Stability of Iowa mutant and wild type Aß-peptide aggregates.

Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid ß-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aß-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

Mutations Alter RNA-Mediated Conversion of Human Prions.

Prion diseases are connected with self-replication and self-propagation of misfolded proteins. The rate-limiting factor is the formation of the initial seed. We have recently studied the early stages in the conversion between functional PrPC and the infectious scrapie PrPSC form, triggered by the binding of RNA. Here, we study how this process is modulated by the prion sequence. We focus on residues 129 and 178, which are connected to the hereditary neurodegenerative disease fatal familial insomnia.

Molecular dynamics simulations of early steps in RNA-mediated conversion of prions.

The rate-limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis-folded state in which the protein not only forms cell-toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA-mediated mis-folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.

Stability of Osaka Mutant and Wild-Type Fibril Models.

Single amino acid mutations in amyloid-beta (Aß) peptides can lead to early onset and increased severity of Alzheimer's disease. An example is the Osaka mutation (Aß1-40E22D), which is more toxic than wild-type Aß1-40. This mutant quickly forms early stage fibrils, one of the hallmarks of the disease, and these fibrils can even seed fibrilization of wild-type monomers. Using molecular dynamic simulations, we show that because of formation of various intra- and intermolecular salt bridges the Osaka mutant fibrils are more stable than wild-type fibrils. The mutant fibril also has a wider water channel with increased water flow than the wild type. These two observations can explain the higher toxicity and aggregation rate of the Osaka mutant over the wild type.

On the lack of polymorphism in Aß-peptide aggregates derived from patient brains.

The amyloid beta (Aß) oligomers and fibrils that are found in neural tissues of patients suffering from Alzheimer's disease may either cause or contribute to the pathology of the disease. In vitro, these Aß-aggregates are characterized by structural polymorphism. However, recent solid state NMR data of fibrils acquired post mortem from the brains of two Alzheimer's patients indicate presence of only a single, patient-specific structure. Using enhanced molecular dynamic simulations we investigate the factors that modulate the stability of Aß-fibrils. We find characteristic differences in molecular flexibility, dynamics of interactions, and structural behavior between the brain-derived Aß-fibril structure and in vitro models. These differences may help to explain the lack of polymorphism in fibrils collected from patient brains, and have to be taken into account when designing aggregation inhibitors and imaging agents for Alzheimer's disease.

Engineering vascularized soft tissue flaps in an animal model using human adipose-derived stem cells and VEGF+PLGA/PEG microspheres on a collagen-chitosan scaffold with a flow-through vascular pedicle.

Insufficient neovascularization is associated with high levels of resorption and necrosis in autologous and engineered fat grafts. We tested the hypothesis that incorporating angiogenic growth factor into a scaffold-stem cell construct and implanting this construct around a vascular pedicle improves neovascularization and adipogenesis for engineering soft tissue flaps. Poly(lactic-co-glycolic-acid/polyethylene glycol (PLGA/PEG) microspheres containing vascular endothelial growth factor (VEGF) were impregnated into collagen-chitosan scaffolds seeded with human adipose-derived stem cells (hASCs). This setup was analyzed in vitro and then implanted into isolated chambers around a discrete vascular pedicle in nude rats. Engineered tissue samples within the chambers were harvested and analyzed for differences in vascularization and adipose tissue growth. In vitro testing showed that the collagen-chitosan scaffold provided a supportive environment for hASC integration and proliferation. PLGA/PEG microspheres with slow-release VEGF had no negative effect on cell survival in collagen-chitosan scaffolds. In vivo, the system resulted in a statistically significant increase in neovascularization that in turn led to a significant increase in adipose tissue persistence after 8 weeks versus control constructs. These data indicate that our model-hASCs integrated with a collagen-chitosan scaffold incorporated with VEGF-containing PLGA/PEG microspheres supported by a predominant vascular vessel inside a chamber-provides a promising, clinically translatable platform for engineering vascularized soft tissue flap. The engineered adipose tissue with a vascular pedicle could conceivably be transferred as a vascularized soft tissue pedicle flap or free flap to a recipient site for the repair of soft-tissue defects.