Regarding the comment about SDS-PAGE potentially inducing artifactual dimers from the natural Aβ species present in AD brain extracts (as opposed to synthetic Aβ peptides): In previous work (Shankar et al., 2008, and Jin et al., 2011), we used just non-denaturing size-exclusion chromatography to obtain fractions of 8 kDa from typical AD brain soluble extracts (made in physiological buffer [TBS]) that contained dimers which we subsequently visualized on SDS-PAGE/Western blots. These data indicate that Aβ dimers of 8 kDa existed in the cortical extracts without any denaturing steps and could then be seen as just dimers (no monomers) on the subsequent blots (see Jin et al., 2011, Fig. 1A). So, even if SDS could induce some artifactual dimers, I definitely don't think all dimers recovered from AD brain soluble extracts are formed artifactually.
References:
Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ.
Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.
Nat Med. 2008 Aug;14(8):837-42.
PubMed.
Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ.
Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration.
Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5819-24.
PubMed.
At Crossbeta, we aim to use oligomeric species for drug discovery purposes, and we have put considerable effort in the characterization and isolation of biologically relevant oligomeric species. To overcome the problem of their transient and heterogeneous nature, we have developed a proprietary procedure for the reproducible preparation of stable Aβ1-42 oligomers that retain their biological activity (neuronal toxicity and inflammation) as well as receptor binding properties. These preparations are free of monomeric Aβ peptide and can be stored for over a month at 4 °C without any noticeable change in physical properties and composition. The availability of stable oligomers has enabled us to successfully run an HTS campaign, and resulted in the identification of several compounds that bind to the oligomers and can have more than 60 percent toxicity-neutralizing activity. Our stabilized, well-defined oligomers also might prove to be of great value in the development of a suitable biomarker. Several groups are currently testing our oligomers as reference standards, and the results so far look very promising.
Comments
Co-Director, Brigham and Women's Hospital's Ann Romney Center for Neurologic Diseases
Regarding the comment about SDS-PAGE potentially inducing artifactual dimers from the natural Aβ species present in AD brain extracts (as opposed to synthetic Aβ peptides): In previous work (Shankar et al., 2008, and Jin et al., 2011), we used just non-denaturing size-exclusion chromatography to obtain fractions of 8 kDa from typical AD brain soluble extracts (made in physiological buffer [TBS]) that contained dimers which we subsequently visualized on SDS-PAGE/Western blots. These data indicate that Aβ dimers of 8 kDa existed in the cortical extracts without any denaturing steps and could then be seen as just dimers (no monomers) on the subsequent blots (see Jin et al., 2011, Fig. 1A). So, even if SDS could induce some artifactual dimers, I definitely don't think all dimers recovered from AD brain soluble extracts are formed artifactually.
References:
Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.
Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ. Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5819-24. PubMed.
View all comments by Dennis SelkoeCrossbeta Biosciences
At Crossbeta, we aim to use oligomeric species for drug discovery purposes, and we have put considerable effort in the characterization and isolation of biologically relevant oligomeric species. To overcome the problem of their transient and heterogeneous nature, we have developed a proprietary procedure for the reproducible preparation of stable Aβ1-42 oligomers that retain their biological activity (neuronal toxicity and inflammation) as well as receptor binding properties. These preparations are free of monomeric Aβ peptide and can be stored for over a month at 4 °C without any noticeable change in physical properties and composition. The availability of stable oligomers has enabled us to successfully run an HTS campaign, and resulted in the identification of several compounds that bind to the oligomers and can have more than 60 percent toxicity-neutralizing activity. Our stabilized, well-defined oligomers also might prove to be of great value in the development of a suitable biomarker. Several groups are currently testing our oligomers as reference standards, and the results so far look very promising.
View all comments by Martijn GebbinkMake a Comment
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