A surrogate marker for very early-stage tau pathology is detectable by molecular magnetic resonance imaging.

The abnormal phosphorylation of tau is a necessary precursor to the formation of tau fibrils, a marker of Alzheimer's disease. We hypothesize that hyperphosphorylative conditions may result in unique cell surface markers. We identify and demonstrate the utility of such surrogate markers to identify the hyperphosphorylative state. Methods: Cell SELEX was used to identify novel thioaptamers specifically binding hyperphosphorylative cells. Cell surface vimentin was identified as a potential binding target of the aptamer. Novel molecular magnetic resonance imaging (M-MRI) probes using these aptamers and a small molecule ligand to vimentin were used for in vivo detection of this pre-pathological state. Results: In a mouse model of pathological tau, we demonstrated in vivo visualization of the hyperphosphorylative state by M-MRI, enabling the identification at a pre-pathological stage of mice that develop frank tau pathology several months later. In vivo visualization of the hyperphosphorylative state by M-MRI was further validated in a second mouse model (APP/PS1) of Alzheimer's disease again identifying the mutants at a pre-pathological stage. Conclusions: M-MRI of the hyperphosphorylative state identifies future tau pathology and could enable extremely early-stage diagnosis of Alzheimer's disease, at a pre-patholgical stage.

Prostacyclin Promotes Degenerative Pathology in a Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is the most common form of dementia in aged populations. A substantial amount of data demonstrates that chronic neuroinflammation can accelerate neurodegenerative pathologies. In AD, chronic neuroinflammation results in the upregulation of cyclooxygenase and increased production of prostaglandin H2, a precursor for many vasoactive prostanoids. While it is well-established that many prostaglandins can modulate the progression of neurodegenerative disorders, the role of prostacyclin (PGI2) in the brain is poorly understood. We have conducted studies to assess the effect of elevated prostacyclin biosynthesis in a mouse model of AD. Upregulated prostacyclin expression significantly worsened multiple measures associated with amyloid-ß (Aß) disease pathologies. Mice overexpressing both Aß and PGI2 exhibited impaired learning and memory and increased anxiety-like behavior compared with non-transgenic and PGI2 control mice. PGI2 overexpression accelerated the development of Aß accumulation in the brain and selectively increased the production of soluble Aß42. PGI2 damaged the microvasculature through alterations in vascular length and branching; Aß expression exacerbated these effects. Our findings demonstrate that chronic prostacyclin expression plays a novel and unexpected role that hastens the development of the AD phenotype.

1-Indanone and 1,3-indandione Derivatives as Ligands for Misfolded α-Synuclein Aggregates.

The development of imaging agents for in vivo detection of alpha-synuclein (α-syn) pathologies faces several challenges. A major gap in the field is the lack of diverse molecular scaffolds with high affinity and selectivity to α-syn fibrils for in vitro screening assays. Better in vitro scaffolds can instruct the discovery of better in vivo agents. We report the rational design, synthesis, and in vitro evaluation of a series of novel 1-indanone and 1,3-indandione derivatives from a Structure-Activity Relationship (SAR) study centered on some existing α-syn fibril binding ligands. Our results from fibril saturation binding experiments show that two of the lead candidates compounds 8 and 32 bind α-syn fibrils with binding constants (Kd ) of 9.0 and 18.8 nM, respectively, and selectivity of greater than 10× for α-syn fibrils compared with amyloid-ß (Aß) and tau fibrils. Our results demonstrate that the lead ligands avidly label all forms of α-syn on PD brain tissue sections, but only the dense core of senile plaques in AD brain tissue, respectively. These results are corroborated by ligand-antibody colocalization data from Syn211, which shows immunoreactivity toward all forms of α-syn aggregates, and Syn303, which displays preferential reactivity toward mature Lewy pathology. Our results reveal that 1-indanone derivatives have desirable properties for the biological evaluation of α-synucleinopathies.

Exercise training ameliorates cerebrovascular dysfunction in a murine model of Alzheimer's disease: role of the P2Y2 receptor and endoplasmic reticulum stress.

