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.

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.

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.