Vascular endothelial growth factor isoforms differentially protect neurons against neurotoxic events associated with Alzheimer's disease

Abstract

Alzheimer's disease (AD) is the most common cause of dementia, the chronic and progressive deterioration of memory and cognitive abilities. AD can be pathologically characterised by neuritic plaques and neurofibrillary tangles, formed by the aberrant aggregation of β-amyloid and tau proteins, respectively. We tested the hypothesis that VEGF isoforms VEGF-A₁₆₅a and VEGF-A₁₆₅b, produced by differential splice site selection in exon 8, could differentially protect neurons from neurotoxicities induced by β-amyloid and tau proteins, and that controlling expression of splicing factor kinase activity could have protective effects on AD-related neurotoxicity in vitro. Using oxidative stress, β-amyloid, and tau hyperphosphorylation models, we investigated the effect of VEGF-A splicing isoforms, previously established to be neurotrophic agents, as well as small molecule kinase inhibitors, which selectively inhibit SRPK1, the major regulator of VEGF splicing. While both VEGF-A₁₆₅a and VEGF-A₁₆₅b isoforms were protective against AD-related neurotoxicity, measured by increased metabolic activity and neurite outgrowth, VEGF-A₁₆₅a was able to enhance neurite outgrowth but VEGF-A₁₆₅b did not. In contrast, VEGF-A₁₆₅b was more effective than VEGF-A₁₆₅a in preventing neurite "dieback" in a tau hyperphosphorylation model. SRPK1 inhibition was found to significantly protect against neurite "dieback" through shifting AS of VEGFA towards the VEGF-A₁₆₅b isoform. These results indicate that controlling the activities of the two different isoforms could have therapeutic potential in Alzheimer's disease, but their effect may depend on the predominant mechanism of the neurotoxicity-tau or β-amyloid.

Keywords: Alzheimer’s disease; SRPK1; VEGF; amyloid-beta; splicing; tau.

Figure 1

VEGF-A isoforms increase metabolic activity of SHSY5Y cells but not N2a cells co-treated with hydrogen peroxide. SHSY5Y cells and N2a cells were treated with H₂O₂ alone or with NGF or 2.5 nM VEGF-A isoforms for 24 h, and a WST-1 assay was performed to measure metabolic activity. Readings were normalized against an assay control (media + WST-1 reagent) and experimental control (cells treated with PBS or NGF alone + WST-1 reagent). (A) Concentration-dependent decrease in percent metabolic activity of SHSY5Y cells with H₂O₂ without (control) or with NGF, VEGF-A₁₆₅a, or VEGF-A₁₆₅b (two-way ANOVA, p < 0.01). (B) Concentration-dependent decrease in percent metabolic activity of N2a cells with H₂O₂ without (control) or with NGF, VEGF-A₁₆₅a, or VEGF-A₁₆₅b (two-way ANOVA, p < 0.0001). N = 8 or N = 12 readings. ^*VEGF-A₁₆₅a different from control; ^##VEGF-A₁₆₅b different from control; and ^*NGF different from control. ^*p < 0.05, ^{##, **}p < 0.01, compared with control, Holm Sidak post hoc tests. N = 12 per group per concentration.

Figure 2

β-amyloid (Aβ) decreased cell survival as measured by metabolic activity of SHSY5Y cells but not in N2a cells. SHSY5Y cells and N2a cells were treated with 1 μM Aβ for 24 and 48 h. WST-1 assay was performed to measure metabolic activity and readings were normalised against an assay control (media + WST-1 reagent) and experimental control (cells treated with vehicle alone + WST-1 reagent). N = 6 readings. (A) After both 24 and 48 h treatment, SHSY5Y cell metabolic activity was significantly decreased with 1 μM Aβ (one-way ANOVA, p < 0.001). (B) N2a cells had a variable response to Aβ; metabolic activity was reduced but not to a significant degree (one-way ANOVA, p > 0.05). (C,D) To confirm the assay worked, cisplatin treatment was shown to decrease metabolic activity in both SHSY5Y cells and N2a cells. ns, not significant; ^*p < 0.05, ^***p < 0.001; ^****p < 0.0001 compared with vehicle, Holm Sidak post hoc test.

Figure 3

VEGF-A₁₆₅a or VEGF-A₁₆₅b treatment recovers Aβ-induced decrease in metabolic activity in SHSY5Y cells. (A) Normalised metabolic activity showed decrease with 1 μM Aβ (one-way ANOVA, p < 0.0001) and was recovered with co-treatment of 2.5 nM VEGF-A₁₆₅a. Treatment with 2.5 nM VEGF-A₁₆₅a alone did not significantly change metabolic activity. (B) Similarly, 2.5 nM VEGF-A₁₆₅b alone did not change metabolic activity but protected against Aβ-induced reduction. N = 4–6. ^*p < 0.05, ^****p < 0.0001 compared with vehicle, Holm Sidak post hoc tests.

