BackgroundLactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ) of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP).Methodology/Principal FindingsLactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y), whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes.Conclusions/SignificanceThese findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.
Parvovirus B19 has potential as a gene therapy vector because of its restricted tropism for human erythroid progenitor cells in the bone marrow. B19 binds to the cell surface through P antigen and we identified activated beta(1) integrins as coreceptors for internalization. Because differentiation with phorbol ester induces beta(1) integrin coreceptor activity, but cell differentiation is not desirable in gene transfer to human progenitor cells and one of the downstream effectors of phorbol esters is the small GTPase Rap1, the role of Rap1 in the recruitment of beta(1) integrins on hematopoietic cells was examined. Expression of a constitutively active Rap1 (63E) was sufficient to recruit beta(1) integrin coreceptors in erythroleukemic K562 cells by inducing high-affinity integrin conformation. A crucial role of actin polymerization in Rap1-mediated beta(1) integrin recruitment was documented by complete inhibition of the 63E Rap1 effect with low-dose cytochalasin D and by the ability of a constitutively active mutant of the actin cytoskeleton regulator Rac1 to sensitize K562 cells to the pharmacological activation of endogenous Rap1, using the Rap1 exchange factor-specific 8-pCPT-2'-O-Me-cAMP [8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate]. Interestingly, in primary human erythroid progenitor cells, 8-pCPT-2'-O-Me-cAMP was sufficient to significantly increase B19-mediated gene transfer, suggesting that these cells possess the cytoskeleton organization capacity required for efficient recruitment of beta(1) integrins by brief pharmacological stimulation of Rap1 GTP loading. Because 8-pCPT-2'-O-Me-cAMP has been implicated in enhanced homing of progenitor cells, these results identify a novel tool with which to optimize ex vivo B19-mediated gene transfer and potentially improve homing of transduced cells by Rap1-beta(1) integrin activation with 8-pCPT-2'-O-Me-cAMP.
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