β-Cell failure in type 2 diabetes: a case of asking too much of too few?

The islet in type 2 diabetes (T2DM) is characterized by a deficit in β-cells, increased β-cell apoptosis, and extracellular amyloid deposits derived from islet amyloid polypeptide (IAPP). In the absence of longitudinal studies, it is unknown if the low β-cell mass in T2DM precedes diabetes onset (is a risk factor for diabetes) or develops as a consequence of the disease process. Although insulin resistance is a risk factor for T2DM, most individuals who are insulin resistant do not develop diabetes. By inference, an increased β-cell workload results in T2DM in some but not all individuals. We propose that the extent of the β-cell mass that develops during childhood may underlie subsequent successful or failed adaptation to insulin resistance in later life. We propose that a low innate β-cell mass in the face of subsequent insulin resistance may expose β-cells to a burden of insulin and IAPP biosynthetic demand that exceeds the cellular capacity for protein folding and trafficking. If this threshold is crossed, intracellular toxic IAPP membrane permeant oligomers (cylindrins) may form, compromising β-cell function and inducing β-cell apoptosis

Structural Features and Domain Organization of Huntingtin Fibrils

Misfolding and aggregation of huntingtin is one of the hallmarks of Huntington disease, but the overall structure of these aggregates and the mechanisms by which huntingtin misfolds remain poorly understood. Here we used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the structural features of huntingtin exon 1 (HDx1) containing 46 glutamine residues in its polyglutamine (polyQ) region. Despite some residual structuring in the N terminus, we find that soluble HDx1 is highly dynamic. Upon aggregation, the polyQ domain becomes strongly immobilized indicating significant tertiary or quaternary packing interactions. Analysis of spin-spin interactions does not show the close contact between same residues that is characteristic of the parallel, in-register structure commonly found in amyloids. Nevertheless, the same residues are still within 20 Å of each other, suggesting that polyQ domains from different molecules come into proximity in the fibrils. The N terminus has previously been found to take up a helical structure in fibrils. We find that this domain not only becomes structured, but that it also engages in tertiary or quaternary packing interactions. The existence of spin-spin interactions in this region suggests that such contacts could be made between N-terminal domains from different molecules. In contrast, the C-terminal domain is dynamic, contains polyproline II structure, and lacks pronounced packing interactions. This region must be facing away from the core of the fibrils. Collectively, these data provide new constraints for building structural models of HDx1 fibrils

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation

Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin

<p>Abstract</p> <p>Background</p> <p>The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD), age-related macular degeneration (AMD), atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins.</p> <p>Results</p> <p>We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE) cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation.</p> <p>Conclusion</p> <p>These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.</p

Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1–4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT

Online 4D ultrasound guidance for real-time motion compensation by MLC tracking

PURPOSE: With the trend in radiotherapy moving toward dose escalation and hypofractionation, the need for highly accurate targeting increases. While MLC tracking is already being successfully used for motion compensation of moving targets in the prostate, current real-time target localization methods rely on repeated x-ray imaging and implanted fiducial markers or electromagnetic transponders rather than direct target visualization. In contrast, ultrasound imaging can yield volumetric data in real-time (3D + time = 4D) without ionizing radiation. The authors report the first results of combining these promising techniques-online 4D ultrasound guidance and MLC tracking-in a phantom. METHODS: A software framework for real-time target localization was installed directly on a 4D ultrasound station and used to detect a 2 mm spherical lead marker inside a water tank. The lead marker was rigidly attached to a motion stage programmed to reproduce nine characteristic tumor trajectories chosen from large databases (five prostate, four lung). The 3D marker position detected by ultrasound was transferred to a computer program for MLC tracking at a rate of 21.3 Hz and used for real-time MLC aperture adaption on a conventional linear accelerator. The tracking system latency was measured using sinusoidal trajectories and compensated for by applying a kernel density prediction algorithm for the lung traces. To measure geometric accuracy, static anterior and lateral conformal fields as well as a 358° arc with a 10 cm circular aperture were delivered for each trajectory. The two-dimensional (2D) geometric tracking error was measured as the difference between marker position and MLC aperture center in continuously acquired portal images. For dosimetric evaluation, VMAT treatment plans with high and low modulation were delivered to a biplanar diode array dosimeter using the same trajectories. Dose measurements with and without MLC tracking were compared to a static reference dose using 3%/3 mm and 2%/2 mm γ-tests. RESULTS: The overall tracking system latency was 172 ms. The mean 2D root-mean-square tracking error was 1.03 mm (0.80 mm prostate, 1.31 mm lung). MLC tracking improved the dose delivery in all cases with an overall reduction in the γ-failure rate of 91.2% (3%/3 mm) and 89.9% (2%/2 mm) compared to no motion compensation. Low modulation VMAT plans had no (3%/3 mm) or minimal (2%/2 mm) residual γ-failures while tracking reduced the γ-failure rate from 17.4% to 2.8% (3%/3 mm) and from 33.9% to 6.5% (2%/2 mm) for plans with high modulation. CONCLUSIONS: Real-time 4D ultrasound tracking was successfully integrated with online MLC tracking for the first time. The developed framework showed an accuracy and latency comparable with other MLC tracking methods while holding the potential to measure and adapt to target motion, including rotation and deformation, noninvasively

Pericyte degeneration causes white matter dysfunction in the mouse central nervous system

Diffuse white-matter disease associated with small-vessel disease and dementia is prevalent in the elderly. The biological mechanisms, however, remain elusive. Using pericyte-deficient mice, magnetic resonance imaging, viral-based tract-tracing, and behavior and tissue analysis, we found that pericyte degeneration disrupted white-matter microcirculation, resulting in an accumulation of toxic blood-derived fibrin(ogen) deposits and blood-flow reductions, which triggered a loss of myelin, axons and oligodendrocytes. This disrupted brain circuits, leading to white-matter functional deficits before neuronal loss occurs. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white-matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white-matter changes. Thus, our data indicate that pericytes control white-matter structure and function, which has implications for the pathogenesis and treatment of human white-matter disease associated with small-vessel disease