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.

The formation of fibrillar deposits of amyloid beta protein (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). A central question is whether Abeta plays a direct role in the neurodegenerative process in AD. The involvement of Abeta in the neurodegenerative process is suggested by the neurotoxicity of the fibrillar form of Abeta in vitro. However, mice transgenic for the Abeta precursor protein that develop amyloid deposits in the brain do not show the degree of neuronal loss or tau phosphorylation found in AD. Here we show that microinjection of plaque-equivalent concentrations of fibrillar, but not soluble, Abeta in the aged rhesus monkey cerebral cortex results in profound neuronal loss, tau phosphorylation and microglial proliferation. Fibrillar Abeta at plaque-equivalent concentrations is not toxic in the young adult rhesus brain. Abeta toxicity in vivo is also highly species-specific; toxicity is greater in aged rhesus monkeys than in aged marmoset monkeys, and is not significant in aged rats. These results suggest that Abeta neurotoxicity in vivo is a pathological response of the aging brain, which is most pronounced in higher order primates. Thus, longevity may contribute to the unique susceptibility of humans to Alzheimer's disease by rendering the brain vulnerable to Abeta neurotoxicity.

Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD.

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Apoptosis plays a role in AIDS pathogenesis in the immune system, but its role in HIV-1-induced neurological disease is unknown. In this study, we examine apoptosis induced by HIV-1 infection of the central nervous system (CNS) in an in vitro model and in brain tissue from AIDS patients. HIV-1 infection of primary brain cultures induced apoptosis in neurons and astrocytes in vitro as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidium iodide staining and by electron microscopy. Apoptosis was not significantly induced until 1-2 wk after the time of peak virus production, suggesting induction by soluble factors rather than by direct viral infection. Apoptosis of neurons and astrocytes was also detected in brain tissue from 10/11 AIDS patients, including 5/5 patients with HIV-1 dementia and 4/5 nondemented patients. In addition, endothelial cell apoptosis was frequently detected in the brain of AIDS patients and was confirmed by electron microscopy. Most of the apoptotic cells were not localized adjacent to HIV-1-infected cells, providing further evidence for induction by soluble factors. In six non-AIDS control patients with normal brain, apoptotic cells were absent or limited to rare astrocytes. However, TUNEL-positive neurons and astrocytes were frequently detected in seven patients with Alzheimer's disease or abundant senile plaques. These studies suggest that apoptosis is a mechanism of CNS injury in AIDS which is likely to be induced by soluble factors. The apoptosis of endothelial cells in the CNS raises the possibility that some of these factors may be blood-derived. (

Deposition of fibrillar amyloid ␤ (fA␤) plays a critical role in Alzheimer's disease (AD). We have shown recently that fA␤-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, here we explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in A␤-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. Our data indicate that treatment of hippocampal neurons with fA␤ increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibited fA␤-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in A␤-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also showed early signs of AD pathology, such as intracellular A␤ and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology.