Medical student misconceptions in cardiovascular physiology.

Students find cardiovascular physiology challenging. Misunderstandings can be due to the nature of the subject, the way it is taught, and prior knowledge, which impede learning of new concepts. Some misunderstood concepts can be corrected with teaching (i.e., preconceptions), whereas others are resistant to instruction (i.e., misconceptions). A set of questions, specifically created by a panel of physiology experts to probe difficult cardiovascular concepts, was used to identify preconceptions, misconceptions, and the effect of education level on question performance. The introductory cardiovascular lecture used in this study was created based on these questions. In-class polling of medical students' (n = 736) performance was performed using the Turning-Point clicker response system during lecture instruction. Results were compared with published data from undergraduates (n = 1,076) who completed the same questions but without prior instruction. To our knowledge, there have been no studies directly comparing performance using the same instrument and large numbers of undergraduate and medical students. A higher education level was associated with increased performance (preconceptions), whereas several concepts resistant to instruction (misconceptions) were identified. Findings suggest that prior knowledge interfered with the acquisition of medical knowledge. Based on these results, potential causes for these misconceptions and remedial teaching suggestions are discussed.

Using lectures to identify student misconceptions: a study on the paradoxical effects of hyperkalemia on vascular smooth muscle.

Medical students have difficulty understanding the mechanisms underlying hyperkalemia-mediated local control of blood flow. Such control mechanisms are crucial in the brain, kidney, and skeletal muscle vasculature. We aimed to identify medical students' misconceptions via assessment of students' in-class knowledge and, subsequently, improve future teaching of this concept. In-class polling was performed with the TurningPoint clicker response system (n = 860) to gauge students' understanding of three physiological concepts related to hyperkalemia: membrane potential (Vm), conductance, and smooth muscle response. Vm includes the concepts of equilibrium potential (Veq) for specific ions, as well as driving force (DF = Vm - Veq). Students understood the concept of DF (~70% answered correctly), suggesting their understanding of Vm. However, students misunderstood that hyperkalemia results in depolarization (~52% answered correctly) and leads to an increase in potassium conductance (~31% answered correctly). Clarification of the type of smooth muscle as vascular increased the percentage of correct responses (~51 to 73%). The data indicate that students lacked knowledge of specific potassium conductance in various muscle types, resulting in divergent responses, such as the canonical depolarization in skeletal muscle versus hyperpolarization in smooth muscle cells during hyperkalemia. Misunderstanding of this crucial concept of conductance is directly related to the students' performance. Furthermore, we connected the paradoxical effect of hyperkalemia to pathological acute and chronic hyperkalemia clinical scenarios.

Effect of a small-group, active learning, tutorial-based, in-course enrichment program on student performance in medical physiology.

Physiology is one of the major foundational sciences for the medical curriculum. This discipline has proven challenging for students to master due to ineffective content acquisition and retention. Preliminary data obtained from a survey completed by "low-performance" students (those maintaining a grade average below the passing mark of 70%) at Morehouse School of Medicine reported that students lacked the ability to adequately recognize and extract important physiological concepts to successfully navigate multiple-choice assessments. It was hypothesized that a specially designed, small-group, active learning, physiology in-course enrichment program would minimize course assessment failure rates by enhancing the ability of low-performance students to effectively identify important course content, successfully perform on multiple-choice assessments, and, thereby, improve overall course performance. Using self-report surveys, study skills and test-taking deficiencies limiting successful comprehension of course material and examination performance were identified. Mini-quiz assessments and assignments in formulating multiple-choice examination questions were given to help students recognize and solidify core concepts and improve test-taking ability. Lastly, self-report surveys evaluated the effectiveness of the enrichment program on overall course performance. Results showed a marked improvement in student confidence levels with regards to approaching multiple-choice assessments, and a significant improvement in grades achieved in the physiology component of the first-year curriculum, as 100% of participants achieved a final passing grade average of ≥70%. It was concluded that students became more proficient in identifying, understanding, and applying core physiological concepts and more successful in mastering multiple-choice questions.

Protein misfolding and aggregation in neurodegenerative diseases: a review of pathogeneses, novel detection strategies, and potential therapeutics.

