Lead Acetate Exposure and Cerebral Amyloid Accumulation: Mechanistic Evaluations in APP/PS1 Mice

Abstract

Background: The role of environmental factors in Alzheimer's disease (AD) pathogenesis remains elusive. Mounting evidence suggests that acute and past exposure to the environmental toxicant lead (Pb) is associated with longitudinal decline in cognitive function, brain atrophy, and greater brain $\beta$-amyloid ($A\beta$) deposition. However, the nature of Pb-induced amyloid deposition and how it contributes to AD development remain unclear.

Objectives: This study investigates the role of Pb in the pathogenesis of cerebral amyloid angiopathy (CAA) and whether plasminogen activator inhibitor-1 (PAI-1) contributes to this process in the APP/PS1 mouse model.

Methods: Female APP/PS1 mice at 8 wk of age were administered either $50\mathrm{mg}/\mathrm{kg}$ Pb-acetate (PbAc) (i.e., $27\mathrm{mg}\mathrm{Pb}/\mathrm{kg}$) or an equivalent molar concentration of sodium acetate (NaAc) via oral gavage once daily for 8 wk. Amyloid deposition and vascular amyloid were determined by immunostaining. In addition, $A\beta$ perivascular drainage, vascular binding assay, and microglial endocytosis were examined to determine underlying mechanisms. Furthermore, magnetic resonance imaging demyelination imaging was performed in vivo measure the level of demyelination. Finally, Y-maze and Morris water maze tests were assessed to evaluate the cognitive function of mice.

Results: APP/PS1 mice (an AD mice model) exposed to PbAc demonstrated more vascular amyloid deposition less neocortical myelination, and lower cognitive function, as well as greater vascular binding to $A\beta 40$, higher $A\beta 40/A\beta 42$ ratios, strikingly lower $A\beta 40$ levels in the perivascular drainage, and microglial endocytosis. Importantly, exposure to a specific PAI-1 inhibitor, tiplaxtinin, which previously was reported to lower CAA pathology in mice, resulted in less CAA-related outcomes following PbAc exposure.

Discussion: Our findings suggest that PbAc induced CAA/AD pathogenesis via the PAI-1 signaling in the APP/PS1 mouse model, and the inhibition of PAI-1 could be a potential therapeutic target for PbAc-mediated CAA/AD disorders. https://doi.org/10.1289/EHP14384.

Figure 1.

Cortical and hippocampal $\mathrm{A}\mathrm{\beta }$ pathology in 16-wk-old female APP/PS1 mice exposed to PbAc for 8 wk in the presence or absence of TIP. (A) Representative brain sections from APP/PS1 mouse showing expression of collagen IV (red) and amyloid deposits (Thio $\mathrm{S}+$; green). Orange: overlay of the red and green signals. Arrowheads indicate vascular associated amyloid. [Scale bars in (A), $50\mathrm{\mu }\mathrm{m}$.] (B,C) Quantification of total amyloid plaques and vascular associated amyloid in brain sections of 16-wk-old APP/PS1 mice treated with PbAc, NaAc, or PbAc $+\text{TIP}$. (D) The ratio of vascular $\mathrm{A}\mathrm{\beta }$ deposits to parenchymal amyloid deposits in brain sections of 16-wk-old APP/PS1 mice treated with PbAc, NaAc, or PbAc $+\text{TIP}$. (E) Quantification of amyloid associated vessels in brain sections of 16-wk-old APP/PS1 mice treated with PbAc. NaAc, or PbAc $+\text{TIP}$. (F,G) Quantification of parenchymal and vascular levels of $\mathrm{A}\mathrm{\beta }40$ and $\mathrm{A}\mathrm{\beta }42$ in the cortex of 14-wk-old female APP/PS1 mice by ELISA. (H) The ratio of $\mathrm{A}\mathrm{\beta }40$ to $\mathrm{A}\mathrm{\beta }42$ in both cortical parenchyma and vasculature in 16-wk-old female APP/PS1 mice exposed to Pb for 8 wk. $n=3–4$ mice per group. Data are presented as $\text{mean}\pm \text{SD}$. Note: $\mathrm{A}\mathrm{\beta }$, beta amyloid; ANOVA, analysis of variance; APP, amyloid precursor protein; CAA, cerebral amyloid angiopathy; NaAc (control), sodium acetate; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; Pb, lead; PbAc, lead acetate; PS1, presenilin 1; SD, standard deviation; Thio S, thioflavin S; TIP, tiplaxtinin. *$p<0.05$, **$p<0.01$.

