Myosin VIIA regulates microvillus morphogenesis and interacts with cadherin Cad99C in Drosophila oogenesis.

Microvilli and related actin-based protrusions permit multiple interactions between cells and their environment. How the shape, length and arrangement of microvilli are determined remains largely unclear. To address this issue and explore the cooperation of the two main components of a microvillus, the central F-actin bundle and the enveloping plasma membrane, we investigated the expression and function of Myosin VIIA (Myo7A), which is encoded by crinkled (ck), and its interaction with cadherin Cad99C in the microvilli of the Drosophila follicular epithelium. Myo7A is present in the microvilli and terminal web of follicle cells, and associates with several other F-actin-rich structures in the ovary. Loss of Myo7A caused brush border defects and a reduction in the amount of the microvillus regulator Cad99C. We show that Myo7A and Cad99C form a molecular complex and that the cytoplasmic tail of Cad99C recruits Myo7A to microvilli. Our data indicate that Myo7A regulates the structure and spacing of microvilli, and interacts with Cad99C in vivo. A comparison of the mutant phenotypes suggests that Myo7A and Cad99C have co-dependent and independent functions in microvilli.

Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model.

When given orally to a transgenic mouse model of Alzheimer disease, cyclohexanehexol stereoisomers inhibit aggregation of amyloid beta peptide (Abeta) into high-molecular-weight oligomers in the brain and ameliorate several Alzheimer disease-like phenotypes in these mice, including impaired cognition, altered synaptic physiology, cerebral Abeta pathology and accelerated mortality. These therapeutic effects, which occur regardless of whether the compounds are given before or well after the onset of the Alzheimer disease-like phenotype, support the idea that the accumulation of Abeta oligomers has a central role in the pathogenesis of Alzheimer disease.

Modulation of amyloid-beta aggregation and toxicity by inosose stereoisomers.

Amyloid-beta (Abeta) aggregation and amyloid formation are key pathological features of Alzheimer's disease, and are considered to be two of the major contributing factors to neurodegeneration and dementia. Identification of small molecule inhibitors that are orally available, have low toxicity and high central nervous system bioavailability is one approach to the potential development of a disease-modifying treatment for Alzheimer's disease. We have previously identified inositol stereoisomers as exhibiting stereospecific inhibition of Abeta aggregation and toxicity in vitro and in vivo. We report here the effects of inosose versus inositol stereoisomers on Abeta fibrillogenesis as determined using CD and fluorescence spectroscopy and negative-stain electron microscopy. The inososes differ from inositols by the oxidation of one of the hydroxyl groups to a ketone. These molecules help in the further elucidation of the structure-activity relationships of inositol-Abeta interactions and identify both allo-inositol and epi-2-inosose as in vitro inhibitors of Abeta aggregation.

Abeta42-peptide assembly on lipid bilayers.

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are composed predominantly of the amyloid-beta peptide (Abeta). Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta42-membrane interactions using in situ atomic force microscopy and fluorescence spectroscopy. Our studies demonstrate the association of Abeta42 with planar bilayers composed of total brain lipids, which results initially in peptide aggregation and then fibre formation. Modulation of the cholesterol content is correlated with the extent of Abeta42-assembly on the bilayer surface. Although Abeta42 was not visualized directly on cholesterol-depleted bilayers, fluorescence anisotropy and fluorimetry demonstrate Abeta42-induced membrane changes. Our results demonstrate that the composition of the lipid bilayer governs the outcome of Abeta interactions.

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis.

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.

The cellular composition of neurogenic periventricular zones in the adult zebrafish forebrain.

A central goal of adult neurogenesis research is to characterize the cellular constituents of a neurogenic niche and to understand how these cells regulate the production of new neurons. Because the generation of adult-born neurons may be tightly coupled to their functional requirement, the organization and output of neurogenic niches may vary across different regions of the brain or between species. We have undertaken a comparative study of six (D, Vd, Vv, Dm, Dl, Ppa) periventricular zones (PVZs) harboring proliferative cells present in the adult forebrain of the zebrafish (Danio rerio), a species known to possess widespread neurogenesis throughout life. Using electron microscopy, we have documented for the first time the detailed cytoarchitecture of these zones, and propose a model of the cellular composition of pallial and subpallial PVZs, as well as a classification scheme for identifying morphologically distinct cell types. Immunolabeling of resin-embedded tissue confirmed the phenotype of three constitutively proliferating (bromodeoxyuridine [BrdU]+) cell populations, including a radial glial-like (type IIa) cell immunopositive for both S100ß and glutamine synthetase (GS). Our data revealed rostrocaudal differences in the density of distinct proliferative populations, and cumulative labeling studies suggested that the cell cycle kinetics of these populations are not uniform between PVZs. Although the peak numbers of differentiated neurons were generated after ~2 weeks among most PVZs, niche-specific decline in the number of newborn neurons in some regions occurred after 4 weeks. Our data suggest that the cytoarchitecture of neurogenic niches and the tempo of neuronal production are regionally distinct in the adult zebrafish forebrain.

Contribution of simple saccharides to the stabilization of amyloid structure.

The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer's amyloid-beta peptides (Abeta). Using circular dichroism spectroscopy to follow the beta-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Abeta. Two of these sugar molecules are excluded from Abeta during aggregation and promote mature fibre growth, while the other binds Abeta promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy.

alpha-Synuclein-synaptosomal membrane interactions: implications for fibrillogenesis.

alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical and a cytosolic form that is randomly structured. It has been suggested that these environmental and structural differences may play a role in aggregation propensity and development of pathological lesions observed in Parkinson's disease (PD). Such effects may be accentuated by mutations observed in familial PD kindreds. In order to test this hypothesis, wild-type and A53T mutant alpha-synuclein interactions with rat brain synaptosomal membranes were examined. Previous data has demonstrated that the A30P mutant has defective lipid binding and therefore was not examined in this study. Electron microscopy demonstrated that wild-type alpha-synuclein fibrillogenesis is accelerated in the presence of synaptosomal membranes whereas the A53T alpha-synuclein fibrillogenesis is inhibited under the same conditions. These results suggested that subtle sequence changes in alpha-synuclein could significantly alter interaction with membrane bilayers. Fluorescence and absorption spectroscopy using environment sensitive probes demonstrated variations in the inherent lipid properties in the presence and absence of alpha-synuclein. Addition of wild-type alpha-synuclein to synaptosomes did not significantly alter the membrane fluidity at either the fatty acyl chains or headgroup space, suggesting that synaptosomes have a high capacity for alpha-synuclein binding. In contrast, synaptosomal membrane fluidity was decreased by A53T alpha-synuclein binding with concomitant packing of the lipid headgroups. These results suggest that alterations in alpha-synuclein-lipid interactions may contribute to physiological changes detected in early onset PD.