Generation of Glioblastoma Patient-Derived Intracranial Xenografts for Preclinical Studies.

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer affecting adults. Therapeutic options for GBM have remained the same for over a decade with no significant improvement. Many therapies that are successful in culture have failed in patients, likely due to the complex microenvironment in the brain, which has yet to be reproduced in any culture model. Furthermore, the high passage number of cultured cells and clonal selection fail to recapitulate the molecular and genomic signatures of GBM. We have established orthotopic patient-derived xenografts (PDX) from 37 GBM patients with human GBM. Of the 69 patient samples analyzed, we were successful in passaging 37 lines three or more generations (53.6%). After phenotypic characterization of the xenografted tumor tissue, two different growth patterns emerged highly invasive or localized. The phenotype was dependent on malignancy and previous treatment of the patient from which the xenograft was derived. Physiologically, mice exhibited symptoms more quickly with each subsequent passage, particularly in the localized tumors. Study of these physiologically relevant human xenografts in mice will enable therapeutic screenings in a microenvironment that more closely resembles GBM and may allow development of individualized patient models which may eventually be used for simulating treatment.

Biological Assessment of Coral Reefs in Southern Puerto Rico: A Technical Approach for Coral Reef Protection Under the U.S. Clean Water Act.

States and other jurisdictions may protect coral reefs using biological water quality standards outlined by the United States Clean Water Act (CWA). Such protection will require long-term, regional monitoring of the resource using biological indicators and a probability-based sampling design. A 60-station survey targeting nearshore linear coral reef was conducted across southern Puerto Rico in December 2011 to document the status of reef inhabitants using a probabilistic, regional sampling design. The quantity, type and condition of stony corals, fish, gorgonians and sponges were documented from each station, providing a robust representation of linear reef status and composition across the region. Fish represented 106 unique taxa and stony corals 32 unique taxa. Benthic organisms (stony corals, sponges and gorgonians) averaged nearly 12 colonies per square meter, more than half of which were gorgonians. Assessment results can be used as a baseline to compare with future regional surveys to quantify change in reef condition over time (trend). Both temporal and spatial changes can be expected after large-scale disturbances like hurricanes Maria and Irma in 2017. The indicators and probabilistic sampling design support the long-term regional monitoring envisioned by the Environmental Protection Agency to implement CWA protections in Puerto Rico and elsewhere.

Regional status assessment of stony corals in the US Virgin Islands.

States may protect coral reefs using biological water quality standards outlined by the Clean Water Act. This requires biological assessments with indicators sensitive to human disturbance and regional, probability-based survey designs. Stony coral condition was characterized on a regional scale for the first time in the nearshore waters of the US Virgin Islands (USVI). Coral composition, abundance, size, and health were assessed at 66 stations in the St. Croix region in fall 2007 and at 63 stations in the St. Thomas and St. John region in winter 2009. Indicators were chosen for their sensitivity to human disturbance. Both surveys were probability-based (random) designs with station locations preselected from areas covered by hardbottom and coral reef substrate. Taxa richness was as high as 21 species but more than half the area of both regions exhibited taxa richness of <10 species in the 25 m(2) transect area. Coral density was as high as 5 colonies m(-2) but more than half the area of both regions had <2 colonies m(-2). Both regions showed similar dominant species based on frequency of occurrence and relative abundance. Because of large colony sizes, Montastrea annularis provided more total surface area and live surface area than more abundant species. The surveys establish baseline regional conditions and provide a foundation for long-term regional monitoring envisioned by the USVI Department of Planning and Natural Resources. The probabilistic sampling design assures the data can be used in Clean Water Act reporting.

Using metabolomic profiling to inform use of surrogate species in ecological risk assessment practices.

The U.S. EPA frequently uses avian or fish toxicity data to set protective standards for amphibians in ecological risk assessments. However, this approach does not always adequately represent aquatic-dwelling and terrestrial-phase amphibian exposure data. For instance, it is accepted that early life stage tests for fish are typically sensitive enough to protect larval amphibians, however, metamorphosis from tadpole to a terrestrial-phase adult relies on endocrine cues that are less prevalent in fish but essential for amphibian life stage transitions. These differences suggest that more robust approaches are needed to adequately elucidate the impacts of pesticide exposure in amphibians across critical life stages. Therefore, in the current study, methodology is presented that can be applied to link the perturbations in the metabolomic response of larval zebrafish (Danio rerio), a surrogate species frequently used in ecotoxicological studies, to those of African clawed frog (Xenopus laevis) tadpoles following exposure to three high-use pesticides, bifenthrin, chlorothalonil, or trifluralin. Generally, D. rerio exhibited greater metabolic perturbations in both number and magnitude across the pesticide exposures as opposed to X. laevis. This suggests that screening ecological risk assessment surrogate toxicity data would sufficiently protect amphibians at the single life stage studied but care needs to be taken to understand the suite of metabolic requirements of each developing species. Ultimately, methodology presented, and data gathered herein will help inform the applicability of metabolomic profiling in establishing the risk pesticide exposure poses to amphibians and potentially other non-target species.