Figure 2.

Cognitive assessments in WT mice exposed to PbAc. (A) Mice memory in WT female mice exposed to NaAc or PbAc were assessed using the Y-maze spontaneous alternation test. (B–D) The cognitive assessments of mice were also performed using the Morris water maze. After 7-wk NaAc or PbAc exposure, WT mice were subjected to the Morris water maze test. (B) Time interval between the start of the test and the learning to reach the platform (latency) for a visible platform test in WT mice. (C) The latency for a hidden (invisible) platform test in WT mice. (D) The percentage time spent searching in the quadrant previously containing the platform in a probe test of WT mice. Data represent $\text{mean}\pm \text{SD}$. $n=6$/group. Note: ANOVA, analysis of variance; NaAc, sodium acetate; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; Pb, lead; PbAc, lead acetate; SD, standard deviation; WT, wild-type.

Figure 3.

Levels of PAI-1 in cerebral vasculatures, $\mathrm{A}\mathrm{\beta }$ bound to the cerebrovasculature, and the perivascular drainage of $\mathrm{A}\mathrm{\beta }$ following PbAc exposures in the presence or absence of TIP. (A) After 3 d treatments with $27\mathrm{mg}/\mathrm{kg}$ PbAc or NaAc in female APP/PS1 mice, cortical blood vessels were isolated and vascular levels of PAI-1 were determined by western blot. Levels of PAI-1 and intensities were quantified and normalized by GAPDH levels ($n=4$/group). (B,C) The cerebrovasculature was isolated from wildtype mice and were pretreated with or without $10\mathrm{\mu }\mathrm{M}$ TIP for 1 h followed by $1\mathrm{\mu }\mathrm{M}$ PbAc treatments for additional 24 h. Next, $1\mathrm{\mu }\mathrm{M}$ $\mathrm{A}\mathrm{\beta }40$ or $\mathrm{A}\mathrm{\beta }42$ was added to the buffer for 30 min and levels of $\mathrm{A}\mathrm{\beta }$ bound with vessels were determined by ELISA. ($n=4$/group). (D) Perivascular drainage was determined by quantitating levels of FITC-labeled $\mathrm{A}\mathrm{\beta }40$ associated with arteries between the injection site and $100-\mathrm{\mu }\mathrm{m}$ posterior to the injection site 60 min after injection of FITC-$\mathrm{A}\mathrm{\beta }40$. Representative brain sections from APP/PS1 mouse showed $\mathrm{A}\mathrm{\beta }$ (green) associated with collagen IV (red) labeled vasculatures. Arrowheads indicate vascular associated $\mathrm{A}\mathrm{\beta }$ (orange: overlay of the red and green signals). (E) Vascular-associated $\mathrm{A}\mathrm{\beta }$ fluorescent intensity was measured in mice exposed to Pb in the presence or absence of TIP and control mice exposed to Na to examine the perivascular drainage. ($n=3$/group). Data are presented as $\text{mean}\pm \text{SD}$. Note: $\mathrm{A}\mathrm{\beta }$, beta amyloid; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NaAc (control), sodium acetate; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; PAI-1, plasminogen activator inhibitor-1; Pb, lead; PbAc, lead acetate; SD, standard deviation; TIP, tiplaxtinin. *$p<0.05$, **$p<0.01$.

Figure 4.

