Thomas L. Williams

Membrane and surface interactions of Alzheimer’s Aβ peptide – insights into the mechanism of cytotoxicity

Alzheimer’s disease is the most common form of dementia and its pathological hallmarks include the loss of neurones through cell death, as well as the accumulation of amyloid fibres in the form of extracellular neuritic plaques. Amyloid fibrils are composed of the amyloid‐β peptide (Aβ), which is known to assemble to form ‘toxic’ oligomers that may be central to disease pathology. Aβ is produced by cleavage from the amyloid precursor protein within the transmembrane region, and the cleaved peptide may retain some membrane affinity. It has been shown that Aβ is capable of specifically binding to phospholipid membranes with a relatively high affinity, and that modulation of the composition of the membrane can alter both membrane–amyloid interactions and toxicity. Various biomimetic membrane models have been used (e.g. lipid vesicles in solution and tethered lipid bilayers) to examine the binding and interactions between Aβ and the membrane surfaces, as well as the resulting permeation. Oligomeric Aβ has been observed to bind more avidly to membranes and cause greater permeation than fibrillar Aβ. We review some of the recent advances in studying Aβ–membrane interactions and discuss their implications with respect to understanding the causes of Alzheimer’s disease.

Shoulder dystocia: a fetal-physician risk

Trauma that occurs as a result of shoulder dystocia is an important cause of neonatal morbidity. If the occurrence of severe shoulder dystocia, resulting in fetal asphyxia and trauma, could be accurately predicted from maternal risk factors, then a cesarean section would be indicated to prevent the poor outcome. The information available in the obstetric literature, however, is contradictory regarding whether shoulder dystocia can be predicted. In the present study, the patients at greatest risk of shoulder dystocia (all 394 mothers delivering neonates with birth weights greater than or equal to 4000 gm over a 2-year period) were examined. A three-way discriminant analysis was used to determine if a model could be developed that could effectively predict those patients who would be included in each of the groups of no shoulder dystocia, shoulder dystocia without trauma (29 patients), and shoulder dystocia with trauma (20 patients). Three factors, including birth weight, prolonged deceleration phase, and length of second stage labor, were found individually to contribute significantly to the classification. However, when examined in detail, it was noted that while 94% of cases with no shoulder dystocia would be detected, only 16% of the cases of shoulder dystocia with trauma would be predicted by this model. We conclude that in the group of pregnancies delivering neonates greater than or equal to 4000 gm, the occurrence of shoulder dystocia cannot be predicted from clinical characteristics or labor abnormalities, and that the occurrence of shoulder dystocia is not evidence of medical malpractice.

Structural Basis for Increased Toxicity of Pathological Aβ42:Aβ40 Ratios in Alzheimer Disease

Background: Amyloid β peptide plays a role in Alzheimer disease. Results: Interaction of amyloid β peptides with 40 and 42 amino acids has consequences for oligomer formation. Conclusion: Increased production of amyloid β peptide with 42 amino acids affects the behavior of the entire amyloid β peptide pool. Significance: This might explain the synaptotoxic effect observed with a shift in amyloid β peptide production. The β-amyloid peptide (Aβ) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aβ42:Aβ40 ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aβ42 in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aβ40 and Aβ42 individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aβ42:Aβ40 ratios by several techniques. We show that Aβ40 and Aβ42 directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aβ40 and Aβ42 mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aβ40 and Aβ42 influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aβ42:Aβ40 in synaptotoxicity assays and in AD patients.

Oligohydramnios: Clinical associations and predictive value for intrauterine growth retardation☆☆☆

Intrauterine growth retardation (IUGR) is difficult to diagnose before birth. Sonographically diagnosed oligohydramnios has been reported to be highly sensitive and reliable in detecting IUGR in carefully prescreened patients. To evaluate the clinical associations of oligohydramnios and its usefulness as a method of antenatal screening for IUGR on a large obstetric service, the consecutive ultrasound examinations of 2,453 viable singleton pregnancies with intact membranes were surveyed. The 96 (3.9%) pregnancies found to be complicated by oligohydramnios were compared with 96 with the same biparietal diameters, but with normal volumes of amniotic fluid. Mothers with oligohydramnios were younger, of lower parity, and at increased clinical risk for IUGR. Of 96 infants from oligohydramnios-complicated pregnancies, 38 (40%) were small for gestational age (SGA), compared with eight (8%) infants from pregnancies without oligohydramnios. Of 46 SGA births, 38 (83%) were preceded by sonographically diagnosed oligohydramnios (p less than 0.0001). IUGR associated with oligohydramnios tended to occur in young hypertensive gravid women, whereas non-oligohydramnios-associated IUGR tended to occur in gravid women with low prepregnancy weight. With correction for the incidences of oligohydramnios and IUGR in the unselected population, it is shown that four in 10 cases of oligohydramnios would result in SGA births (40% predictive value of an abnormal test), but that only 16% of SGA births would be preceded by sonographically diagnosed oligohydramnios. Eighty-four percent of the cases would be missed. These results suggest that, although the presence of oligohydramnios should increase the clinicians index of suspicion for IUGR, routine sonographic screening to detect oligohydramnios is not warranted.

A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer’s disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.ResultsWe have investigated the formation of dityrosine cross-links in Aβ42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aβ oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aβ in cell cultures treated with oligomeric Aβ42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients.ConclusionsAβ dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer’s disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aβ and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD.

