative damage is one of the hallmarks of the aging process. The current study evaluated effects of two proprietary antioxidant-based ingredients, rosemary extract and spearmint extract containing carnosic acid and rosmarinic acid, respectively, on learning and memory in the SAMP8 mouse model of accelerated aging. The two rosemary extracts contained carnosic acid (60% or 10% carnosic acid) and one spearmint extract contained 5% rosmarinic acid. Three doses of actives in each extract were tested: 32, 16, 1.6 or 0mg/kg. After 90days of treatment mice were tested in T-maze foot shock avoidance, object recognition and lever press. Rosemary extract containing 60% carnosic acid improved acquisition and retention in T-maze foot shock, object recognition and lever press. Rosemary extract with 10% carnosic acid improved retention in T-maze foot shock avoidance and lever press. Spearmint with 5% rosmarinic acid improved acquisition and retention in T-maze foot shock avoidance and object recognition. 4-hydroxynonenal (HNE) was reduced in the brain cortex after treatment with all three extracts (P

Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the "triangle of death" composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277-279.

Down syndrome (DS) is the most common genetic cause of intellectual disability that is associated with an increased risk to develop early-onset Alzheimer-like dementia (AD). The brain neuropathological features include alteration of redox homeostasis, mitochondrial deficits, inflammation, accumulation of both amyloid beta-peptide oligomers and senile plaques, as well as aggregated hyperphosphorylated tau protein-containing neurofibrillary tangles, among others. It is worth mentioning that some of the triplicated genes encoded are likely to cause increased oxidative stress (OS) conditions that are also associated with reduced cellular responses. Published studies from our laboratories propose that increased oxidative damage occurs early in life in DS population and contributes to age-dependent neurodegeneration. This is the result of damaged, oxidized proteins that belong to degradative systems, antioxidant defense system, neuronal trafficking. and energy metabolism. This review focuses on a key element that regulates redox homeostasis, the transcription factor Nrf2, which is negatively regulated by BACH1, encoded on chromosome 21. The role of the Nrf2/BACH1 axis in DS is under investigation, and the effects of triplicated BACH1 on the transcriptional regulation of Nrf2 are still unknown. In this review, we discuss the physiological relevance of BACH1/Nrf2 signaling in the brain and how the dysfunction of this system affects the redox homeostasis in DS neurons and how this axis may contribute to the transition of DS into DS with AD neuropathology and dementia. Further, some of the evidence collected in AD regarding the potential contribution of BACH1 to neurodegeneration in DS are also discussed.

Extracellular vesicles (EVs) generated from redox active anticancer drugs are released into the extracellular environment. These EVs contain oxidized molecules and trigger inflammatory responses by macrophages. Using a mouse model of doxorubicin (DOX)-induced tissue injury, we previously found that the major sources of circulating EVs are from heart and liver, organs that are differentially affected by DOX. Here, we investigated the effects of EVs from cardiomyocytes and those from hepatocytes on macrophage activation. EVs from H9c2 rat cardiomyocytes (H9c2 EVs) and EVs from FL83b mouse hepatocytes (FL83 b EVs) have different levels of protein-bound 4-hydroxynonenal and thus different immunostimulatory effects on mouse RAW264.7 macrophages. H9c2 EVs but not FL83 b EVs induced both pro-inflammatory and anti-inflammatory macrophage activation, mediated by NFκB and Nrf-2 pathways, respectively. DOX enhanced the effects of H9c2 EVs but not FL83 b EVs. While EVs from DOX-treated H9c2 cells (H9c2 DOXEVs) suppressed mitochondrial respiration and increased glycolysis of macrophages, EVs from DOX-treated FL83b cells (FL83b DOXEVs) enhanced mitochondrial reserve capacity. Mechanistically, the different immunostimulatory functions of H9c2 EVs and FL83 b EVs are regulated, in part, by the redox status of the cytoplasmic thioredoxin 1 (Trx1) of macrophages. H9c2 DOXEVs lowered the level of reduced Trx1 in cytoplasm while FL83b DOXEVs did the opposite. Trx1 overexpression alleviated the effect of H9c2 DOXEVs on NFκB and Nrf-2 activation and prevented the upregulation of their target genes. Our findings identify EVs as a novel Trx1-mediated redox mediator of immune response, which greatly enhances our understanding of innate immune responses during cancer therapy. Keywords: Extracellular vesicles, Macrophage activation, Thioredoxin 1, NFκB, Nrf-2