PAI-1 and LRP-1 expression and intracellular levels of $\mathrm{A}\mathrm{\beta }40$ and $\mathrm{A}\mathrm{\beta }42$ in cultured BV2 cells exposed to PbAc or NaAc in the presence of TIP. (A) Expression levels of PAI-1 in BV2 cells exposed to $1\mathrm{\mu }\mathrm{M}$ PbAc for 24 h were determined by western blot. (B,C) Intracellular levels of $\mathrm{A}\mathrm{\beta }40$ and $\mathrm{A}\mathrm{\beta }42$ and the ratio of $\mathrm{A}\mathrm{\beta }40$ to $\mathrm{A}\mathrm{\beta }42$ in BV2 exposed to $1\mathrm{\mu }\mathrm{M}$ PbAc for 24 h in the presence and absence of TIP. (D) Expression levels of LRP1 in BV2 cells exposed to $1\mathrm{\mu }\mathrm{M}$ PbAc for 24 h were determined by western blot. Data are presented as $\text{mean}\pm \text{SD}$, $n=6$/group. Note: $\mathrm{A}\mathrm{\beta }$, beta amyloid; ANOVA, analysis of variance; LRP1, low-density lipoprotein receptor-related protein 1; NaAc (control), sodium acetate; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; PAI-1, plasminogen activator inhibitor-1; Pb, lead; PbAc, lead acetate; SD, standard deviation; TIP, tiplaxtinin. *$p<0.05$.

Figure 5.

Myelin expression and neuronal cell numbers in 8-wk PbAc-treated APP/PS1 mice. (A) Representative 20× images of ${\text{NeuN}}^{+}{\text{DAPI}}^{+}$ staining in the neocortex. (B) Quantitation of ${\text{NeuN}}^{+}{\text{DAPI}}^{+}$ cells in hippocampal and cortical areas of female APP/PS1 mice exposed to NaAc or PbAc ($n=3$/group, $p>0.05$). (C) Representative 20× images of MBP staining in hippocampal and cortical areas of female APP/PS1 mice exposed to NaAc or PbAc. (D,E) MBP expression in hippocampal (D) and cortical areas (E) of female APP/PS1 mice exposed to NaAc or PbAc in the presence or absence of TIP were measured by quantitating MBP fluorescent intensities; data are presented as $\text{mean}\pm \text{SD}$, $n=3$/group. Note: ANOVA, analysis of variance; APP, amyloid precursor protein; CTX, cortex; DAPI, 4′,6-diamidino-2-phenylindole; HPC, hippocampus; NaAc, sodium acetate; NeuN, neuronal nuclei; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; Pb, lead; PbAc, lead acetate; PS1, presenilin 1; SD, standard deviation; TIP, tiplaxtinin. *$p<0.05$.

Figure 6.

MRI imaging of myelin in APP/PS1 mice exposed to PbAc in the presence or absence of tiplaxtinin. Direct myelin imaging with UTE time: (A) Representative myelin/water fraction image. (B) Cortex mean myelin/water fraction in neocortex areas detected by MRI Data represent $\text{mean}\pm \text{SD}$, $n=3–4$/group. Note: APP, amyloid precursor protein; MRI, magnetic resonance imaging; NS, not significant, analyzed by a two-sample Student’s $t$-test; Pb, lead; PS1, presenilin 1; SD, standard deviation; TIP, tiplaxtinin; UTE, ultra-short echo. **$p<0.01$.

Figure 7.

Cognitive assessments in APP/PS1 mice exposed to PbAc in the presence or absence of TIP. (A) Mice memory in APP/PS1 mice exposed to NaAc or PbAc in the presence or absence of TIP was assessed using the Y-maze spontaneous alternation test. (B–D) The cognitive assessments of mice were also performed using the Morris water maze. After 7-wk NaAc or PbAc exposure in the presence or absence of TIP, APP/PS1 mice were subjected to the Morris water maze test. (B) Time interval between the start of the test and learning to reach the platform (latency) for a visible platform test in APP/PS1 mice. (C) The latency for a hidden (invisible) platform test in APP/PS1 mice. (D) The percentage time spent searching in the quadrant previously containing the platform in a probe test of APP/PS1 mice. Data represent $\text{mean}\pm \text{SD}$. Control: $n=9$, $\text{Control}+\text{TIP}$: $n=5$, Pb: $n=11$, $\mathrm{Pb}+\text{TIP}$: $n=9$. Note: ANOVA, analysis of variance; APP, amyloid precursor protein; NaAc (control), sodium acetate; NS, not significant, analyzed by a one-way ANOVA with post hoc comparisons using Dunnett’s test; Pb, lead; PbAc, lead acetate; PS1, presenilin 1; SAP, spontaneous alternation performance; SD, standard deviation; TIP, tiplaxtinin. *$p<0.05$, **$p<0.01$.