Cerebrovascular dysfunction is a critical risk factor for the pathogenesis of Alzheimer's disease (AD). The purinergic P2Y2 receptor and endoplasmic reticulum (ER) stress are tightly associated with vascular dysfunction and the pathogenesis of AD. However, the protective effects of exercise training on P2Y2 receptor- and ER stress-associated cerebrovascular dysfunction in AD are mostly unknown. Control (C57BL/6, CON) and AD (APP/PS1dE9, AD) mice underwent treadmill exercise training (EX). 2-MeS-ATP-induced dose-dependent vasoreactivity was determined by using a pressurized posterior cerebral artery (PCA) from 10-12-mo-old mice. Human brain microvascular endothelial cells (HBMECs) were exposed to laminar shear stress (LSS) at 20 dyn/cm2 for 30 min, 2 h, and 24 h. The expression of P2Y2 receptors, endothelial nitric oxide synthase (eNOS), and ER stress signaling were quantified by Western blot analysis. Notably, exercise converted ATP-induced vasoconstriction in the PCA from AD mice to vasodilation in AD+EX mice to a degree commensurate to the vascular reactivity observed in CON mice. Exercise reduced the expression of amyloid peptide precursor (APP) and increased the P2Y2 receptor and Akt/eNOS expression in AD mice brain. Mechanistically, LSS increased the expression of both P2Y2 receptor and eNOS protein in HBMECs, but these increases were blunted by a P2Y2 receptor antagonist in HBMECs. Exercise also reduced the expression of aberrant ER stress markers p-IRE1, p/t-eIF2α, and CHOP, as well as Bax/Bcl-2, in AD mice brain. Collectively, our results demonstrate for the first time that exercise mitigates cerebrovascular dysfunction in AD through modulating P2Y2 receptor- and ER stress-dependent endothelial dysfunction.NEW & NOTEWORTHY A limited study has investigated whether exercise training can improve cerebrovascular function in Alzheimer's disease. The novel findings of the study are that exercise training improves cerebrovascular dysfunction through enhancing P2Y2 receptor-mediated eNOS signaling and reducing ER stress-associated pathways in AD. These data suggest that exercise training, which regulates P2Y2 receptor and ER stress in AD brain, is a potential therapeutic strategy for Alzheimer's disease.

Multiplex protein-specific microscopy with ultraviolet surface excitation.

Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as microscopy with ultraviolet surface excitation (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.

Three-Dimensional Microscopy by Milling with Ultraviolet Excitation.

Analysis of three-dimensional biological samples is critical to understanding tissue function and the mechanisms of disease. Many chronic conditions, like neurodegenerative diseases and cancers, correlate with complex tissue changes that are difficult to explore using two-dimensional histology. While three-dimensional techniques such as confocal and light-sheet microscopy are well-established, they are time consuming, require expensive instrumentation, and are limited to small tissue volumes. Three-dimensional microscopy is therefore impractical in clinical settings and often limited to core facilities at major research institutions. There would be a tremendous benefit to providing clinicians and researchers with the ability to routinely image large three-dimensional tissue volumes at cellular resolution. In this paper, we propose an imaging methodology that enables fast and inexpensive three-dimensional imaging that can be readily integrated into current histology pipelines. This method relies on block-face imaging of paraffin-embedded samples using deep-ultraviolet excitation. The imaged surface is then ablated to reveal the next tissue section for imaging. The final image stack is then aligned and reconstructed to provide tissue models that exceed the depth and resolution achievable with modern three-dimensional imaging systems.

Robust Tracing and Visualization of Heterogeneous Microvascular Networks.

Advances in high-throughput imaging allow researchers to collect three-dimensional images of whole organ microvascular networks. These extremely large images contain networks that are highly complex, time consuming to segment, and difficult to visualize. In this paper, we present a framework for segmenting and visualizing vascular networks from terabyte-sized three-dimensional images collected using high-throughput microscopy. While these images require terabytes of storage, the volume devoted to the fiber network is ≈ 4 percent of the total volume size. While the networks themselves are sparse, they are tremendously complex, interconnected, and vary widely in diameter. We describe a parallel GPU-based predictor-corrector method for tracing filaments that is robust to noise and sampling errors common in these data sets. We also propose a number of visualization techniques designed to convey the complex statistical descriptions of fibers across large tissue sections-including commonly studied microvascular characteristics, such as orientation and volume.

