Lactic Acid Induces Aberrant Amyloid Precursor Protein Processing by Promoting Its Interaction with Endoplasmic Reticulum Chaperone Proteins

Lactic 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).Lactic 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.These 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

Lactic acid decreases extra- and intracellular pH.

<p>(A) Intracellular pH was measured using the pH-sensitive dye BCECF-AM. Fluorescence of SH-Y5Y cells exposed to 6 mM and 12 mM LA for 6 h and untreated control cells were scanned using a Leica TCS SP5 Laser Scanning Confocal Microscope. (B) The pH of culture media was measured with an UltraBasic UB-10 pH/mV meter (A. Daigger & Company, Chicago, IL, USA). Mean pH levels are noted by horizontal lines. (C) Viability of cells exposed to different concentrations of lactic acid was determined using an MTT Cell Viability Assay. (D) The percentage of dead (necrotic) cells at different concentrations of lactic acid was determined using Annexin V-FITC and Propidium iodide fluorescence.</p

Lactic acid alters APP processing – lowering alpha secretase cleavage in SH-SY5Y cells.

<p>(A) APP processing was examined by immunoblot analysis of cell lysates and culture medium using the monoclonal antibody 6E10, which binds to amino acids 3-8 of APP (EFRHDS) and therefore detects full-length APP, sAPPα, and Aβ. Human SH-SY5Y neuroblastoma cells were cultured in the absence and presence of 12 mM lactic acid (LA) for 6 h. Accumulation of APP immunoreactivity in cells exposed to lactic acid and decreased levels of sAPPα in medium were detected. (B) Immunoprecipitation using 6E10 antibody followed by immunoblot confirmed the increased presence of APP in SH-Y5Y cells following exposure to 12 mM lactic acid (upper panel). An abundantly expressed control protein, heat shock protein 90 (HSP90), could not be detected in the immuno-precipitate (middle and lower panels).</p

Lactic acid and HCl stimulate the secretion of Aβ40 and 42.

<p>The levels of Aβ40 (A) and 42 (B) were measured by ELISA in culture medium of SH-SY5Y cells exposed to 12 mM lactic acid for 6 h. (C) Aβ40 levels were measured in culture medium of SH-SY5Y cells exposed to 12 mM HCl for 6 h. The intracellular pH of cells exposed to 12 mM HCl dropped to approximately pH 5.5 (not shown). Mean levels of Aβ40 and 42 are noted by horizontal lines.</p

Lactic acid induces APP aggregation.

<p>(A) Fluorescence microscopy studies of SH-SY5Y cells were performed using the 6E10 antibody, which detects full-length APP, sAPPα, and Aβ peptides, respectively. Cells were untreated (Control) or exposed to 12 mM lactic acid (LA) for 24 h, fixed and stained with 6E10 and DAPI and examined by fluorescence microscopy. Perinuclear APP-positive material was detected in a subset of cells exposed to lactic acid. (B) Blinded evaluation of cells for the presence of APP-positive inclusions by two independent investigators revealed their presence in ∼2.5% of control cells, 6% of lactic-acid-treated and ∼8% of HCl-treated cells. (C) Fluorescence microscopy study of control and lactic acid-treated SH-SY5Y cells for APP (red) and intermediate filaments (green) using 6E10 and anti-vimentin antibodies, respectively. DNA was visualized with DAPI stain (blue). Following lactic acid treatment, the occurrence of vimentin filaments surrounding the inclusion-like structures reminiscent of aggresomes (inset) is observed.</p

Lactic acid increases the expression of ER stress–dependent chaperone proteins Grp78 and Grp94 and enhances their interaction with APP.

<p>(A) Immunoblot analysis of SH-SY5Y cell lysates in the absence and presence of 12 mM lactic acid was performed using an antibody to the ER retrieval sequence KDEL, which is present in both, Grp78 and Grp94 proteins. Increased expression of Grp94 and to a lesser extent Grp78, was detected upon lactic acid treatment. (B) Immunoprecipitation using the anti-KDEL antibody revealed the increased presence of APP in complex with KDEL proteins following exposure of cells to lactic acid. An abundantly expressed control protein, heat shock protein 90 (HSP90, lower right panel), could not be detected in the immunoprecipitate (lower left panel). (C) Fluorescence microscopy study of APP using 6E10 (green) and KDEL proteins (red) documented co-localization (yellow) of APP and KDEL proteins in SH-SY5Y cells exposed to lactic acid.</p