Lymphocyte toxicity of prion fragments.

Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects.

Cytotoxic and membrane perturbation effects of a novel amyloid forming model peptide poly(leucine-glutamic acid).

In the present study we have elucidated the toxicity of a novel amyloid forming model peptide, Poly (leucine-glutamic acid). The toxicity of the fibrils prepared from this peptide was analyzed in peripheral blood lymphocytes (PBL). The MTT reduction assay revealed that the viability of PBL decreases significantly upon treatment with Poly(leucine-glutamic acid) (Poly [LE]). Enhanced DCFH-DA fluorescence in treated cells suggests that peptide toxicity is probably mediated by the formation of free radicals. In vivo and in vitro biochemical studies indicated that Poly [LE] inactivates the antioxidant system of cells. Perturbation of Poly [LE] in a membrane lipid environment was assessed by circular dichroism (CD) using phosphotidyl choline-cholesterol bilayers. The CD results revealed that LE enhances its beta sheet content in a bilayer environment. Sequestration of Poly [LE] in lipid rafts demonstrates that it has a binding cleft similar to Abeta in lymphocyte raft domains. Nuclear membrane binding studies showed that Poly [LE] binds to nuclear membranes and may cause genotoxicity.

Red cell perturbations by amyloid beta-protein.

Amyloid beta-protein (A beta) accumulation in brain is thought to be important in causing the neuropathology of Alzheimer's disease (AD). A beta interactions with both neurons and microglial cells play key roles in AD. Since vascular deposition of A beta is also implicated in AD, the interaction of red cells with these toxic aggregates gains importance. However, the effects of A beta interactions with red blood cells are less well understood. Synthetic amyloid beta-protein (1-40) was labeled with biotin and preincubated at 37 degrees C for 4, 14 and 72 h to produce fibrils. Flow cytometry was used to study the binding of these fibrils to red cells. The amyloid fibrils had a high affinity for the red cell with increased binding for the larger fibrils produced by longer preincubation. Bovine serum albumin (BSA) did not reverse the binding, but actually resulted in a more efficient binding of the A beta fibrils to the red cells. The interaction of A beta with red cells increased the mean cell volume and caused the cells to become more spherical. This effect was greater for the longer fibrils. At the same time the interaction of A beta with red cells produced an increase in their fluorescence measured after 16-h incubation at 37 degrees C. This increase in fluorescence is attributed to the formation of fluorescent heme degradation products. The effect of prior hemoglobin oxidation, catalase inhibition and glutathione peroxidase inhibition indicated that the amyloid-induced oxidative damage to the red cell involved hydrogen peroxide-induced heme degradation. These results suggest that amyloid interactions with the red cell may contribute to the pathology of AD.