Protein folding is a complex, multisystem process characterized by heavy molecular and cellular footprints. Chaperone machinery enables proper protein folding and stable conformation. Other pathways concomitant with the protein folding process include transcription, translation, post-translational modifications, degradation through the ubiquitin-proteasome system, and autophagy. As such, the folding process can go awry in several different ways. The pathogenic basis behind most neurodegenerative diseases is that the disruption of protein homeostasis (i.e. proteostasis) at any level will eventually lead to protein misfolding. Misfolded proteins often aggregate and accumulate to trigger neurotoxicity through cellular stress pathways and consequently cause neurodegenerative diseases. The manifestation of a disease is usually dependent on the specific brain region that the neurotoxicity affects. Neurodegenerative diseases are age-associated, and their incidence is expected to rise as humans continue to live longer and pursue a greater life expectancy. We presently review the sequelae of protein misfolding and aggregation, as well as the role of these phenomena in several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, transmissible spongiform encephalopathies, and spinocerebellar ataxia. Strategies for treatment and therapy are also conferred with respect to impairing, inhibiting, or reversing protein misfolding.

Evaluation of Metabolic and Synaptic Dysfunction Hypotheses of Alzheimer's Disease (AD): A Meta-Analysis of CSF Markers.

BACKGROUND: Alzheimer's disease (AD) is currently incurable and a majority of investigational drugs have failed clinical trials. One explanation for this failure may be the invalidity of hypotheses focusing on amyloid to explain AD pathogenesis. Recently, hypotheses which are centered on synaptic and metabolic dysfunction are increasingly implicated in AD. OBJECTIVE: Evaluate AD hypotheses by comparing neurotransmitter and metabolite marker concentrations in normal versus AD CSF. METHODS: Meta-analysis allows for statistical comparison of pooled, existing cerebrospinal fluid (CSF) marker data extracted from multiple publications, to obtain a more reliable estimate of concentrations. This method also provides a unique opportunity to rapidly validate AD hypotheses using the resulting CSF concentration data. Hubmed, Pubmed and Google Scholar were comprehensively searched for published English articles, without date restrictions, for the keywords "AD", "CSF", and "human" plus markers selected for synaptic and metabolic pathways. Synaptic markers were acetylcholine, gamma-aminobutyric acid (GABA), glutamine, and glycine. Metabolic markers were glutathione, glucose, lactate, pyruvate, and 8 other amino acids. Only studies that measured markers in AD and controls (Ctl), provided means, standard errors/deviation, and subject numbers were included. Data were extracted by six authors and reviewed by two others for accuracy. Data were pooled using ratio of means (RoM of AD/Ctl) and random effects meta-analysis using Cochrane Collaboration's Review Manager software. RESULTS: Of the 435 identified publications, after exclusion and removal of duplicates, 35 articles were included comprising a total of 605 AD patients and 585 controls. The following markers of synaptic and metabolic pathways were significantly changed in AD/controls: acetylcholine (RoM 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.74, 0.58-0.94, p<0.01), pyruvate (0.48, 0.24-0.94, p=0.03), glutathione (1.11, 1.01- 1.21, p=0.03), alanine (1.10, 0.98-1.23, p=0.09), and lower levels of significance for lactate (1.2, 1.00-1.47, p=0.05). Of note, CSF glucose and glutamate levels in AD were not significantly different than that of the controls. CONCLUSION: This study provides proof of concept for the use of meta-analysis validation of AD hypotheses, specifically via robust evidence for the cholinergic hypothesis of AD. Our data disagree with the other synaptic hypotheses of glutamate excitotoxicity and GABAergic resistance to neurodegeneration, given observed unchanged glutamate levels and decreased GABA levels. With regards to metabolic hypotheses, the data supported upregulation of anaerobic glycolysis, pentose phosphate pathway (glutathione), and anaplerosis of the tricarboxylic acid cycle using glutamate. Future applications of meta-analysis indicate the possibility of further in silico evaluation and generation of novel hypotheses in the AD field.

Amyloid Plaque-Associated Oxidative Degradation of Uniformly Radiolabeled Arachidonic Acid.