Glioma stem cells activate platelets by plasma-independent thrombin production to promote glioblastoma tumorigenesis.

Background: The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example, glioblastoma (GBM) where metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcomes in many cancers. While platelets are known to promote the progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Methods: Male and female NSG mice were transplanted with GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. Results: We show that GSCs activate platelets by endogenous production of all the factors of the intrinsic and extrinsic coagulation cascades in a plasma-independent manner. Therefore, GSCs produce thrombin resulting in platelet activation. We further demonstrate that the endogenous coagulation cascades of these cancer stem cells are tumorigenic: they activate platelets to promote stemness and proliferation in vitro and pharmacological inhibition delays tumor growth in vivo. Conclusions: Our findings uncover a specific preferential relationship between platelets and GSCs that drive GBM malignancies and identify a therapeutically targetable novel interaction.

A novel origin for granulovacuolar degeneration in aging and Alzheimer's disease: parallels to stress granules.

The phosphorylated ribosomal protein S6 (pS6) is associated with the 40S ribosomal subunit in eukaryotes and is thought to have a role in RNA storage, degradation, and re-entry into translation. In this study, we found pS6 localized to granulovacuolar degeneration (GVD) within the pyramidal neurons. Immunohistochemical analysis found that nearly 20-fold more neurons contain pS6-positive granules in Alzheimer's disease (AD) hippocampus compared with age-matched controls. Further, pS6-positive granules were more common in neurons not containing neurofibrillary tangles (NFTs), were never associated with extracellular NFTs or in apoptotic neurons, and contained less RNA than neighboring pyramidal neurons not containing pS6-positive granules. In model systems, pS6 is a specific marker for stress granules, and another stress granule protein, p54/Rck, was also found to be a component of GVD in the current study. Stress granules are transient, intracellular, dense aggregations of proteins and RNAs that accumulate as a stress response, protecting cells from apoptosis and inappropriate transcriptional activity, often described as a form of 'molecular triage.' The RNA oxidation modification 8-hydroxyguanosine (8OHG) is strikingly increased in AD, yet this study reports that those neurons with pS6 granules display reduced RNA oxidation demonstrated by lower levels of 8OHG. Since chronic oxidative stress is central to AD pathogenesis, and RNA is a specific oxidative stress target and is intimately associated with stress granule biogenesis in model systems, we suggest that GVD in human brain parallel stress granules, and may in fact be more representative of early disease pathogenesis than traditionally believed. This proposed origin for GVD as a neuroprotective response, may represent a morphologic checkpoint between cell death and reversible cellular stress that proceeds in the absence of other inclusions.

CD3 in Lewy pathology: does the abnormal recall of neurodevelopmental processes underlie Parkinson's disease.

CD3ζ is a subunit of the CD3 molecule that, until recently, appeared restricted to T cells and natural killer cells. However, experimental studies have demonstrated a role of CD3ζ in dendritic outgrowth in the visual system as well as in synaptic plasticity. Given the increasing evidence for uncharacteristic recapitulation of neurodevelopmental processes in neurodegenerative diseases, in this study, we evaluated brains from subjects with Parkinson's disease and Lewy body dementia for evidence of aberrant CD3 expression. Our data shows marked CD3ζ in association with the α-synuclein containing pathological lesions, i.e., Lewy bodies and Lewy neurites, in the brains of subjects with Parkinson's disease and Lewy body dementia. This finding raises the novel concept of CD3 dysregulation in these disorders as a pathogenic factor and also furthers the increasing evidence that the recall of aberrant neurodevelopmental processes underlies the pathogenesis of neurodegenerative diseases.

Cross-Taxa Distinctions in Mechanisms of Developmental Effects for Aquatic Species Exposed to Trifluralin.