Background Type II diabetes is a vascular risk factor for cognitive impairment and increased risk of dementia. Disruption of the blood–retinal barrier (BRB) and blood–brain barrier (BBB) are hallmarks of subsequent retinal edema and central nervous system dysfunction. However, the mechanisms by which diet or metabolic syndrome induces dysfunction are not understood. A proposed mechanism is an increase in reactive oxygen species (ROS) and oxidative stress. Inhibition of mitochondrial carbonic anhydrase (mCA) decreases ROS and oxidative stress. In this study, topiramate, a mCA inhibitor, was examined for its ability to protect the BRB and BBB in diet-induced obese type II diabetic mice. Methods BBB and BRB permeability were assessed using 14C-sucrose and 99mTc-albumin in CD-1 mice fed a low-fat (control) or a high-fat diet. Topiramate administration was compared to saline controls in both preventative and efficacy arms examining BRB and BBB disruption. Body weight and blood glucose were measured weekly and body composition was assessed using EchoMRI. Metabolic activity was measured using a comprehensive laboratory animal monitoring system. Brain tissues collected from the mice were assessed for changes in oxidative stress and tight junction proteins. Results High-fat feeding caused increased entry of 14C-sucrose and 99mTc-albumin into the brains of diet-induced obese type II diabetic mice. Increased permeability to 14C-sucrose was observed in the hypothalamus and hippocampus, and attenuated by topiramate treatment, while increased permeability to 99mTc-albumin occurred in the whole brain and was also attenuated by topiramate. Treatment with topiramate decreased measures of oxidative stress and increased expression of the tight junction proteins ZO-1 and claudin-12. In the retina, we observed increased entry of 99mTc-albumin simultaneously with increased entry into the whole brain during the preventative arm. This occurred prior to increased entry to the retina for 14C-sucrose which occurred during the efficacy arm. Treatment with topiramate had no effect on the retina. Conclusions Blood–brain barrier and blood–retinal barrier dysfunction were examined in a mouse model of diet-induced obese type II diabetes. These studies demonstrate that there are spatial and temporal differences in 14C-sucrose and 99mTc-albumin permeability in the brain and retina of diet-induced obese type II diabetic mice. Topiramate, a mitochondrial carbonic anhydrase inhibitor, is efficacious at both preventing and treating BBB disruption in this diet-induced obese type II diabetic mouse model.

Dysregulation of insulin signaling pathway with reduced downstream neuronal survival and plasticity mechanisms is a fundamental abnormality observed in Alzheimer's disease (AD) brain. This phenomenon, known as brain insulin resistance, is associated with poor cognitive performance and is driven by the uncoupling of insulin receptor (IR) from its direct substrate (IRS1). Considering that Down syndrome (DS) and AD neuropathology share many common features, we investigated metabolic aspects of neurodegeneration, i.e., brain insulin resistance, in DS and whether it would contribute to early onset AD in DS population. Changes of levels and activation of main brain proteins belonging to the insulin signaling pathway (i.e., IR, IRS1, PTEN, GSK3β, PKCζ, AS160, GLUT4) were evaluated. Furthermore, we analyzed whether changes of these proteins were associated with alterations of: (i) proteins regulating brain energy metabolism; (ii) APP cleavage; and (ii) regulation of synaptic plasticity mechanisms in post-mortem brain samples collected from people with DS before and after the development of AD pathology (DSAD) compared with their age-matched controls. We found that DS cases were characterized by key markers of brain insulin resistance (reduced IR and increased IRS1 inhibition) early in life. Furthermore, downstream from IRS1, an overall uncoupling among the proteins of insulin signaling was observed. Dysregulated brain insulin signaling was associated with reduced hexokinase II (HKII) levels and proteins associated with mitochondrial complexes levels as well as with reduced levels of syntaxin in DS cases. Tellingly, these alterations precede the development of AD neuropathology and clinical presentations in DS. We propose that markers of brain insulin resistance rise earlier with age in DS compared with the general population and may contribute to the cognitive impairment associated with the early development of AD in DS.

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.

Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen.The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis.EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA.Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.