Point Mutations in Aβ Result in the Formation of Distinct Polymorphic Aggregates in the Presence of Lipid Bilayers

A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity

Laser capture microdissection of human pancreatic islets reveals novel eQTLs associated with type 2 diabetes.

Genome wide association studies (GWAS) for type 2 diabetes (T2D) have identified genetic loci that often localise in non-coding regions of the genome, suggesting gene regulation effects. We combined genetic and transcriptomic analysis from human islets obtained from brain-dead organ donors or surgical patients to detect expression quantitative trait loci (eQTLs) and shed light into the regulatory mechanisms of these genes. Pancreatic islets were isolated either by laser capture microdissection (LCM) from surgical specimens of 103 metabolically phenotyped pancreatectomized patients (PPP) or by collagenase digestion of pancreas from 100 brain-dead organ donors (OD). Genotyping (> 8.7 million single nucleotide polymorphisms) and expression (> 47,000 transcripts and splice variants) analyses were combined to generate cis-eQTLs. After applying genome-wide false discovery rate significance thresholds, we identified 1,173 and 1,021 eQTLs in samples of OD and PPP, respectively. Among the strongest eQTLs shared between OD and PPP were CHURC1 (OD p-value=1.71 × 10 <sup>-24</sup> ; PPP p-value = 3.64 × 10 <sup>-24</sup> ) and PSPH (OD p-value = 3.92 × 10 <sup>-26</sup> ; PPP p-value = 3.64 × 10 <sup>-24</sup> ). We identified eQTLs in linkage-disequilibrium with GWAS loci T2D and associated traits, including TTLL6, MLX and KIF9 loci, which do not implicate the nearest gene. We found in the PPP datasets 11 eQTL genes, which were differentially expressed in T2D and two genes (CYP4V2 and TSEN2) associated with HbA1c but none in the OD samples. eQTL analysis of LCM islets from PPP led us to identify novel genes which had not been previously linked to islet biology and T2D. The understanding gained from eQTL approaches, especially using surgical samples of living patients, provides a more accurate 3-dimensional representation than those from genetic studies alone

Multivesicular exocytosis in rat pancreatic beta cells

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 μmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

Effects of external tetraethylammonium ions and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells.

1. The whole-cell and outside-out patch configurations of the patch-clamp technique were used to study the mechanisms of block produced by external tetraethylammonium ions (TEA+) and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells. 2. In whole-cell recordings, TEA+ blocks the delayed outward current (which reflects the activity of delayed rectifying K+ channels by greater than 85%) in a concentration-dependent manner. The block appeared to be 1:1 with a Kd of approximately 1.4 mM at a membrane potential of 0 mV. The value of Kd varied with the membrane potential and there was an e-fold increase for a 70 mV depolarization. 3. Single-channel recordings revealed that delayed rectifying K+ channels have a unitary conductance of 8.5 pS ([K+]1 = 155 mM; [K+]o = 5.6 mM) and a single-channel K+ permeability of 2.8 X 10(-14) cm3 s-1. 4. First latency histograms of channel openings during voltage pulses from -70 to 0 mV peaked after 4 ms. A reaction scheme involving two closed states adequately but not perfectly described the distribution of the first latencies. The openings of the channels were grouped in bursts and the distribution of the closed times required two exponentials with time constants of 2.0 and 13 ms, respectively. The distribution of the open times could be described by a single exponential with a time constant of 25 ms. 5. Channel block produced by TEA+ (1 mM) was associated with a 40% decrease of the single-channel current amplitudes and a reduction in single-channel K+ permeability to 1.9 X 10(-14) cm3 s-1 but did not measurably affect the single-channel kinetics suggesting that the blocking reaction is very rapid. 6. Quinine blocked the whole-cell delayed outward current in a concentration-dependent manner. Half-maximal inhibition was attained at approximately 4 microM and the binding appeared to be 2:1. 7. Single-channel recordings indicated that the inhibition produced by quinine (10 microM) resulted from a decrease in the duration of the openings to a mean value of 6.7 ms. The time constants for the distribution of the closures were increased by approximately 30%. Quinine did not affect the amplitude of the openings. The rate constant of the blocking reaction (kB) was 15 mM-1 ms-1 at 0 mV

Demonstration of A-currents in pancreatic islet cells.

Voltage-activated K+ currents resistant to TEA but blockable by 4-AP were recorded from mouse pancreatic islet cells. These currents first become observable during depolarizations to voltages more positive than -40 mV, reaching a peak amplitude of 120 +/- 34 pA at +6 mV (n = 4), display rapid turn on (tau = 3.3 +/- 1.1 ms at +6 mV) and inactivate completely within 250 ms (tau = 65 +/- 5 at +6 mV). The current is subject to steady-state inactivation. The midpoint (Vh) of the inactivation curve (h infinity) was observed at -72 +/- 2 mV. The properties of this current resemble those reported for the A-current in neurons