Oxidative stress is a frequently observed feature of Alzheimer's disease, but its pathological significance is not understood. To explore the relationship between oxidative stress and amyloid plaques, uniformly radiolabeled arachidonate was introduced into transgenic mouse models of Alzheimer's disease via intracerebroventricular injection. Uniform labeling with carbon-14 is used here for the first time, and made possible meaningful quantification of arachidonate oxidative degradation products. The injected arachidonate entered a fatty acid pool that was subject to oxidative degradation in both transgenic and wild-type animals. However, the extent of its degradation was markedly greater in the hippocampus of transgenic animals where amyloid plaques were abundant. In human Alzheimer's brain, plaque-associated proteins were post-translationally modified by hydroxynonenal, a well-known oxidative degradation product of arachidonate. These results suggest that several recurring themes in Alzheimer's pathogenesis, amyloid ß proteins, transition metal ions, oxidative stress, and apolipoprotein isoforms, may be involved in a common mechanism that has the potential to explain both neuronal loss and fibril formation in this disease.

Small molecules and Alzheimer's disease: misfolding, metabolism and imaging.

Small molecule interactions with amyloid proteins have had a huge impact in Alzheimer's disease (AD), especially in three specific areas: amyloid folding, metabolism and brain imaging. Amyloid plaque amelioration or prevention have, until recently, driven drug development, and only a few drugs have been advanced for use in AD. Amyloid proteins undergo misfolding and oligomerization via intermediates, eventually forming protease resistant amyloid fibrils. These fibrils accumulate to form the hallmark amyloid plaques and tangles of AD. Amyloid binding compounds can be grouped into three categories, those that: i) prevent or reverse misfolding, ii) halt misfolding or trap intermediates, and iii) accelerate the formation of stable and inert amyloid fibrils. Such compounds include hydralazine, glycosaminoglycans, curcumin, beta sheet breakers, catecholamines, and ATP. The versatility of amyloid binding compounds suggests that the amyloid structure may serve as a scaffold for the future development of sensors to detect such compounds. Metabolic dysfunction is one of the earliest pathological features of AD. In fact, AD is often referred to as type 3 diabetes due to the presence of insulin resistance in the brain. A recent study indicates that altering metabolism improves cognitive function. While metabolic reprogramming is one therapeutic avenue for AD, it is more widely used in some cancer therapies. FDA approved drugs such as metformin, dichloroacetic acid (DCA), and methylene blue can alter metabolism. These drugs can therefore be potentially applied in alleviating metabolic dysfunction in AD. Brain imaging has made enormous strides over the past decade, offering a new window to the mind. Recently, there has been remarkable development of compounds that have the ability to image both types of pathological amyloids: tau and amyloid beta. We have focused on the low cost, simple to use, near infrared fluorescence (NIRF) imaging probes for amyloid beta (Aß), with specific attention on recent developments to further improve contrast, specificity, and sensitivity. With advances in imaging technologies, such fluorescent imaging probes will open new diagnostic avenues.

Islet amyloid polypeptide (IAPP): a second amyloid in Alzheimer's disease.

Amyloid formation is the pathological hallmark of type 2 diabetes (T2D) and Alzheimer's disease (AD). These diseases are marked by extracellular amyloid deposits of islet amyloid polypeptide (IAPP) in the pancreas and amyloid ß (Aß) in the brain. Since IAPP may enter the brain and disparate amyloids can cross-seed each other to augment amyloid formation, we hypothesized that pancreatic derived IAPP may enter the brain to augment misfolding of Aß in AD. The corollaries for validity of this hypothesis are that IAPP [1] enters the brain, [2] augments Aß misfolding, [3] associates with Aß plaques, and most importantly [4] plasma levels correlate with AD diagnosis. We demonstrate the first 3 corollaries that: (1) IAPP is present in the brain in human cerebrospinal fluid (CSF), (2) synthetic IAPP promoted oligomerization of Aß in vitro, and (3) endogenous IAPP localized to Aß oligomers and plaques. For the 4th corollary, we did not observe correlation of peripheral IAPP levels with AD pathology in either an African American cohort or AD transgenic mice. In the African American cohort, with increased risk for both T2D and AD, peripheral IAPP levels were not significantly different in samples with no disease, T2D, AD, or both T2D and AD. In the Tg2576 AD mouse model, IAPP plasma levels were not significantly elevated at an age where the mice exhibit the glucose intolerance of pre-diabetes. Based on this negative data, it appears unlikely that peripheral IAPP cross-seeds or "infects" Aß pathology in AD brain. However, we provide novel and additional data which demonstrate that IAPP protein is present in astrocytes in murine brain and secreted from primary cultured astrocytes. This preliminary report suggests a potential and novel association between brain derived IAPP and AD, however whether astrocytic derived IAPP cross-seeds Aß in the brain requires further research.

