Gram-negative bacterial molecules associate with Alzheimer disease pathology.

ObjectiveWe determined whether Gram-negative bacterial molecules are associated with Alzheimer disease (AD) neuropathology given that previous studies demonstrate Gram-negative Escherichia coli bacteria can form extracellular amyloid and Gram-negative bacteria have been reported as the predominant bacteria found in normal human brains.MethodsBrain samples from gray and white matter were studied from patients with AD (n = 24) and age-matched controls (n = 18). Lipopolysaccharide (LPS) and E coli K99 pili protein were evaluated by Western blots and immunocytochemistry. Human brain samples were assessed for E coli DNA followed by DNA sequencing.ResultsLPS and E coli K99 were detected immunocytochemically in brain parenchyma and vessels in all AD and control brains. K99 levels measured using Western blots were greater in AD compared to control brains (p < 0.01) and K99 was localized to neuron-like cells in AD but not control brains. LPS levels were also greater in AD compared to control brain. LPS colocalized with Aβ1-40/42 in amyloid plaques and with Aβ1-40/42 around vessels in AD brains. DNA sequencing confirmed E coli DNA in human control and AD brains.ConclusionsE coli K99 and LPS levels were greater in AD compared to control brains. LPS colocalized with Aβ1-40/42 in amyloid plaques and around vessels in AD brain. The data show that Gram-negative bacterial molecules are associated with AD neuropathology. They are consistent with our LPS-ischemia-hypoxia rat model that produces myelin aggregates that colocalize with Aβ and resemble amyloid-like plaques

Interpretable classification of Alzheimer's disease pathologies with a convolutional neural network pipeline.

Neuropathologists assess vast brain areas to identify diverse and subtly-differentiated morphologies. Standard semi-quantitative scoring approaches, however, are coarse-grained and lack precise neuroanatomic localization. We report a proof-of-concept deep learning pipeline that identifies specific neuropathologies-amyloid plaques and cerebral amyloid angiopathy-in immunohistochemically-stained archival slides. Using automated segmentation of stained objects and a cloud-based interface, we annotate > 70,000 plaque candidates from 43 whole slide images (WSIs) to train and evaluate convolutional neural networks. Networks achieve strong plaque classification on a 10-WSI hold-out set (0.993 and 0.743 areas under the receiver operating characteristic and precision recall curve, respectively). Prediction confidence maps visualize morphology distributions at high resolution. Resulting network-derived amyloid beta (Aβ)-burden scores correlate well with established semi-quantitative scores on a 30-WSI blinded hold-out. Finally, saliency mapping demonstrates that networks learn patterns agreeing with accepted pathologic features. This scalable means to augment a neuropathologist's ability suggests a route to neuropathologic deep phenotyping

Inflammation Combined with Ischemia Produces Myelin Injury and Plaque-Like Aggregates of Myelin, Amyloid-β and AβPP in Adult Rat Brain.

BackgroundIschemia, white matter injury, and Alzheimer's disease (AD) pathologies often co-exist in aging brain. How one condition predisposes to, interacts with, or perhaps causes the others remains unclear.ObjectivesTo better understand the link between ischemia, white matter injury, and AD, adult rats were administered lipopolysaccharide (LPS) to serve as an inflammatory stimulus, and 24 h later subjected to 20-min focal cerebral ischemia (IS) followed by 30-min hypoxia (H).MethodsMyelin and axonal damage, as well as amyloid-β (Aβ) and amyloid-β protein precursor (AβPP) deposition were examined by Western blot and immunocytochemistry following LPS/IS/H. Findings were compared to the 5XFAD mouse AD brain.ResultsMyelin/axonal injury was observed bilaterally in cortex following LPS/IS/H, along with an increase in IL-1, granzyme B, and LPS. AβPP deposition was present in ischemic striatum in regions of myelin loss. Aβ(1-42) and AβPP were deposited in small foci in ischemic cortex that co-localized with myelin aggregates. In the 5XFAD mouse AD model, cortical amyloid plaques also co-localized with myelin aggregates.ConclusionsLPS/IS/H produce myelin injury and plaque-like aggregates of myelin. AβPP and Aβ co-localize with these myelin aggregates

Repurposing the KCa3.1 inhibitor senicapoc for Alzheimer's disease.

ObjectiveMicroglia play a pivotal role in the initiation and progression of Alzheimer's disease (AD). We here tested the therapeutic hypothesis that the Ca2+-activated potassium channel KCa3.1 constitutes a potential target for treating AD by reducing neuroinflammation.MethodsTo determine if KCa3.1 is relevant to AD, we tested if treating cultured microglia or hippocampal slices with Aβ oligomer (AβO) activated KCa3.1 in microglia, and if microglial KCa3.1 was upregulated in 5xFAD mice and in human AD brains. The expression/activity of KCa3.1 was examined by qPCR, Western blotting, immunohistochemistry, and whole-cell patch-clamp. To investigate the role of KCa3.1 in AD pathology, we resynthesized senicapoc, a clinically tested KCa3.1 blocker, and determined its pharmacokinetic properties and its effect on microglial activation, Aβ deposition and hippocampal long-term potentiation (hLTP) in 5xFAD mice.ResultsWe found markedly enhanced microglial KCa3.1 expression/activity in brains of both 5xFAD mice and AD patients. In hippocampal slices, microglial KCa3.1 expression/activity was increased by AβO treatment, and its inhibition diminished the proinflammatory and hLTP-impairing activities of AβO. Senicapoc exhibited excellent brain penetrance and oral availability, and in 5xFAD mice, reduced neuroinflammation, decreased cerebral amyloid load, and enhanced hippocampal neuronal plasticity.InterpretationOur results prompt us to propose repurposing senicapoc for AD clinical trials, as senicapoc has excellent pharmacological properties and was safe and well-tolerated in a prior phase-3 clinical trial for sickle cell anemia. Such repurposing has the potential to expedite the urgently needed new drug discovery for AD

Metabolic flux analysis of the neural cell glycocalyx reveals differential utilization of monosaccharides.