Characterizing the Assembly of the Sup35 Yeast Prion Fragment, GNNQQNY: Structural Changes Accompany a Fiber-to-Crystal Switch

Amyloid-like fibrils can be formed by many different proteins and peptides. The structural characteristics of these fibers are very similar to those of amyloid fibrils that are deposited in a number of protein misfolding diseases, including Alzheimers disease and the transmissible spongiform encephalopathies. The elucidation of two crystal structures from an amyloid-like fibril-forming fragment of the yeast prion, Sup35, with sequence GNNQQNY, has contributed to knowledge regarding side-chain packing of amyloid-forming peptides. Both structures share a cross-beta steric zipper arrangement but vary in the packing of the peptide, particularly in terms of the tyrosine residue. We investigated the fibrillar and crystalline structure and assembly of the GNNQQNY peptide using x-ray fiber diffraction, electron microscopy, intrinsic and quenched tyrosine fluorescence, and linear dichroism. Electron micrographs reveal that at concentrations between 0.5 and 10 mg/mL, fibers form initially, followed by crystals. Fluorescence studies suggest that the environment of the tyrosine residue changes as crystals form. This is corroborated by linear dichroism experiments that indicate a change in the orientation of the tyrosine residue over time, which suggests that a structural rearrangement occurs as the crystals form. Experimental x-ray diffraction patterns from fibers and crystals also suggest that these species are structurally distinct. A comparison of experimental and calculated diffraction patterns contributes to an understanding of the different arrangements accessed by the peptide.

The Effect of Alzheimer’s Aβ Aggregation State on the Permeation of Biomimetic Lipid Vesicles

Alzheimers disease is characterized by the aggregation and deposition of the Aβ peptide. This 40 or 42 residue peptide is the product of the proteolysis of the amyloid precursor protein membrane protein and is able to assemble to form ordered, stable amyloid fibrils as well as small, soluble, and potentially cytotoxic oligomers. The toxicity of the oligomers may be associated with the ability to bind to and affect the integrity of lipid membranes. In this novel work, we have monitored and compared the ability of the potent Aβ42 peptide, the less amyloidogenic Aβ40 peptide, and a variant with reduced amyloidogenicity to bind to and affect the integrity of membranes using dye-filled synthetic vesicles. We reveal that the potency of the assemblies reduces with incubation time of the peptide and that maximal effect occurs when a particular species is apparent by electron microscopy. We have investigated the effect of lipid vesicle composition, and our results suggest that charge on the vesicles is important and that binding may partly be mediated by the GM1 ganglioside receptors expressed in the outer leaflet of vertebrate membranes.

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimers disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.

Neonatal manifestations of maternal phencyclidine exposure

The purpose of this study was to determine the effects of maternal phencyclidine use on the fetus. Ninety-four neonates with maternal phencyclidine exposure were compared with 94 controls. Maternal phencyclidine use was assessed by questionnaire and repeated urine testing. Mothers of study and control patients were matched for demographic characteristics. Infants were assessed between 24-72 hours postnatally by a single examiner blind to the maternal history. The results showed that study infants had a mean of 5.02 +/- 2.93 abnormalities while controls had a mean of 4.13 +/- 2.65 abnormalities (p less than 0.01). Furthermore, study infants were more likely than controls to have poor attention, hypertonia, and depressed neonatal reflexes (p less than 0.05). The contribution of seven drug classes to the total number of abnormalities was assessed using stepwise multiple regression. Only phencyclidine accounted for a significant proportion of the variance (f = 4.38; p less than 0.05). The results of this study suggest that maternal phencyclidine use may lead to abnormal neonatal neurologic findings and behavior.The purpose of this study was to determine the effects of maternal phencyclidine use on the fetus. Ninety-four neonates with maternal phencyclidine exposure were compared with 94 controls. Maternal phencyclidine use was assessed by questionnaire and repeated urine testing. Mothers of study and control patients were matched for demographic characteristics. Infants were assessed between 24-72 hours postnatally by a single examiner blind to the maternal history. The results showed that study infants had a mean of 5.02 +/- 2.93 abnormalities while controls had a mean of 4.13 +/- 2.65 abnormalities (p less than 0.01). Furthermore, study infants were more likely than controls to have poor attention, hypertonia, and depressed neonatal reflexes (p less than 0.05). The contribution of seven drug classes to the total number of abnormalities was assessed using stepwise multiple regression. Only phencyclidine accounted for a significant proportion of the variance (f = 4.38; p less than 0.05). The results of this study suggest that maternal phencyclidine use may lead to abnormal neonatal neurologic findings and behavior.

Stabilization of native amyloid β-protein oligomers by Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP).

Oligomeric assemblies are postulated to be proximate neurotoxic species in human diseases associated with aberrant protein aggregation. Their heterogeneous and transient nature makes their structural characterization difficult. Size distributions of oligomers of several amyloidogenic proteins, including amyloid β-protein (Aβ) relevant to Alzheimers disease (AD), have been previously characterized in vitro by photo-induced cross-linking of unmodified proteins (PICUP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Due to non-physiological conditions associated with the PICUP chemistry, Aβ oligomers cross-linked by PICUP may not be representative of in vivo conditions. Here, we examine an alternative Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP), which utilizes naturally occurring divalent copper ions and hydrogen peroxide and does not require photo activation. Our results demonstrate that CHICUP and PICUP applied to the two predominant Aβ alloforms, Aβ40 and Aβ42, result in similar oligomer size distributions. Thioflavin T fluorescence data and atomic force microscopy images demonstrate that both CHICUP and PICUP stabilize Aβ oligomers and attenuate fibril formation. Relative to noncross-linked peptides, CHICUP-treated Aβ40 and Aβ42 cause prolonged disruption to biomimetic lipid vesicles. CHICUP-stabilized Aβ oligomers link the amyloid cascade, metal, and oxidative stress hypotheses of AD into a more comprehensive understanding of the molecular basis of AD pathology. Because copper and hydrogen peroxide are elevated in the AD brain, CHICUP-stabilized Aβ oligomers are biologically relevant and should be further explored as a new therapeutic target.