Adenosine triphosphate (ATP) reduces amyloid-ß protein misfolding in vitro.

Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-ß protein (Aß) levels. Since levels of ATP decrease over the course of the disease and Aß is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aß (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aß mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aß42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aß fibrils in solution. Experimentally, both ATP and ADP reduced Aß misfolding at physiological intracellular concentrations, with thresholds at ~500 µM and 1 mM respectively. This inhibition of Aß misfolding is specific; requiring Tyr10 of Aß and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aß and reduction of Aß misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aß mirrors lowered extracellular ATP levels. Last, the findings suggest that Aß conformation change may be a sensor of metabolic dysfunction.

Ruthenium red colorimetric and birefringent staining of amyloid-ß aggregates in vitro and in Tg2576 mice.

Alzheimer's disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-ß (Aß) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aß fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aß aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aß deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aß plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aß plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aß fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aß fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aß aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Probing and trapping a sensitive conformation: amyloid-ß fibrils, oligomers, and dimers.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with pathological misfolding of amyloid-ß protein (Aß). The recent interest in Aß misfolding intermediates necessitates development of novel detection methods and ability to trap these intermediates. We speculated that two regions of Aß may allow for detection of specific Aß species: the N-terminal and 22-35, both likely important in oligomer interaction and formation. We determined via epitomics, proteomic assays, and electron microscopy that the Aß(42) species (wild type, ΔE22, and MetOx) predominantly formed fibrils, oligomers, or dimers, respectively. The 2H4 antibody to the N-terminal of Aß, in the presence of 2% SDS, primarily detected fibrils, and an antibody to the 22-35 region detected low molecular weight Aß species. Simulated molecular modeling provided insight into these SDS-induced structural changes. We next determined if these methods could be used to screen anti-Aß drugs as well as identify compounds that trap Aß in various conformations. Immunoblot assays determined that taurine, homotaurine (Tramiprosate), myoinositol, methylene blue, and curcumin did not prevent Aß aggregation. However, calmidazolium chloride trapped Aß at oligomers, and berberine reduced oligomer formation. Finally, pretreatment of AD brain tissues with SDS enhanced 2H4 antibody immunostaining of fibrillar Aß. Thus we identified and characterized Aßs that adopt specific predominant conformations (modified Aß or via interactions with compounds), developed a novel assay for aggregated Aß, and applied it to drug screening and immunohistochemistry. In summary, our novel approach facilitates drug screening, increases the probability of success of antibody therapeutics, and improves antibody-based detection and identification of different conformations of Aß.

Amyloid-ß metabolite sensing: biochemical linking of glycation modification and misfolding.

Glycation is the reaction of a reducing sugar with proteins and lipids, resulting in myriads of glycation products, protein modifications, cross-linking, and oxidative stress. Glycation reactions are also elevated during metabolic dysfunction such as in Alzheimer's disease (AD) and Down's syndrome. These reactions increase the misfolding of the proteins such as tau and amyloid-ß (Aß), and colocalize with amyloid plaques in AD. Thus, glycation links metabolic dysfunction and AD and may have a causal role in AD. We have characterized the reaction of Aß with reactive metabolites that are elevated during metabolic dysfunction. One metabolite, glyceraldehyde-3-phosphate, is a normal product of glycolysis, while the others are associated with pathology. Our data demonstrates that lipid oxidation products malondialdehyde, hydroxynonenal, and glycation metabolites (methylglyoxal, glyceraldehyde, and glyceraldehyde-3-phosphate) modify Aß42 and increase misfolding. Using mass spectrometry, modifications primarily occurred at the amino terminus. However, the metabolite methylglyoxal modified Arg5 in the Aß sequence. 4-Hydroxy-2-nonenal modifications were similar to our previous publication. To place such modifications into an in vivo context, we stained AD brain tissue for endproducts of glycation, or advanced glycation endproducts (AGE). Similar to previous findings, AGE colocalized with amyloid plaques. In summary, we demonstrate the glycation of Aß and plaques by metabolic compounds. Thus, glycation potentially links metabolic dysfunction and Aß misfolding in AD, and may contribute to the AD pathogenesis. This association can further be expanded to raise the tantalizing concept that such Aß modification and misfolding can function as a sensor of metabolic dysfunction.