Saccharides in our diet are major sources of carbon for the formation of biomass such as proteins, lipids, nucleic acids and glycans. Among the dietary monosaccharides, glucose occupies a central role in metabolism, but human blood contains regulated levels of other monosaccharides as well. Their influence on metabolism and how they are utilized have not been explored thoroughly. Applying metabolic flux analysis on glycan synthesis can reveal the pathways that supply glycosylation precursors and provide a snapshot of the metabolic state of the cell. In this study, we traced the incorporation of six 13C uniformly labeled monosaccharides in the N-glycans, O-glycans and glycosphingolipids of both pluripotent and neural NTERA-2 cells. We gathered detailed isotopologue data for hundreds of glycoconjugates using mass spectrometry methods. The contributions of de novo synthesis and direct incorporation pathways for glucose, mannose, fructose, galactose, N-acetylglucosamine and fucose were determined based on their isotope incorporation. Co-feeding studies revealed that fructose incorporation is drastically decreased by the presence of glucose, while mannose and galactose were much less affected. Furthermore, increased sialylation slowed down the turnover of glycans, but fucosylation attenuated this effect. Our results demonstrated that exogenous monosaccharide utilization can vary markedly depending on the cell differentiation state and monosaccharide availability, and that the incorporation of carbons can also differ among different glycan structures. We contend that the analysis of metabolic isotope labeling of glycans can yield new insights about cell metabolism

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

Alzheimer’s disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers

Syntheses of 3-[(Alkylamino)methylene]-6-methyl-1Hpyridine-2,4-diones, Fluorescence Probes 3-Substituted 7-Methyl-6H-pyrano[3,2-c]pyridine-2,5-diones, and Tetrahydro-6H-2,10-dioxa-9-azaanthracen-1-ones

Various condensation and ring-closing reactions were used for the syntheses of 3-[(alkylamino)methylene]-6-methylpyridine-2,4(1H,3H)-diones, bicyclic pyridinones, and tricyclic morpholinopyrones. For instance, 3-[(dialkylamino)methylene]-6-methylpyridine-2,4(1H,3H)-diones were synthesized from the condensation of dialkylamines and 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one. 3-Formyl-4-hydroxy-6-methylpyridin-2(1H)-one, derived from 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one, was used to construct a number of bicyclic pyridinones via a one-pot Knoevenagal and intramolecular lactonization reaction. Tricyclic morpholinopyrones were assembled from a dialkylation reaction involving a dinucleophile, 3-amino-4-hydroxy-6-methyl-2H-pyran-2-one, and a dielectrophile, trans-3,6-dibromocyclohexene. Depending on the reaction conditions, isomers of the tricyclic molecules can be selectively produced, and their chemical structures were unequivocally determined using single-crystal X-ray analyses and 2D COSY spectroscopy. The fluorescently active bicyclic pyridinone compounds show longer absorption (368–430 nm; maximum) and emission wavelengths (450–467 nm) than those of 7-amino-4-methylcoumarin (AMC; λ[subscript abs,max] = 350 nm; λ[subscript em] = 430 nm) suggesting these molecules, such as 3-(2-aminoacetyl)-7-methyl-2H-pyrano[3,2-c]pyridine-2,5(6H)-dione, can be employed as fluorescence activity based probes for tracing biological pathways

Syntheses, neural protective activities, and inhibition of glycogen synthase kinase-3beta of substituted quinolines.

A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3β (GSK-3β) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid β-induced MC65 cells and enzymatic inhibitory activities against GSK-3β, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer’s disease mouse model

Neuropathologic features in the hippocampus and cerebellum of three older men with fragile X syndrome

<p>Abstract</p> <p>Background</p> <p>Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, and is the most common single-gene disorder known to be associated with autism. Despite recent advances in functional neuroimaging and our understanding of the molecular pathogenesis, only limited neuropathologic information on FXS is available.</p> <p>Methods</p> <p>Neuropathologic examinations were performed on post-mortem brain tissue from three older men (aged 57, 64 and 78 years) who had received a clinical or genetic diagnosis of FXS. In each case, physical and cognitive features were typical of FXS, and one man was also diagnosed with autism. Guided by reports of clinical and neuroimaging abnormalities of the limbic system and cerebellum of individuals with FXS, the current analysis focused on neuropathologic features present in the hippocampus and the cerebellar vermis.</p> <p>Results</p> <p>Histologic and immunologic staining revealed abnormalities in both the hippocampus and cerebellar vermis. Focal thickening of hippocampal CA1 and irregularities in the appearance of the dentate gyrus were identified. All lobules of the cerebellar vermis and the lateral cortex of the posterior lobe of the cerebellum had decreased numbers of Purkinje cells, which were occasionally misplaced, and often lacked proper orientation. There were mild, albeit excessive, undulations of the internal granular cell layer, with patchy foliar white matter axonal and astrocytic abnormalities. Quantitative analysis documented panfoliar atrophy of both the anterior and posterior lobes of the vermis, with preferential atrophy of the posterior lobule (VI to VII) compared with age-matched normal controls.</p> <p>Conclusions</p> <p>Significant morphologic changes in the hippocampus and cerebellum in three adult men with FXS were identified. This pattern of pathologic features supports the idea that primary defects in neuronal migration, neurogenesis and aging may underlie the neuropathology reported in FXS.</p