Standard ecological risk assessment practices often rely on larval and juvenile fish toxicity data as representative of the amphibian aquatic phase. Empirical evidence suggests that endpoints measured in fish early life stage tests are often sufficient to protect larval amphibians. However, the process of amphibian metamorphosis relies on endocrine cues that affect development and morphological restructuring and are not represented by these test endpoints. The present study compares developmental endpoints for zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis), 2 standard test species, exposed to the herbicide trifluralin throughout the larval period. Danio rerio were more sensitive and demonstrated a reduction in growth measurements with increasing trifluralin exposure. Size of X. laevis at metamorphosis was not correlated with exposure concentration; however, time to metamorphosis was delayed relative to trifluralin concentration. Gene expression patterns indicate discrepancies in response by D. rerio and X. laevis, and dose-dependent metabolic activity suggests that trifluralin exposure perturbed biological pathways differently within the 2 species. Although many metabolites were correlated with exposure concentration in D. rerio, nontargeted hepatic metabolomics identified a subset of metabolites that exhibited a nonmonotonic response to trifluralin exposure in X. laevis. Linking taxonomic distinctions in cellular-level response with ecologically relevant endpoints will refine assumptions used in interspecies extrapolation of standard test effects and improve assessment of sublethal impacts on amphibian populations. Environ Toxicol Chem 2020;39:1797-1812. Published 2020. This article is a US government work and is in the public domain in the USA.

Proteins inform survival-based differences in patients with glioblastoma.

BACKGROUND: Improving the care of patients with glioblastoma (GB) requires accurate and reliable predictors of patient prognosis. Unfortunately, while protein markers are an effective readout of cellular function, proteomics has been underutilized in GB prognostic marker discovery. METHODS: For this study, GB patients were prospectively recruited and proteomics discovery using liquid chromatography-mass spectrometry analysis (LC-MS/MS) was performed for 27 patients including 13 short-term survivors (STS) (≤10 months) and 14 long-term survivors (LTS) (≥18 months). RESULTS: Proteomics discovery identified 11 941 peptides in 2495 unique proteins, with 469 proteins exhibiting significant dysregulation when comparing STS to LTS. We verified the differential abundance of 67 out of these 469 proteins in a small previously published independent dataset. Proteins involved in axon guidance were upregulated in STS compared to LTS, while those involved in p53 signaling were upregulated in LTS. We also assessed the correlation between LS MS/MS data with RNAseq data from the same discovery patients and found a low correlation between protein abundance and mRNA expression. Finally, using LC-MS/MS on a set of 18 samples from 6 patients, we quantified the intratumoral heterogeneity of more than 2256 proteins in the multisample dataset. CONCLUSIONS: These proteomic datasets and noted protein variations present a beneficial resource for better predicting patient outcome and investigating potential therapeutic targets.

Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

A simple and rapid matrix-assisted laser desorption/ionization time of flight mass spectrometry method to screen fish plasma samples for estrogen-responsive biomarkers.

In the present study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry coupled with a short-term fish assay. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria consisting of vehicle control and the following estrogen agonist treatments: 17beta-estradiol (0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.5, and 1.0 microg/L, 4-tert-pentylphenol (100 microg/L), methoxychlor (6 and 12 microg/L), and bisphenol A (100 and 1,000 microg/L). Treatments with chlorpyrifos (80 microg/L) and endosulfan (0.6 microg/L) served as nonestrogenic negative controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus. Plasma was obtained from individuals, diluted and applied to an inert surface, and analyzed by MALDI. Multiple protein peaks, ranging from 2.9 to 12.9 kDa, were identified as markers of estrogenic effects when comparing estrogen-treated and control fish using interpercentile reference values. A binary classification tree model was constructed from plasma protein profiles of the vehicle control and the 0.2 microg/L of 17beta-estradiol treatments and then used to evaluate all samples. Treatments with the estrogen agonists 17beta-estradiol, 4-tert-pentylphenol, methoxychlor, and bisphenol-A generated reproducible diagnostic biomarkers based on the presence of specific estrogen-responsive plasma proteins. The controls and nonestrogenic compounds chlorpyrifos and endosulfan did not produce this estrogen-responsive protein profile. A no-observed-effect level for 17beta-estradiol at 0.025 microg/L was estimated from concentration-response exposures. The MALDI method described here provides a straightforward, sensitive, and specific tool to screen chemicals for estrogenic activity.

Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts.

