The P3A peptide is neurotoxic in cell culture and in vivo (9-12). Aggregation of (3A appears to be necessary to achieve a toxic state (13)(14)(15)(16)(17), although the precise physical form ofthe peptide that mediates toxicity is unknown. We recently demonstrated that diabetes-associated amylin is toxic to insulinproducing islet cells ofthe pancreas and showed that the toxic activity is mediated by the fibrillar form of the peptide (18) (1-42), and Fib-pA-(1-40) were stable in culture medium, whereas Am-pA-(1-40) dissolved gradually. Therefore, Am-PA-(1-40) was not used for comparison with fibrils. Amylin was dissolved in ddH20 to 350 uM and diluted immediately into islet cell cultures to 8 ,M. Electron microscopy and Congo red staining were performed as described (18).Cell Culture. Primary rat hippocampal cultures were prepared as described (9) and plated at 175 cells per r2 in 16-mm poly(L-lysine)-coated wells. After 4 days in culture, the medium was changed to serum-free Dulbecco's modified Eagle's medium with N2 and B27 supplements (GIBCO). One day later, peptides were added for a 3-day incubation. Neuronal viability was determined as described (9) in five 0.4-mm2 fields per well in triplicate wells (>300 neurons scored in controls). Rat pancreatic islet cell cultures were prepared, and cell viability was determined by propidium iodide staining as described (18). For immunocytochemistry, cultures were fixed in PBS containing 4% paraformaldehyde and 0.12 M sucrose, blocked with 5% (vol/vol) bovine serum albumin, and then incubated with primary antibody for 12 hr at 40C [microtubule-associated protein 2 (MAP-2) monoclonal antibody AP-20 from Sigma, 1:500; synaptophysin monoclonal antibody from Boehringer Mannheim, 1:200]. Cells were incubated with biotinylated anti-mouse IgG (1:200) and developed by using the ABC kit (Vector Laboratories) and diaminobenzidine. Presynaptic terminals were scored in neurons maintained for 8 days in culture and immunocytochemically stained for synaptophysin by counting synaptophysin dots in the initial 30 ,um of a dendrite in 30 identified viable neurons (>450 synapses scored in controls).Abbreviations: f3A, P-amyloid; Fib-,MA fibrillar /3A; Am-P8A amorphous pA; MAP-2, microtubule-associated protein 2.

The 37-amino-acid polypeptide amylin is the principal constituent of the amyloid deposits that form in the islets of Langerhans in patients with type-2 diabetes mellitus, but its role in the pathogenesis of this disease is unresolved. In view of the fact that the beta-amyloid protein that forms fibrils in Alzheimer's disease is toxic to neurons, we have investigated whether amylin fibrils could be toxic to pancreatic islet cells. We show here that human amylin is toxic to insulin-producing beta-cells of the adult pancreas of rats and humans. This toxicity is mediated by the fibrillar form of the amylin peptide and requires direct contact of the fibrils with the cell surface. The mechanism of cell death involves RNA and protein synthesis and is characterized by plasma membrane blebbing, chromatin condensation and DNA fragmentation, indicating that amylin induces islet cell apoptosis. These findings indicate that amylin fibril formation in the pancreas may cause islet cell dysfunction and death in type-2 diabetes mellitus.

A central issue in the pathogenesis of Alzheimer's disease (AD) is the relationship between amyloid deposition and neurofibrillary tangle formation. To determine whether amyloid fibril formation affects the phosphorylation state of tau, primary cultures of fetal rat hippocampal and human cortical neurons were treated with beta-amyloid (beta A) in a soluble, amorphous-aggregated, or fibrillar form. Fibrillar beta A, but not soluble or amorphous-aggregated beta A, markedly induces the phosphorylation of tau at Ser-202 and Ser-396/Ser-404, resulting in a shift in the tau M(r) in human cortical neurons. Hyperphosphorylated tau accumulates in the somatodendritic compartment of fibrillar beta A-treated neurons in a soluble form that is not associated with microtubules and is incapable of binding to microtubules in vitro. Dephosphorylation of beta A-induced tau restores its capacity to bind to microtubules. Thus, amyloid fibril formation alters the phosphorylation state of tau, resulting in the loss of microtubule binding capacity and somatodendritic accumulation, properties also exhibited by tau in the AD brain. Amyloid fibril formation may therefore be a cause of abnormal tau phosphorylation in AD.