7B2 chaperone knockout in APP model mice results in reduced plaque burden.

Impairment of neuronal proteostasis is a hallmark of Alzheimer's and other neurodegenerative diseases. However, the underlying molecular mechanisms leading to pathogenic protein aggregation, and the role of secretory chaperone proteins in this process, are poorly understood. We have previously shown that the neural-and endocrine-specific secretory chaperone 7B2 potently blocks in vitro fibrillation of Aß42. To determine whether 7B2 can function as a chaperone in vivo, we measured plaque formation and performed behavioral assays in 7B2-deficient mice in an hAPPswe/PS1dE9 Alzheimer's model mouse background. Surprisingly, immunocytochemical analysis of cortical levels of thioflavin S- and Aß-reactive plaques showed that APP mice with a partial or complete lack of 7B2 expression exhibited a significantly lower number and burden of thioflavin S-reactive, as well as Aß-immunoreactive, plaques. However, 7B2 knockout did not affect total brain levels of either soluble or insoluble Aß. While hAPP model mice performed poorly in the Morris water maze, their brain 7B2 levels did not impact performance. Since 7B2 loss reduced amyloid plaque burden, we conclude that brain 7B2 can impact Aß disposition in a manner that facilitates plaque formation. These results are reminiscent of prior findings in hAPP model mice lacking the ubiquitous secretory chaperone clusterin.

Plasmonic nanoparticle-based expansion microscopy with surface-enhanced Raman and dark-field spectroscopic imaging.

Fluorescence-based expansion microscopy (ExM) is a new technique which can yield nanoscale resolution of biological specimen on a conventional fluorescence microscope through physical sample expansion up to 20 times its original dimensions while preserving structural information. It however inherits known issues of fluorescence microscopy such as photostability and multiplexing capabilities, as well as an ExM-specific issue in signal intensity reduction due to a dilution effect after expansion. To address these issues, we propose using antigen-targeting plasmonic nanoparticle labels which can be imaged using surface-enhanced Raman scattering spectroscopy (SERS) and dark-field spectroscopy. We demonstrate that the nanoparticles enable multimodal imaging: bright-field, dark-field and SERS, with excellent photostability, contrast enhancement and brightness.

A Novel Liposomal Nanoparticle for the Imaging of Amyloid Plaque by Magnetic Resonance Imaging.

Amyloid binding molecules with greater hydrophilicity than existing ligands were synthesized. The lead candidate ET6-21 bound amyloid fibrils, and amyloid deposits in dog brain and human brain tissue ex vivo. The ligand was used to prepare novel amyloid-targeted liposomal nanoparticles. The preparation was tested in the Tg2576 and TetO/APP mouse models of amyloid deposition. Gd chelates and Indocyanine green were included in the particles for visualization by MRI and near-infrared microscopy. Upon intravenous injection, the particles successfully traversed the blood-brain barrier in these mice, and bound to the plaques. Magnetic resonance imaging (T1-MRI) conducted 4 days after injection demonstrated elevated signal in the brains of mice with amyloid plaques present. No signal was observed in amyloid-negative mice, or in amyloid-positive mice injected with an untargeted version of the same agent. The MRI results were confirmed by immunohistochemical and fluorescent microscopic examination of mouse brain sections, showing colocalization of the fluorescent tags and amyloid deposits.

Characterization of Polymyxin B Biodistribution and Disposition in an Animal Model.

