Nucleation-Dependent Polymerization Is an Essential Component of Amyloid-Mediated Neuronal Cell Death

Abstract

Accumulating evidence suggests that amyloid protein aggregation is pathogenic in many diseases, including Alzheimer's disease. However, the mechanisms by which protein aggregation mediates cellular dysfunction and overt cell death are unknown. Recent reports have focused on the potential role of amyloid oligomers or protofibrils as a neurotoxic form of amyloid-␤ (A␤) and related amyloid aggregates. Here we describe studies indicating that overt neuronal cell death mediated by A␤ 1-40 is critically dependent on ongoing A␤ 1-40 polymerization and is not mediated by a single stable species of neurotoxic aggregate. The extent and rate of neuronal cell death can be controlled by conditions that alter the rate of A␤ polymerization. The results presented here indicate that protofibrils and oligomeric forms of A␤ most likely generate neuronal cell death through a nucleation-dependent process rather than acting as direct neurotoxic ligands. These findings bring into question the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide formazan assay (MTT assay) as a reporter of A␤-mediated neuronal cell death and suggest that diffusible A␤ protofibrils and oligomers more likely mediate subtle alterations of synaptic function and long-term potentiation rather than overt neuronal cell death. These results have been extended to A␤ 1-42 , the non-A␤ component of Alzheimer's disease amyloid plaques, and human amylin, suggesting that nucleationdependent polymerization is a common mechanism of amyloid-mediated neuronal cell death. Our findings indicate that ongoing amyloid fibrillogenesis may be an essential mechanistic process underlying the pathogenesis associated with protein aggregation in amyloid disorders.

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…Similarly, using SEC-isolated Aβ42 protofibrils, we have shown that monomeric Aβ40 inhibited Aβ42 protofibril toxicity toward cultured neurons by altering their aggregation properties and inhibiting fibril formation. These observations reinforced the importance of an ongoing Aβ-nucleated polymerization as a critical factor for Aβ-induced toxicity 21,66 .…”

Soluble Oligomers Require a Ganglioside to Trigger Neuronal Calcium Overload

“…The different behavior of A-oligomers (non-toxic) with respect to A+ oligomers (toxic) was confirmed in human SH-SY5Y neuroblastoma cells by using the MTT assay (Fig. 1B), taking into account some concerns regarding a nonspecific interference of A␤ peptide with MTT reduction [54]. However, A-oligomers become significantly toxic in cells enriched in GM1 content by a factor of ca.…”

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

“…We first compared the toxic properties of the purified α-syn species in human dopaminergic neuroblastoma cell lines (M17 and SH-S5Y5 cells). Using the uptake of the vital dye propidium iodide (PI) as a marker for membrane disruption and toxicity, in combination with flow cytometry quantification, we observed that neither α-syn monomers ( Although several studies have shown that different aggregated forms of α-syn exhibit toxicity in various cellular 17,20,23,32 and in vivo models, 12,32 increasing evidence from our group 27,33 and others 34 suggest that amyloid toxicity could be linked to the process of amyloid formation rather than to specific aggregated forms of amyloidogenic proteins. To determine whether this applies to α-syn-induced toxicity, we assessed the effect of inducing fibril growth by the addition of α-syn PFFs (10%) to monomeric α-syn solutions.…”