Oligomeric amyloid decreases basal levels of brain-derived neurotrophic factor (BDNF) mRNA via specific downregulation of BDNF transcripts IV and V in differentiated human neuroblastoma cells

Abstract

Alzheimer's disease (AD) is a senile dementia characterized by amyloid plaques, neurofibrillary tangles, and synaptic and cell loss. The "amyloid cascade" hypothesis suggests that amyloid-beta (Abeta), the peptide deposited as amyloid plaques, is the primary insult in AD. However, debate continues over the mechanism of Abeta toxicity and whether fibrillar or oligomeric Abeta is the active species of the peptide that ultimately causes the synaptic loss and dementia associated with AD. Brain-derived neurotrophic factor (BDNF) is required for survival and function of cells compromised in AD. Decreased BDNF causes defects in long-term potentiation and memory and correlates with cognitive decline. We previously demonstrated that BDNF reduction occurs early in the course of AD, suggesting that decreased BDNF may promote neuronal dysfunction in AD. We also demonstrated that three of seven human BDNF transcripts are specifically downregulated in AD. What pathological feature(s) of AD leads to the decreased BDNF is unknown. In this study, we administered both fibrillar and oligomeric conformations of Abeta(1-42) to differentiated SH-SY5Y, a human neuroblastoma cell line, and measured both phosphorylated cAMP response element-binding protein (CREB), a regulator of BDNF transcription, and BDNF total mRNA. We found that oligomeric but not fibrillar preparations of Abeta(1-42) significantly decrease both phosphorylated CREB and total BDNF mRNA. Furthermore, oligomeric Abeta(1-42) decreases BDNF transcripts IV and V in these cells, demonstrating that Abeta(1-42) downregulates the major BDNF transcript decreased in vivo in the AD brain. Thus, oligomeric Abeta(1-42) could compromise neuronal function, causing memory loss and cognitive dysfunction by downregulation of BDNF in AD.

Figure 1.

Human BDNF gene with mRNA transcripts. A, Diagram showing the entire BDNF gene with the noncoding exons in white spliced to the coding exon in black. The asterisk (*) indicates splice variants, of which three are known, two on exon II and one on exon VI for a total of 10 transcripts. B, PCR products for all seven BDNF transcripts from retinoic acid-differentiated SH-SY5Y cells at 9 d in vitro. Lanes numbered I–VII correspond to the transcripts in A. PCR primer sequences and size of fragments are found in Table 1. The splice variants, although present, are not significant products in lanes 2 and 6. A 100 bp ladder is shown on the left.

Figure 2.

Measurement of total BDNF mRNA after 3, 5, and 9 d of growth in different media. BDNF is significantly increased in N-2 supplements and 10 μm RA compared with N-2 supplements alone or 10% FBS at 3, 5, and 9 d (*p < 0.01; **p < 0.001). In addition, comparing BDNF mRNA levels for N-2 and RA across the time points, there is a significant increase in BDNF mRNA between days 3 and 9 (*p = 0.036) and between days 5 and 9 (*p = 0.022). Error bars represent SEM; n = 3.

Figure 3.

Western blotting of inactivated caspase-3 and -7. A, No difference in levels of inactivated caspase-3 and -7 among differently treated RA-differentiated SY5Y cells (one-way ANOVA, p = 0.354 for caspase-3, and p = 0.921 for caspase-7). Analysis is based on three independent experiments; n = 9. Error bars represent SEM. B, Western blot shows only inactivated caspase-3 and -7 were detected after treatment with Aβ_1–42. As a positive control, SY5Y cells were treated with 25 μm etoposide, an apoptosis-inducing reagent. After 24 h, no inactivated caspase-3 or -7 was detected.

Figure 4.

Western blot of fibrillar and oligomeric Aβ_1–42 speciation. The oligomeric preparation (lane 1) contains monomers (4 kDa), small oligomeric trimers (∼12 kDa), and the larger oligomeric amyloid (40–98 kDa), but no fibrils. The fibrillar amyloid (lane 2) contains both monomeric amyloid and fibrillar amyloid (>250 kDa), but no oligomers. The procedures for amyloid speciation were also performed on the scrambled peptide, containing the same amino acid composition as Aβ_1–42, which served as a negative control and did not cross-react with the amyloid antibody (lanes 3 and 4).

Figure 5.

BDNF mRNA levels in RA-differentiated SY5Y cells exposed to oligomeric or fibrillar Aβ. Five micromolar Aβ_1–42, treated to form either oligomeric or fibrillar species, was added to RA-differentiated SH-SY5Y cells for 48 h. Only oligomeric amyloid caused a significant decrease (**p < 0.001) in BDNF mRNA (exon VIII) compared with no peptide and scrambled oligomeric amyloid controls. There was no change in BDNF exposed to fibrillar amyloid compared with either no peptide or scrambled fibril controls. The analysis is based on two independent experiments; n = 6. Error bars represent SEM.

Figure 6.

Measurement of BDNF transcripts I, II, IV, and V in RA-differentiated SY5Y cells exposed to oligomeric or fibrillar Aβ. A, A significant increase (*p = 0.034) in BDNF transcript I after oligomeric amyloid administration compared with both control conditions. B, No change in BDNF transcript II levels among the five experimental conditions. C, D, Significant decreases in BDNF transcripts IV and V (**p = 0.001 and *p = 0.015, respectively) after oligomeric amyloid administration compared with both controls. There was no change in any BDNF transcript level after fibrillar amyloid administration compared with either control. Transcripts III, VI, and VII were below the levels of detection. The analysis is based on two independent experiments; n = 6. Error bars represent SEM.

Figure 7.

Western blots of T-CREB and P-CREB levels in RA-differentiated SY5Y cells treated with oligomeric or fibrillar Aβ. RA-differentiated SY5Y cells administered oligomeric amyloid demonstrate significantly decreased phosphorylated CREB levels (*p = 0.005). Levels of total CREB were unchanged among all five conditions (one-way ANOVA, p = 0.237). The analysis is based on three independent experiments; n = 9. Error bars represent SEM.