Amyloids as Sensors and Protectors (ASAP) hypothesis.

This paper propounds the Amyloids as Sensors and Protectors (ASAP) hypothesis. In this novel hypothesis, we provide evidence that amyloids are capable of sensing dysfunction, and after misfolding, initiate protective cellular responses. Amyloid proteins are initially protective, but chronic stress and overstimulation of the amyloid sensor leads to pathology. This proposed ASAP hypothesis has two sequential stages: (i) sensing, and then (ii) protection. Sensing involves a conformational change of amyloids in response to the cellular environment. The protection aspect translates conformational change into a cellular response via several mechanisms. The most obvious mechanism is that protein misfolding triggers the protective unfolded protein response, and thus downregulates protein translation and increases chaperone proteins. Other documented responses include metabolic pathways and microRNAs. This ASAP hypothesis has precedence, as amyloid sensors exist (evidenced by CPEB and Sup35), and both prion and amyloid-ß sensing redox stress and metals. Our hypothesis expands on previous observations to link sensing with inciting protective cellular response. Furthermore, we substantiate the ASAP hypothesis with previously published evidence from several amyloid diseases. This novel hypothesis links disparate findings in amyloid diseases: metabolic dysfunction, unfolding protein response/chaperones, modification of amyloids, and nutrient or caloric sensing. While this hypothesis can be applied to Alzheimer's disease, it goes beyond the Alzheimer's context. Thus all amyloid proteins can potentially act as sensors of misfolding-causing stress. Finally, this hypothesis will allow for the sensor mechanism and metabolic dysfunction to serve as biomarkers of the diseases as well as therapeutic targets early in disease pathology.

Vascular and metabolic dysfunction in Alzheimer's disease: a review.

Alzheimer's disease (AD) is thought to start years or decades prior to clinical diagnosis. Overt pathology such as protein misfolding and plaque formation occur at later stages, and factors other than amyloid misfolding contribute to the initiation of the disease. Vascular and metabolic dysfunctions are excellent candidates, as they are well-known features of AD that precede pathology or clinical dementia. While the general notion that vascular and metabolic dysfunctions contribute to the etiology of AD is becoming accepted, recent research suggests novel mechanisms by which these/such processes could possibly contribute to AD pathogenesis. Vascular dysfunction includes reduced cerebrovascular flow and cerebral amyloid angiopathy. Indeed, there appears to be an interaction between amyloid ß (Aß) and vascular pathology, where Aß production and vascular pathology both contribute to and are affected by oxidative stress. One major player in the vascular pathology is NAD(P)H oxidase, which generates vasoactive superoxide. Metabolic dysfunction has only recently regained popularity in relation to its potential role in AD. The role of metabolic dysfunction in AD is supported by the increased epidemiological risk of AD associated with several metabolic diseases such as diabetes, dyslipidemia and hypertension, in which there is elevated oxidative damage and insulin resistance. Metabolic dysfunction is further implicated in AD as pharmacological inhibition of metabolism exacerbates pathology, and several metabolic enzymes of the glycolytic, tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathways are damaged in AD. Recent studies have highlighted the role of insulin resistance, in contributing to AD. Thus, vascular and metabolic dysfunctions are key components in the AD pathology throughout the course of disease. The common denominator between vascular and metabolic dysfunction emerging from this review appears to be oxidative stress and Aß. This review also provides a framework for evaluation of current and future therapeutics for AD.

Oxidative stress and cell membranes in the pathogenesis of Alzheimer's disease.

