Amyloid beta resistance in nerve cell lines is mediated by the Warburg effect

Abstract

Amyloid beta (Aβ) peptide accumulation in the brains of patients with Alzheimer's disease (AD) is closely associated with increased nerve cell death. However, many cells survive and it is important to understand the mechanisms involved in this survival response. Recent studies have shown that an anti-apoptotic mechanism in cancer cells is mediated by aerobic glycolysis, also known as the Warburg effect. One of the major regulators of aerobic glycolysis is pyruvate dehydrogenase kinase (PDK), an enzyme which represses mitochondrial respiration and forces the cell to rely heavily on glycolysis, even in the presence of oxygen. Recent neuroimaging studies have shown that the spatial distribution of aerobic glycolysis in the brains of AD patients strongly correlates with Aβ deposition. Interestingly, clonal nerve cell lines selected for resistance to Aβ exhibit increased glycolysis as a result of activation of the transcription factor hypoxia inducible factor 1. Here we show that Aβ resistant nerve cell lines upregulate Warburg effect enzymes in a manner reminiscent of cancer cells. In particular, Aβ resistant nerve cell lines showed elevated PDK1 expression in addition to an increase in lactate dehydrogenase A (LDHA) activity and lactate production when compared to control cells. In addition, mitochondrial derived reactive oxygen species (ROS) were markedly diminished in resistant but not sensitive cells. Chemically or genetically inhibiting LDHA or PDK1 re-sensitized resistant cells to Aβ toxicity. These findings suggest that the Warburg effect may contribute to apoptotic-resistance mechanisms in the surviving neurons of the AD brain. Loss of the adaptive advantage afforded by aerobic glycolysis may exacerbate the pathophysiological processes associated with AD.

Figure 1. LDHA activity and lactate levels are elevated in Aβ-resistant cells.

A) LDHA activity was significantly greater in PC12 Aβ resistant cells lines, R1 and R7, as compared to the parental line both in the absence and presence of Aβ (*, P<0.01). As a control, PC12 parental cells exposed to 24 hr hypoxia (1%O₂) also exhibited significantly greater LDHA activity when compared to untreated parental cells (*, P<0.01). B) LDHA activity was also significantly greater in B12 Aβ resistant cells lines, R2 and R4, as compared to the parental line under the same conditions (* P<0.01). B12 parental cells exposed to 24 hr hypoxia (1%O₂) also exhibited greater LDHA activity when compared to untreated parental cells (*, P<0.01). (C) Extracellular lactate was significantly elevated in PC12 Aβ-resistant lines, R1 and R7 and (D) B12 Aβ resistant lines, R2 and R4, when compared to their respective parental cells cultured under similar conditions (*; P<0.01). Data represent the average ± SD of three independent experiments. Data was analyzed by a one-way ANOVA followed by a Tukey test.

Figure 2. PDK1 is upregulated in Aβ resistant cells.

A) PDK1 levels were significantly elevated in both PC12 resistant lines, R1 and R7, when compared to the parental cell line (*,P<0.01). These elevated levels were maintained following 24 and 48 hr Aβ treatment (* 20 µM). B) PDK1 levels were also significantly greater in both B12 resistant lines, R2 and R4, when compared to the parental line (*,P<0.01; **,P<0.05). These increased levels were also maintained with Aβ treatment. An additional band of approximately 30 kDa that was more prominent than full length PDK1 (∼48 kDa). This additional band also showed the same elevated trend as full length PDK1 in the R2 and R4 lines when compared to the parental under similar conditions. The smaller band may represent a cleavage product of PDK1. Densitometric analysis of PDK1 band densities relative to actin are found in the lower panel. Relative intensity was calculated by comparing the PDK1/actin ratio of the resistant lines to the same ratio in the parental cell line. Data represent the average ± SD of three independent experiments.

Figure 3. Decreased mitochondrial reactive oxygen species in Aβ resistant cells.

