Guangyuan Lu

Inhibition of γ-secretase activity reduces Aβ production, reduces oxidative stress, increases mitochondrial activity and leads to reduced vulnerability to apoptosis : Implications for the treatment of Alzheimer's disease

It has been argued that gamma-secretase should be considered as a pharmacological target, as there are few mechanism-based experimental and clinical studies on gamma-secretase treatment. In this study, we found that N2a cells bearing APP695 or its Swedish mutant exhibited increased basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as reduced level of the mitochondrial membrane potential and ATP. When the activity of gamma-secretase was inhibited by expression of the D385A PS1 variant, cells (N2a/Swe.D385A) showed reduced basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as increased mitochondrial membrane potential and ATP level. In addition, N2a/Swe.D385A cells showed reduced vulnerability to H(2)O(2)-induced apoptosis. The Bcl-2 and JNK/ERK pathways were proven to be involved in the change of vulnerability to H(2)O(2)-induced apoptosis. Moreover, we discovered that inhibition of gamma-secretase by DAPT would lead to a reduction of ROS levels and stabilization of mitochondrial function in APP (N2a/APP695) and APP Swedish mutant (N2a/APPswe) transfected cells. At last, it was shown that Abeta antibody and antiserum prevented increase of ROS and reduction of mitochondrial membrane potential in N2a/Swe.DeltaE9 cells but not in N2a/Swe.D385A cells, which indicated that reduced formation of Abeta was the reason for reduction of ROS formation and increase of mitochondrial membrane potential when PS-1 activity was impaired in N2a/Swe.D385A cells. We concluded that neurotoxicity was positively correlated with the activity of gamma-secretase, which suggested inhibition of gamma-secretase is a rational pharmacological target for Alzheimers disease treatment.

Preparation and Characterization of a Multilayer Biomimetic Scaffold for Bone Tissue Engineering

In scaffold based bone tissue engineering, both the pore size and the mechanical properties of the scaffold are of great importance. However, an increase in pore size is generally accompanied by a decrease in mechanical properties. In order to achieve both suitable mechanical properties and porosity, a multilayer scaffold is designed to mimic the structure of cancellous bone and cortical bone. A porous nano-hydroxyapatite—chitosan composite scaffold with a multilayer structure is fabricated and encased in a smooth compact chitosan membrane layer to prevent fibrous tissue ingrowth. The exterior tube is shown to have a small pore size (15—40 μm in diameter) for the enhancement of mechanical properties, while the core of the multilayer scaffold has a large pore size (predominantly 70—150 μm in diameter) for nutrition supply and bone formation. Compared with the uniform porous scaffold, the multilayer scaffold with the same size shows an enhanced mechanical strength and larger pore size in the center. More cells are shown to grow into the center of the multilayer scaffold in vitro than into the uniform porous scaffold under the same seeding condition. Finally, the scaffolds are implanted into a rabbit fibula defect to evaluate the osteoconductivity of the scaffold and the efficacy of the scaffold as a barrier to fibrous tissue ingrowth. At 12 weeks post operation, affluent blood vessels and bone formation are found in the center of the scaffold and little fibrous tissue is noted in the defect site.

Porous Chitosan Microcarriers for Large Scale Cultivation of Cells for Tissue Engineering: Fabrication and Evaluation*

Abstract Porous chitosan microspheres with diameters ranging from 180 μm to 280 μm were successfully prepared, using an anti-phase suspension method combined with temperature controlled freeze-extraction. The mean pore diameter could be regulated from 5 μm to 60 μm by varying the freezing temperature through the cooling rate. Results with in vitro chondrocyte cultures showed that cells could attach, proliferate, and spread on these porous microspheres as well as inside the microcarriers. The materials and cell co-cultures were characterized using both optical and scanning electron microscopy. These results show that the porous chitosan microspheres are promising candidates for tissue culture for use as an injectable tissue engineering scaffold.

Abnormal cleavage of APP impairs its functions in cell adhesion and migration.

Amyloid precursor protein (APP) is expressed ubiquitously but its wrong cleavage only occurs in central nervous system. In this research, overexpression of wild type human APP695 was found to stimulate the adhesion and migration of N2a cells. In the cells co-transfected by familial Alzheimers disease (FAD)-linked Swedish mutant of APP695 gene plus big up tri, openE9 deleted presenilin1 gene (N2a/Swe. big up tri, open9), however, this stimulating function was impaired compared to that in the cells co-transfected by Swedish mutant of APP695 gene plus dominant negative mutant of presenilin1 D385A gene (N2a/Swe.385). Furthermore, it was also found that the phosphorylation of FAK Tyr-861 and GSK-3beta Ser-9 was reduced in N2a/Swe.Delta9 cells, which can be possibly taken as a reasonable explanation for the underlying mechanism. Our results suggest that impaired cell adhesion and migration induced by abnormal cleavage of APP could contribute to the pathological effects in FAD brain.