Amyloid ß proteins and oxidative stress are believed to have central roles in the development of Alzheimer's disease. Lipid membranes are among the most vulnerable cellular components to oxidative stress, and membranes in susceptible regions of the brain are compositionally distinct from those in other tissues. This review considers the evidence that membranes are either a source of neurotoxic lipid oxidation products or the target of pathogenic processes involving amyloid ß proteins that cause permeability changes or ion channel formation. Progress toward a comprehensive theory of Alzheimer's disease pathogenesis is discussed in which lipid membranes assume both roles and promote the conversion of monomeric amyloid ß proteins into fibrils, the pathognomonic histopathological lesion of the disease.

Hydralazine modifies Aß fibril formation and prevents modification by lipids in vitro.

Lipid oxidative damage and amyloid ß (Aß) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aß misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aß production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aß misfolding and (2) Aß protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aß aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aß, and Aß was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aß and also did not prevent Aß aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aß formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aß misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.

Lipid oxidation and modification of amyloid-ß (Aß) in vitro and in vivo.

Oxidative damage and amyloid-ß (Aß) protein misfolding are prominent features of Alzheimer's disease (AD). In vitro studies indicated a direct linkage between these two features, where lipid oxidation products augmented Aß misfolding. We tested this linkage further, mimicking specific conditions present in amyloid plaques. In vitro lipid oxidation and lipid modification of Aß were thus performed with elevated levels of copper or physiological levels of calcium. These in vitro experiments were then confirmed by in vivo immunohistochemical and chemical tagging of oxidative damage in brains from the PSAPP mouse model of AD. Our in vitro findings indicate that: 1) high levels of copper prevent lipid oxidation; 2) physiological concentrations of calcium reduce 4 hydroxy-2-nonenal (HNE) modification of Aß; and 3) anti-Aß and HNE antibody epitopes are differentially masked. In vivo we demonstrated increased lipid oxidation around plaques but 4) a lack of immunological colocalization of HNE-adducts with Aß. Thus, the lack of colocalization of Aß and HNE-adduct immunostaining is most likely due to a combination of metals inhibiting HNE modification of Aß, quenching lipid oxidation and a masking of HNE-Aß histopathology. However, other forms of oxidative damage colocalize with Aß in plaques, as demonstrated using a chemical method for identifying oxidative damage. Additionally, these findings suggest that HNE modification of Aß may affect therapeutic antibodies targeting the amino terminal of Aß and that metals effect on lipid oxidation and lipid modification of Aß could raise concerns on emerging anti-AD treatments with metal chelators.

TNFalpha-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish S-adenosylhomocysteine hydrolase.

Hepatic steatosis and liver degeneration are prominent features of the zebrafish ducttrip (dtp) mutant phenotype. Positional cloning identified a causative mutation in the gene encoding S-adenosylhomocysteine hydrolase (Ahcy). Reduced Ahcy activity in dtp mutants led to elevated levels of S-adenosylhomocysteine (SAH) and, to a lesser degree, of its metabolic precursor S-adenosylmethionine (SAM). Elevated SAH in dtp larvae was associated with mitochondrial defects and increased expression of tnfa and pparg, an ortholog of the mammalian lipogenic gene. Antisense knockdown of tnfa rescued hepatic steatosis and liver degeneration in dtp larvae, whereas the overexpression of tnfa and the hepatic phenotype were unchanged in dtp larvae reared under germ-free conditions. These data identify an essential role for tnfa in the mutant phenotype and suggest a direct link between SAH-induced methylation defects and TNF expression in human liver disorders associated with elevated TNFalpha. Although heterozygous dtp larvae had no discernible phenotype, hepatic steatosis was present in heterozygous adult dtp fish and in wild-type adult fish treated with an Ahcy inhibitor. These data argue that AHCY polymorphisms and AHCY inhibitors, which have shown promise in treating autoimmunity and other disorders, may be a risk factor for steatosis, particularly in patients with diabetes, obesity and liver disorders such as hepatitis C infection. Supporting this idea, hepatic injury and steatosis have been noted in patients with recently discovered AHCY mutations.

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product.

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid beta proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain omega-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Abeta proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid beta proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.