Despite dose-limiting nephrotoxicity concerns, polymyxin B has resurged as the treatment of last resort for multidrug-resistant Gram-negative bacterial infections. However, the pharmacokinetic, pharmacodynamic, and nephrotoxic properties of polymyxin B still are not thoroughly understood. The objective of this study was to provide additional insights into the overall biodistribution and disposition of polymyxin B in an animal model. Sprague-Dawley rats were dosed with intravenous polymyxin B (3 mg/kg of body weight). Drug concentrations in the serum, urine, bile, and tissue (brain, heart, lungs, liver, spleen, kidneys, and skeletal muscle) samples over time were assayed by a validated methodology. Among all the organs evaluated, polymyxin B distribution was highest in the kidneys. The mean renal tissue/serum polymyxin B concentration ratios were 7.45 (95% confidence interval [CI], 4.63 to 10.27) at 3 h and 19.62 (95% CI, 5.02 to 34.22) at 6 h postdose. Intrarenal drug distribution was examined by immunostaining. Using a ratiometric analysis, proximal tubular cells showed the highest accumulation of polymyxin B (Mander's overlap coefficient, 0.998) among all cell types evaluated. Less than 5% of the administered dose was recovered in urine over 48 h, but all 4 major polymyxin B components were detected in the bile over 4 h. These findings corroborate previous results that polymyxin B is highly accumulated in the kidneys, but the elimination likely is via a nonrenal route. Biliary excretion could be one of the routes of polymyxin B elimination, and this should be further explored. The elucidation of mechanism(s) of drug uptake in proximal tubular cells is ongoing.

Formaldehyde scavengers function as novel antigen retrieval agents.

Antigen retrieval agents improve the detection of formaldehyde-fixed proteins, but how they work is not well understood. We demonstrate that formaldehyde scavenging represents a key characteristic associated with effective antigen retrieval; under controlled temperature and pH conditions, scavenging improves the typical antigen retrieval process through reversal of formaldehyde-protein adduct formation. This approach provides a rational framework for the identification and development of more effective antigen retrieval agents.

Long-term treadmill exercise attenuates tau pathology in P301S tau transgenic mice.

BACKGROUND: Recent epidemiological evidence suggests that modifying lifestyle by increasing physical activity could be a non-pharmacological approach to improving symptoms and slowing disease progression in Alzheimer's disease and other tauopathies. Previous studies have shown that exercise reduces tau hyperphosphorylation, however, it is not known whether exercise reduces the accumulation of soluble or insoluble tau aggregates and neurofibrillary tangles, which are both neuropathological hallmarks of neurodegenerative tauopathy. In this study, 7-month old P301S tau transgenic mice were subjected to 12-weeks of forced treadmill exercise and evaluated for effects on motor function and tau pathology at 10 months of age. RESULTS: Exercise improved general locomotor and exploratory activity and resulted in significant reductions in full-length and hyperphosphorylated tau in the spinal cord and hippocampus as well as a reduction in sarkosyl-insoluble AT8-tau in the spinal cord. Exercise did not attenuate significant neuron loss in the hippocampus or cortex. Key proteins involved in autophagy-microtubule-associated protein 1A/1B light chain 3 and p62/sequestosome 1 -were also measured to assess whether autophagy is implicated in the exercised-induced reduction of aggregated tau protein. There were no significant effects of forced treadmill exercise on autophagy protein levels in P301S mice. CONCLUSIONS: Our results suggest that forced treadmill exercise differently affects the brain and spinal cord of aged P301S tau mice, with greater benefits observed in the spinal cord versus the brain. Our work adds to the growing body of evidence that exercise is beneficial in tauopathy, however these benefits may be more limited at later stages of disease.

Regular exercise prevents non-cognitive disturbances in a rat model of Alzheimer's disease.

Previously, we reported that in a rat model of sporadic Alzheimer's disease (AD) generated by exogenous administration of Aß1₋42 (250 pmol/d for 2 wk) via mini-osmotic pump, the animals exhibited learning and memory impairment, which could be attributed to the deleterious alterations in the levels of cognition-related signalling molecules. We showed that 4 wk of treadmill exercise totally prevented these impairments. Here, we evaluated the effect of exercise on non-cognitive function and basal synaptic transmission in the Cornu Ammonis 1 (CA1) area using the same AD model. Our results indicated that the anxiety behaviour of Aß-treated rats was prevented by 4 wk of treadmill exercise. Exercised/Aß-infused rats spent a longer time in the centre area of the open field (OF), elevated plus maze (EPM) paradigms and the light area of the light-dark (LD) box, which were similar to those of control and exercise rats. Furthermore, under basal conditions the aberrant up-regulation of calcineurin (PP2B) and reduction of phosphorylated Ca²âº/calmodulin dependent protein kinase II (p-CaMKII) levels induced by AD-like pathology were normalised by the exercise regimen. We conclude that regular exercise may exert beneficial effects on both cognitive and non-cognitive functions in this AD model.

