Synthesis of Hybrid Inositol Glycan Analogues

Inositol glycans (IGs) are small oligosaccharides exhibiting insulin-like metabolic activities in insulin-sensitive cells. The signal transduction pathways activated by IGs in these cells are still under study, but it is known that there is cross talk between the IG-signaling pathway and the insulin-signaling pathway downstream of the insulin receptor. Therefore, IGs may have potential for use in the treatment of type II diabetes mellitus. However, natural IGs are heterogeneous and difficult to isolate. Hence, synthetic IGs and their analogues have been chemically synthesized and evaluated for insulin-mimetic properties by various research groups. Unfortunately, the most biologically active IG analogues are structurally complex and difficult to synthesize. The present work reports the progress towards designing and preparing biologically active IG analogues with short and relatively simple synthetic pathways. The strategy is to synthesize a small library of hybrid inositol glycan analogues (HIGAs) where each HIGA consists of an inositol core covalently tethered to a variety of readily available non-carbohydrate moieties. Synthesis of one such HIGA (compound 16) was successfully accomplished. Initial results from mass spectrometric analysis provide evidence of molecular mass of compound 16

Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein

Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of β-amyloid (Aβ) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Aβ proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Aβ proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Aβ40 (F-Aβ40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Aβ proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Aβ40 or F-Aβ42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Aβ40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Aβ40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Aβ proteins and help explain the vulnerability of cortical and hippocampal neurons to Aβ toxicity

Figure 4

<p>A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ40 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B; (D) Sparse co-localization of F-Aβ40 and AF633-Trf as indicated by the white masked areas. E–G: Uptake of F-Aβ40 and AF633-Trf in differentiated PC12 cells subjected conditions that inhibit clathrin mediated endocytosis (hypotonic shock for 5 min followed by incubation with potassium free salt solution for 30 min). (E) Uptake of F-Aβ40; (F) Substantial reduction in AF633-Trf; (G). Superimposition of images E and F on the differential interference contrast (DIC) image to show the condition of the cells.</p

Figure 9

<p>A–D: Uptake of fluorescein labeled Aβ42 (F-Aβ42) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ42 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B show limited co-localization of F-Aβ42 and AF633-Trf. (D) A magnified portion of image C (enclosed in the white rectangle) to indicate the lack of co-localization of both fluorophores. I–II: Histograms of fluorescence intensity in differentiated PC12 cells treated with (I) F-Aβ42; and (II) AF633-Trf, at 37°C and 4°C.</p

Figure 5

<p>A–C: Co-localization of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf) in PC12 cells following: (A) 15 min; (B) 45 min; and (C) 60 min incubation. White masked areas indicate the extent of co-localization of F-Aβ40 and AF633-Trf. D: Cellular internalization of F-Aβ40 and AF633-Trf established by the XY, XZ, and YZ projections of differentiated PC12 cells treated with the fluorophores for 60 min. Optical sections (planes 1–45) were obtained from the coverslip bottom to the cell surface with a 0.6 µm Z-step interval.</p

Cellular uptake of a fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf) in wild type mouse brain slices after 30 min incubation.

<p>A–B: Uptake of F-Aβ40 by a subpopulation of (A) cortical neurons (20×) and; (B) hippocampal neurons (20×). C–I: Effect of temperature on the uptake of F-Aβ40 and AF633-Trf by the pyramidal neurons (63× and 3× optical zoom). C–F: Uptake of F-Aβ40 and AF633-Trf at 37°C (C) F-Aβ40 uptake; (D) AF633-Trf uptake; (E) Superimposition of images C and D; (F) Limited co-localization of F-Aβ40 and AF633-Trf, indicated by white masked areas, was found only around the edges of pyramidal neurons. G–I: Uptake of F-Aβ40 and AF633-Trf at 4°C (G) F-Aβ40 uptake; (H) Inhibition of AF633-Trf internalization; (I) Superimposition of images G and H.</p

Figure 11

<p>A–D: F-Aβ40 uptake into the acidic compartments of bovine brain microvascular endothelial (BBME) cells labeled by Lysotracker Red® (60×). (A) Uptake of F-Aβ40 (B) Uptake of Lysotracker Red®; (C) Superimposition of images A and B; (D) A magnified portion of image C (enclosed in the white rectangle) to show co-localization of both fluorophores. I–II: Histograms of fluorescence intensity in BBME cells treated with (I) F-Aβ40: (A) Untreated control, (B) at 4°C, (C) at 37°C; and (II) AF633-Trf: (A) Untreated control, (B) at 4°C, (C) at 37°C.</p

Figure 10

<p>A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, in rat primary hippocampal (RPH) neurons following 30 min incubation at 37°C. (A) F-Aβ40 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B; (D) Overlay of fluorescence images on the DIC image of RPH neurons. E–G: Uptake of F-Aβ40 and AF633-Trf in RPH neurons at 4°C. (E) Uptake of F-Aβ40; (F) No significant neuronal uptake of AF633-Trf at 4°C; (G) Superimposition of images D and E on the DIC image of RPH neurons; H–J: Uptake of F-Aβ40 and AF633-Trf in RPH neurons treated with 10 mM Sodium Azide and 50 mM 2-deoxy glucose, agents that are known to deplete cellular ATP. (H) Uptake of F-Aβ40; (I) No significant cellular uptake of AF633-Trf was observed; (J) Superimposition of images H and I on the DIC image of neurons.</p

Figure 2

<p>(I) Effect of donor concentration and temperature on the uptake of ¹²⁵I-Aβ40 in wild type (WT) mouse brain slices. (II) Effect of endocytotic inhibitor dansyl cadaverine on the uptake of ¹²⁵I-Aβ40 (450 ng/ml) in WT mouse brain slices. (III) Histograms of fluorescence intensity in differentiated PC12 cells exposed to various concentrations of F-Aβ40. (A) Untreated cells; (B) Cells incubated with 0.65 µM F-Aβ40; (C) Cells incubated with 1.3 µM F-Aβ40; (D) Cells incubated with 3.2 µM F-Aβ40; (E) Cells incubated with 3.2 µM F-Aβ40+32 µM unlabeled Aβ40.</p