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Jeffery W. Kelly | |
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The amyloid β-peptide (Aβ) refers to a family of peptides ranging from 39- to 43-residues that are formed through endoproteolytic processing of a 770-residue trans-membrane precursor protein in both the exocytic and endocytic pathways. The process of Aβ amyloidogenesis (Figure 1) appears to cause Alzheimer's disease based on biochemical and genetic evidence. Some cases of early onset Alzheimer's disease are associated with mutations that alter the sequence of Aβ or affect the processing of the precursor protein into a higher concentration of longer and more amyloidogenic sequences of Aβ. However, the vast majority of Alzheimer's disease cases are sporadic, not associated with any known mutations. Therefore, the occurrence of Alzheimer's disease in some individuals but not others is difficult to explain, because factors other than known mutations may initiate or enhance the formation of Aβ fibrils and neurodegeneration.
Inflammation is associated with the early stages of Alzheimer's disease. Reactive oxygen species produced during inflammation (during for instance a viral infection or an injury), including ozone, superoxide and hydroperoxides, can convert normal metabolites (e.g., cholesterol, arachidonic acid and unsaturated lipids) into highly reactive metabolites. These oxidative metabolites contain functional groups (e.g., aldehydes) that covalently modify proteins and alter their physical properties, especially natively unfolded proteins like Aβ and α-synuclein, the latter being associated with Parkinson's disease. In collaboration with Dr. Lerner and Dr. Wentworth (The Scripps Research Institute, Department of Chemistry), we discovered a new class of aberrant metabolites originating from cholesterol ozonolysis that exist in human brains. We found that these and other metabolites covalently modify Aβ and greatly increase its amyloidogenicity, suggesting that metabolite-initiated Aβ amyloidogenesis may contribute to the onset of sporadic Alzheimer's disease. We expanded this study by synthesizing Aβ conjugates that are site-specifically modified with the cholesterol aldehyde (3-β-hydroxy-5-oxo-5,6-secocholestan-6-al) at Asp1, Lys16, or Lys28, to evaluate the contribution of the different sites rather than studying mixtures. Modification at the different sites lowers the critical concentration for aggregation in all cases, making aggregation much more efficient at lower total Aβ concentrations. In contrast, the effect of metabolite modification on aggregation kinetics depended strongly on the location of the modification within Aβ. Aβ modified at Lys16 formed amorphous aggregates fastest and at the lowest concentrations (within 2 h at 20 nM), followed by the Lys28 and Asp1 conjugates. Furthermore, Aβ aggregates modified at Lys16 were more toxic to primary rat cortical neurons than unmodified Aβ under identical conditions and at the same concentration. Our results show that Aβ modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Aβ—seemingly explaining how Aβ could aggregate in humans when its physiological concentration is below its critical concentration.
The Aβ peptide aggregates into a mixture of quarternary structures, including cross β-sheet fibrils, and in Alzheimer's patients, there is better correlation between disease severity and the concentration of spherical aggregates, annular structures, protofibrils and other soluble oligomeric species than the concentration of fibrillar amyloid. Previously we have shown that mutating the Phe19-Phe20 backbone amide bond to an E-olefin bond allows the formation of spherical aggregates to the exclusion of fibrils. Comparing the toxicity of these aggregates to those formed by wild type Aβ or amide-to-ester mutant Aβ, we showed that all were similarly toxic to PC12 neuronal cells despite their different morphologies. This suggests that a common, but low abundance aggregate morphology mediates toxicity, or that several different aggregate morphologies are similarly toxic.
We seek to understand the structures of Aβ resulting in toxicity in tissues, and the roles that metabolites and catabolism (turnover) play in pathology. We are also interested to design immune strategies against Alzheimer's disease.
In collaboration with the Dillin Laboratory at the Salk Institute, we are very interested in understanding the role of aging signaling pathways in explaining the age onset of Alzheimer's disease and the role of stress-responsive signaling pathways in providing protection from proteotoxicity-associated phenotypes in C. elegans and murine models of Alzheimer's disease. Aggregation-mediated Aβ1-42 toxicity was dramatically reduced in a C. elegans model of Alzheimer's disease when aging was slowed by decreased insulin/insulin growth factor-1—like signaling (IIS). The downstream transcription factors, heat shock factor 1 and DAF-16, appear to regulate opposing disaggregation and aggregation activities to promote cellular survival in response to constitutive toxic protein aggregation. Because the IIS pathway is central to the regulation of longevity and youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated proteotoxicity.
We also reduced IIS in Alzheimer's model mice and discovered that these animals are protected from Alzheimer's like disease symptoms, including reduced behavioral impairment, neuroinflammation, and neuronal loss. This protection is correlated with the hyperaggregation of Aβ, as it was in the C. elegans model, leading to tightly packed, ordered plaques, suggesting that one aspect of the protection conferred by reduced IGF signaling is the sequestration of soluble Aβ oligomers into dense aggregates of lower toxicity. These findings indicate that insulin/insulin growth factor-1—like signaling-regulated mechanism that protects from Aβ toxicity is conserved from worms to mammals and point to the modulation of IIS as a promising strategy for the development of Alzheimer’s disease therapy.