A) PC12 Aβ resistant lines R1 and R7 exhibited a significant reduction in mitochondrial reactive oxygen species (ROS) compared to the parental cell line under normal culture conditions (*, P<0.01). This decrease in ROS was maintained with 48 hr Aβ (20 µM) exposure. Conversely, mitochondrial ROS significantly increased in the parental line when exposed to Aβ (**, P<0.05). B) The B12 resistant lines, R2 and R4 also exhibited decreased mitochondrial ROS when compared to parental cells under similar conditions (*, P<0.01). A similar increase in mitochondrial ROS in B12 parental cells was also observed following treatment with Aβ for 48 hr (**, P<0.05). Cells were stained with MitoTracker Red (100 nM) and nuclei were stained with Hoescht (10 µg/ml) and visualized by fluorescence microscopy at 400X magnification and quantified with ImageJ software. Data represent the average ± SD of three independent experiments. Data was analyzed by a one-way ANOVA followed by a Tukey test.

Figure 4. Chemically inhibiting LDHA or PDK1 decreases cell viability in Aβ resistant cells.

A significant decrease in cell viability in both PC12 (A) and B12 (B) resistant lines was observed after 48 hr concomitant exposure to Aβ (20 µM) and 20 mM oxamate (ox), a chemical inhibitor of LDHA (*P<0.01). Similarly, 48 hr Aβ exposure significantly decreased cell viability in both PC12 (C) and B12 (D) resistant lines when cells were co-treated with 2.5 mM dichloroacetate (DCA), a chemical inhibitor of PDK1 (*P<0.01). Interestingly, the cell viability of the parental lines does not appear to decrease with treatment of either inhibitor and Aβ. Cell viability was determined by the reduction of the tetrazolium salt MTT. Data are representative of three separate experiments. Data was analyzed by a one-way ANOVA followed by a Tukey test.

Figure 5. Attenuated LDHA or PDK1 expression increases sensitivity of resistant cell lines to Aβ.

A) Immunoblot analysis of PC12 R7 resistant cells stably transfected with LDHA-specific shRNA vectors revealed two clonal cell lines (clones 71-7,72-11) exhibited a significant decrease in LDHA protein levels when compared to an R7 cell line transfected with a non-specific shRNA (SCR) (*P<0.01). B) Immunoblot analysis also confirmed a significant decrease in PDK1 protein in two PDK-1 shRNA stably transfected R7 cell lines, (clones 29-1 and 30-10) when compared to the control cell line (*P<0.01). C) A significant decrease in the cell viability of the R7 clones with attenuated expression of either LDHA or PDK1 when exposed to Aβ (20 µM) for 48 hr when compared to the control (* P<0.01). D) Immunoblots of R2 cell lines (clones 71-23 and 72-5) confirming significantly decreased LDHA expression when compared to a control cell line (SCR) (*P<0.01). E) R2 clonal cell lines, (clones 29-12 and 30-4) stably expressing PDK-1 shRNA showed a significant decrease in the PDK1 full length protein (∼48 kDa) as well as a decrease in the proposed PDK1 cleavage product (∼30 kDa) when compared to the control (*P<0.01). F) Both R2 LDHA and PDK1 knockdown cell lines exposed to 48 hr Aβ (20 µM) treatment showed a significant decrease in cell viability when compared to R2 control (* P<0.01). Densitometric analysis of LDHA and PDK1 band densities relative to actin are found below the corresponding blot. Relative intensity was calculated by comparing the LDHA/actin or PDK1/actin ratio of the resistant lines to the same ratio in the scrambled shRNA cell line. Data represent the average ± SD of three independent experiments. Data was analyzed by a one-way ANOVA followed by a Tukey test.

Figure 6. Proposed role of the Warburg effect in Aβ resistant cells.

The stabilization of hypoxia inducible factor 1 α (HIF1α) and subsequent increased activity of HIF-1 in amyloid beta (Aβ) resistant cells stimulates increased expression of glucose transporters (Glut1), and glycolytic enzymes increasing the conversion of glucose to pyruvate. Additionally, HIF-1 promotes the reduction of pyruvate to lactate through the upregulation of lactate dehydrogenase A (LDHA). HIF-1 also actively suppresses the production of acetyl CoA through the mitochondria via increased expression of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits pyruvate dehydrogenase (PDH) resulting in decreased flux through the tricarboxcylic acid (TCA) cycle and repressed oxidative phosphorylation (OXPHOS). The decrease in electron transport activity decreases the generation of reactive oxygen species (ROS) in the mitochondria and renders cells more resistant to apoptosis in the presence of Aβ.