In this study, we evaluated the effect of lipoic acid (LA) and N-acetyl cysteine (NAC) on oxidative [4-hydroxy-2-nonenal, N(epsilon)-(carboxymethyl)lysine and heme oxygenase-1] and apoptotic (caspase 9 and Bax) markers in fibroblasts from patients with Alzheimer disease (AD) and age-matched and young controls. AD fibroblasts showed the highest levels of oxidative stress, and the antioxidants, lipoic acid (1 mM) and/or N-acetyl cysteine (100 microM) exerted a protective effect as evidenced by decreases in oxidative stress and apoptotic markers. Furthermore, we observed that the protective effect of LA and NAC was more pronounced when both agents were present simultaneously. AD-type changes could be generated in control fibroblasts using N-methylprotoporphyrin to inhibit cytochrome oxidase assembly indicating that the the oxidative damage observed was associated with mitochondrial dysfunction. The effects of N-methylprotoporphyrine were reversed or attenuated by both lipoic acid and N-acetyl cysteine. These data suggest mitochondria are important in oxidative damage that occurs in AD. As such, antioxidant therapies based on lipoic acid and N-acetyl cysteine supplementation may be promising.

Increased expression of the remodeling- and tumorigenic-associated factor osteopontin in pyramidal neurons of the Alzheimer's disease brain.

Osteopontin (OPN) is a glycophosphoprotein expressed by several cell types and has pro-adhesive, chemotactic, and cytokine-like properties. OPN is involved in a number of physiologic and pathologic events including angiogenesis, apoptosis, inflammation, oxidative stress, remyelination, wound healing, bone remodeling, cell migration and tumorigenesis. Since these functions of OPN, and the events that it regulates, are involved with neurodegeneration, we examined whether OPN was differentially expressed in the hippocampus of the Alzheimer's disease (AD) compared with age-matched (59-93 years) control brain. We report for the first time the immunocytochemical localization of OPN in the cytoplasm of pyramidal neurons. In AD brains, there was a significant 41 % increase in the expression of neuron OPN compared with age-matched control brain. No staining of other neuronal cell types was observed. Additionally, there was a significant positive correlation between OPN staining intensity and both amyloid-beta load (r(2) = 0.25; P < 0.05; n = 20) and aging (r(2) = 0.32; P < 0.01; n = 20) among all control and AD subjects. Controlling for age indicated that OPN expression was significantly influenced by amyloid-beta load, but not age. While the functional consequences of this amyloid-beta associated increase in OPN expression are unclear, it is notable that OPN is primarily localized to those neurons that are known to be vulnerable to AD-related neurite loss, degeneration and death. Given that the induction of OPN expression (and amyloid-beta generation) is associated with remodeling and tumorigenesis, our results suggest that OPN may play a role in the aberrant re-entry of neurons into the cell cycle and/or neuronal remyelination in AD.

A proteomic (SELDI-TOF-MS) approach to estrogen agonist screening.

A small fish model and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 17beta-estradiol (E2), methoxychlor (MXC), bisphenol-A (BPA), 4-tert-pentylphenol (TPP), endosulfan (ES), and chlorpyriphos (CP). Fish plasma was applied to weak cation exchange (CM10) ProteinChip arrays, processed, and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen-responsive mass spectral biomarker peaks were identified by comparison of E2-treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1 to 13 kDa were up- or downregulated in E2-treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist, and nonagonist stressor-treated males and normal female fish plasma. One of the biomarkers, mass-to-charge ratio 3025.5, was identified by high-resolution tandem mass spectrometry as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens MXC, BPA, and TPP elicited protein expression profiles consistent with the estrogen expression model. Estrogen-responsive peaks were not detected in plasma from fish in the seawater, vehicle, ES, or CP treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Vascular oxidative stress in Alzheimer disease.

Alzheimer disease and cerebrovascular dementia are two common causes of dementia and, by present diagnostic criteria, are mutually exclusive using vascular pathology as an arbitrary demarcation in differential diagnosis. However, evidence from epidemiological, neuropathological, clinical, pharmacological, and functional studies suggest considerable overlap in risk factors and pathological changes suggesting shared common pathogenic mechanisms between these two diseases such that vascular factors play a vital role in the pathogenesis of Alzheimer disease. A high energy demand and lack of an endogenous fuel reserve make the brain highly dependent upon a continuous blood supply where disruption of cerebral blood vessels and blood flow can have serious consequences on neural activities. Indeed, many studies implicate metabolic defects in Alzheimer disease, such a reduced brain metabolism is one of the best documented abnormalities in the disease. Notably, since endothelial reactive oxygen species such as nitric oxide act as vasodilators at low concentrations, increased production coupled with elevated reactive oxygen species scavenging of nitric oxide, can lead to reduced bioavailability of nitric oxide and increased oxidative stress that damage sensitive vascular cells. In this respect, we and others have demonstrated that oxidative stress is one of the earliest pathological changes in the brain of Alzheimer disease patients and plays a critical role in the vascular abnormalities underlying metabolic defects in Alzheimer disease. Here, we discuss vascular factors in relation to Alzheimer disease and review hypoperfusion as a potential cause by triggering mitochondrial dysfunction and increased oxidative stress initiating the pathogenic process.