Figure 4

Reduced neurite outgrowth with increased concentration of OA. (A) Neuron-specific marker βIII tubulin identified neurites in SHSY5Y cells shown in green. Cell nuclei stained with Hoechst shown in blue. Merged images in final row. N = 18 images per condition. (B) Decreased number of live cells with increased concentration of OA. Nuclei automatically counted on FIJI software with a macro. (C) Neurite length was quantified using simple tracer and the sum neurite length was divided by number of nuclei. The outgrowth in SHSY5Y cells treated with OA was plotted as percentage of DMSO control. Points = mean, error bars = SEM. ^****p < 0.0001 compared with vehicle. One way ANOVA with Holm Sidak post hoc tests. N = 3 with six images analysed per repeat.

Figure 5

VEGF-A₁₆₅a increased neurite outgrowth in SHSY5Y cells co-treated with OA. (A) neuron-specific marker βIII tubulin identified neurites in green. Cell nuclei stained with Hoechst shown in blue. N = 18 images per condition. (B) Using FIJI software, neurite length was quantified using simple tracer, and the sum neurite length was divided by number of nuclei counted with a macro. Plot showed that average neurite length is significantly and consistently higher with VEGF-A₁₆₅ in control, 1 and 3 nM OA treatments (two-way ANOVA, p < 0.0001). With 10 nM OA, VEGF-A₁₆₅ did not have an effect. By itself, VEGF-A₁₆₅b did not increase average neurite length but maintained outgrowth in SHSY5Y cells co-treated with 1 and 3 nM OA where average length was significantly higher than PBS control (two-way ANOVA, p < 0.0001). With 10 nM OA, VEGF-A₁₆₅b did not have an effect. (C) Neurite outgrowth was plotted as percentage of DMSO control with and without VEGF-A₁₆₅ treatment. Points = mean, error bars = SEM. ^*p < 0.05 compared with PBS, ^**p < 0.01 compared with PBS, ^***p < 0.001 compared with PBS, ^###p < 0.001 compared with VEGF-A₁₆₅b. Two way ANOVA with Holm Sidak post hoc tests. N = 3 with six images analysed per repeat.

Figure 6

Standard curves for the (A) VEGF-A₁₆₅b ELISA and (B) VEGF-A₁₆₅a ELISA, which show detection window for protein expression. Points on each graph are average of two independent ELISA plates, run for SHSY5Y cell lysates and media samples.

Figure 7

Sphinx31 treatment significantly reduces VEGF-A₁₆₅a:VEGF-A₁₆₅b ratio expression in SHSY5Y cells. (A) Sphinx31 does not produce measurable change in VEGF-A₁₆₅a expression in cell lysate. (B) Cell lysate shows a significant increase in VEGF-A₁₆₅b Sphinx31 (p < 0.001). (C) The ratio of VEGF-A₁₆₅a:VEGF-A₁₆₅b is significantly reduced in cell lysate with Sphinx31 treatment, but this does not occur in a concentration-dependent manner (p < 0.001 one-way ANOVA). (D) VEGF-A₁₆₅a in cell media remains stable with 1–3 μM Sphinx31 but is significantly reduced with 10 μM treatment (p < 0.0001, one-way ANOVA). (E) There was an increase in VEGF-A₁₆₅b in the media with Sphinx31 treatment. Measured increase with 3 μM is statistically significant (p < 0.05, one-way ANOVA). (F) The ratio of VEGF-A₁₆₅a:VEGF-A₁₆₅b is dose dependently reduced in media after treatment with Sphinx31 (p < 0.001 and p < 0.05, one-way ANOVA). N = 3 biological replicates and N = 2 technical replicates.

Figure 8

Sphinx31 significantly ameliorates OA-induced decline in neurite outgrowth from SHSY5Y cells. (A) neuron-specific marker βIII tubulin identified neurites in green. Cell nuclei stained with Hoechst shown in blue. Neurite outgrowth was calculated by tracing neurites on Image J and dividing the sum length per image by the number of cells. The top row of images shows OA vehicle alone, the middle row shows co-treatment with Sphinx31, the bottom row treatment with Sphinx31 and anti-VEGF-A₁₆₅b. (B) Sphinx31 co-treatment significantly ameliorates decline in neurite outgrowth induced by 1 and 3 nM OA, which was inhibited by treatment with anti-VEGF-A₁₆₅b (two-way ANOVA, p < 0.01 and p < 0.0001, respectively). N = 2 with six images analysed per repeat. Error bars = SEM.

Figure 9

OA and Sphinx31 treatments do not significantly alter the 4R:3R tau ratio in SHSY5Y cells. (A) SHSY5Y cells were treated with 3 nM OA and/or 3 μM Sphinx31 for 24 h. MAPT PCR was carried out using primers spanning exon 10 (forward primer in exon 9, reverse primer in exon 11) to quantify relative expression of 4R and 3R tau isoforms. Images show tau isoform expression and housekeeping gene GAPDH matched to each sample. (B) Sphinx31 treatment does not significantly alter 4R:3R tau ratio (one-way ANOVA, p > 0.05). N = 4, error bars show SEM.

Figure 10

Schematic summarising proposed action of VEGF-A₁₆₅a and VEGF-A₁₆₅b via VEGFR2 binding and full or partial agonism, respectively. Green path represents VEGF-A₁₆₅a activation, blue path represents VEGF-A₁₆₅b activation.