Elevated prostacyclin biosynthesis in mice impacts memory and anxiety-like behavior.

Prostacyclin is an endogenous lipid metabolite with properties of vasodilation and anti-platelet aggregation. While the effects of prostacyclin on the vascular protection have been well-documented, the role of this eicosanoid in the central nervous system has not been extensively studied. Recently, a transgenic mouse containing a hybrid enzyme, of cyclooxygenase-1 linked to prostacyclin synthase, was developed that produces elevated levels of prostacyclin in vivo. The goal of this study was to investigate whether increased prostacyclin biosynthesis could affect behavioral phenotypes in mice. Our results uncovered that elevated levels of prostacyclin broadly affect both cognitive and non-cognitive behaviors, including decreased anxiety-like behavior and improved learning in the fear-conditioning memory test. This study demonstrates that prostacyclin plays an important, but previously unrecognized, role in central nervous system function and behavior.

A novel function for proSAAS as an amyloid anti-aggregant in Alzheimer's disease.

Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by an abnormal aggregation of misfolded beta-sheet rich proteins such as ß-amyloid (Aß). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti-aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12-month-old APdE9 mice, and in the cortex of a human AD-affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co-immunoprecipitated with Aß immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aß(1-42) at molar ratios of 1 : 10, and this anti-aggregation effect was dose dependent. Structure-function studies showed that residues 97-180 were sufficient for the anti-aggregation function against Aß. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral-mediated proSAAS over-expression, blocked the neurocytotoxic effect of Aß(1-42) in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimer's disease pathology.

Chronic treatment with DCPCX, an adenosine A(1) antagonist, worsens long-term memory.

Alzheimer's disease is characterized by progressive cognitive disturbances and neurotransmitter dysfunction. Previous studies targeting the adrenergic A1 pathway suggest that this plays a role in cognitive impairment in Alzheimer's disease. Previous studies have reported that acute treatment with A1 antagonists appears to improve behavioral deficits in rodent models of memory and behavioral impairment. In this study, we addressed whether the chronic administration of 8-cyclopentyl-1,3-dipropylxanthine, a potent and selective adenosine A1 antagonist, could reverse the memory deficits found in aged APPswe/PS1dE9 mice. Chronic treatment did not improve memory in the APPswe/PS1dE9 mouse model and resulted in reduced exploratory behavior, suggestive of reduced anxiety, and a worsening of long-term memory in nontransgenic mice. These results have important implications for understanding the mechanisms of A1 receptor modulation as a target in Alzheimer's disease therapy.

Treadmill exercise prevents learning and memory impairment in Alzheimer's disease-like pathology.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by progressive memory loss. In contrast, accumulating evidence suggests a neuroprotective role of regular exercise in aging associated memory impairment. In this study, we investigated the ability of regular exercise to prevent impairments of short-term memory (STM) and early long-term potentiation (E-LTP) in area CA1 of the hippocampus in a rat model of AD (i.c.v. infusion of 250 pmol/day Aß1-42 peptides). We utilized behavioral assessment, in vivo electrophysiological recording, and immunoblotting in 4 groups of adult Wistar rats: control, treadmill exercise (Ex), ß-amyloid-infused (Aß), and amyloid-infused/treadmill exercised (Ex/Aß). Our findings indicated that Aß rats made significantly more errors in the radial arm water maze (RAWM) compared to all other groups and exhibited suppressed E-LTP in area CA1, which correlated with deleterious alterations in the levels of memory and E-LTP-related signaling molecules including calcineurin (PP2B), brain derivedneurotrophic factor (BDNF) and phosphorylated CaMKII (p-CaMKII). Compared to controls, Ex and Ex/Aß rats showed a similar behavioral performance and a normal E-LTP with no detrimental changes in the levels of PP2B, BDNF, and p- CaMKII. We conclude that treadmill exercise maybe able to prevent cognitive impairment associated with AD pathology.