Tau modifiers as therapeutic targets for Alzheimer's disease.

Fibrillogenesis is a major feature of Alzheimer's disease (AD) and other neurodegenerative diseases. Fibers are correlated with disease severity and they have been implicated as playing a direct role in disease pathophysiology. In studies of tau, instead of finding causality with tau fibrils, we found that tau is associated with reduction of oxidative stress. Biochemical findings show that tau oxidative modifications are regulated by phosphorylation and that tau found in neurofibrillary tangles is oxidatively modified, suggesting that tau is closely linked to the biology, not toxicity, of AD.

Nanoparticle iron chelators: a new therapeutic approach in Alzheimer disease and other neurologic disorders associated with trace metal imbalance.

Accumulating evidence suggests that oxidative stress may be a major etiologic factor in initiating and promoting neurodegeneration in Alzheimer disease. Contributing to this, there is a dyshomeostasis of metal ions in Alzheimer disease with abnormally high levels of redox-active metals, particularly iron, in affected areas of the brain. Although it is unclear whether metal excesses are the sole cause of oxidative stress and neurodegeneration or a by-product of neuronal loss, the finding that metal chelators can partially solubilize amyloid-beta deposits in Alzheimer disease suggests a promising therapeutic role for chelating agents. However, the blood-brain barrier and toxicity of known chelators limit their utility. In this study, we suggest that covalent conjugation of iron chelators with nanoparticles may help overcome the limitations in blood-brain barrier permeability of existing chelation therapy. Using in vitro studies, we have shown that a chelator-nanoparticle system and the chelator-nanoparticle system complexed with iron, when incubated with human plasma, preferentially adsorb apolipoprotein E and apolipoprotein A-I, that would facilitate transport into and out of the brain via mechanisms used for transporting low-density lipoprotein. Our studies suggest a unique approach, utilizing nanoparticles, to transport chelators and chelator-metal complexes in both directions across the blood-brain barrier, thus providing safer and more effective chelation treatment in Alzheimer disease and other neurodegenerative diseases.

Sequestration of p27 within the cytoplasm of cardiac myocytes in chronic ischemic heart disease: pathogenic implications for ischemic cardiomyopathy.

A number of recent studies have suggested that cardiac myocytes, previously considered post-mitotic, re-enter the cell cycle and possess the ability to proliferate with certain pathogenic stimuli. To study this further, we examined cellular proliferation in myocardial tissue from subjects with chronic ischemic heart disease-associated myocardial infarction and subsequent congestive heart failure. We found striking increases in cytoplasmic phospho-p27, a well-known mitotic regulator, compared to controls by both immunocytochemical and immunoblot analyses. However, we found no evidence for cardiac myocyte proliferation in either disease or control subjects using both mitotic counting (no mitotic figures were observed) and Ki-67 immunocytochemistry, which demonstrated a 0% proliferation index. That increased cytoplasmic phospho-p27 is not accompanied by division prompts us to speculate that ectopic cell cycle activation occurs in the face of minimal to absent myocyte proliferation per se. Based on these findings, and the parallel findings in post-mitotic neurons in neurodegenerative disease, we suggest that cell-cycle activation in ischemic heart disease is a deleterious event that perpetuates disease pathogenesis culminating in myocardial failure.

Telomeres and telomerase in Alzheimer's disease: epiphenomena or a new focus for therapeutic strategy?

New approaches to the elucidation of Alzheimer disease, even those with solid data, are often ignored or dismissed as epiphenomenal when they differ from the mainstream or theoretical expectations. Here we present a new piece to the puzzle, decreases in telomere length, and telomerase expression in neuronal populations known to be vulnerable to degeneration and death in Alzheimer's disease. We can present the answers to the question "what," but the "why," "when," and "how" are not so easily answered. The goal of this report is to prompt discussion and more intensive study of this finding toward a new focus of therapeutic strategy.

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations.

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.