Circulating Small Extracellular Vesicle-Derived miR-342-5p Ameliorates Beta-Amyloid Formation via Targeting Beta-site APP Cleaving Enzyme 1 in Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a common neurodegenerative disorder with progressive cognitive impairment in the elderly. Beta-amyloid (Aβ) formation and its accumulation in the brain constitute one of the pathological hallmarks of AD. Until now, how to modulate Aβ formation in hippocampal neurons remains a big challenge. Herein, we investigated whether the exosomal transfer of microRNA (miR) relates to amyloid pathology in the recipient neuron cells. We isolated circulating small extracellular vesicles (sEVs) from AD patients and healthy controls, determined the miR-342-5p level in the sEVs by RT-PCR, and evaluated its diagnostic performance in AD. Then, we took advantage of biomolecular assays to estimate the role of miR-342-5p in modulating the amyloid pathway, including amyloid precursor protein (APP), beta-site APP cleaving enzyme 1 (BACE1), and Aβ42. Furthermore, we subjected HT22 cells to the sEVs from the hippocampal tissues of transgenic APP mice (Exo-APP) or C57BL/6 littermates (Exo-CTL), and the Exo-APP enriched with miR-342-5p mimics or the control to assess the effect of the sEVs' delivery of miR-342-5p on Aβ formation. We observed a lower level of miR-342-5p in the circulating sEVs from AD patients compared with healthy controls. MiR-342-5p participated in Aβ formation by modulating BACE1 expression, specifically binding its 3'-untranslated region (UTR) sequence. Exo-APP distinctly promoted Aβ42 formation in the recipient cells compared to Exo-CTL. Intriguingly, miR-342-5p enrichment in Exo-APP ameliorated amyloid pathology in the recipient cells. Our study indicated that miR-342-5p was dysregulated in human circulating sEVs from AD patients; sEV transfer of miR-342-5p ameliorates Aβ formation by modulating BACE1 expression. These findings highlight the promising potential of exosomal miRNAs in AD clinical therapy.

Keywords: Alzheimer’s disease; BACE1; amyloid-beta; extracellular vesicle; miRNA; neurons.

Figure 1

Circulating sEV miRNA-342-5p was downregulated in patients with Alzheimer’s disease (AD) compared with healthy controls (HC). (a) Nanoparticle Tracking Analysis (NTA) histograms of purified circulating sEVs from AD patients. The horizontal axis represents the particle diameters (nm), and the longitudinal axis depicts particle concentrations (particles/mL) at each diameter. (b) NTA histograms of purified circulating sEVs from healthy controls; (c) tetraspanins CD63 and CD9, and Alix, the exosomal biomarkers were detected using Western blot assay. (d) The transmission electron microscopy (TEM) images exhibited the morphology of sEVs from healthy controls (left) and AD patients (right). Scale bars represent 100 nm. (e) RT-PCR assay was performed to quantify the level of miR-342-5p in circulating sEVs from healthy controls or AD patients, p = 0.0291. (f) The ratios of miR-342-5p to miR-191-5p in sEVs from 16 AD patients and 16 healthy controls were calculated, p = 0.0027. (g) MiR-425-3p levels in sEVs were evaluated in the HC and AD groups, p = 0.7774. (h) The ratios of miR-342-5P to miR-425-3p were calculated, p = 0.5588. (i) The receiver operating characteristic (ROC) curve and area under the curve (AUC) were applied to estimate the diagnostic performance of sEVs miR-342-5p in distinguishing AD patients from normal individuals.

Figure 2

MiRNA-342-5p modulated the expression of Bace1 by targeting its 3′-UTR in mouse hippocampal HT-22 neurons. (a) MiR-342-5p was detected by RT-PCR assay after the cells were transfected with miR-342-5p inhibitors or mimics or their corresponding negative control (NC). *, p < 0.05, ***, p < 0.001. (b) BACE1 was detected by RT-PCR assay after the cells were transfected with the above RNA oligonucleotides. *, p < 0.05. (c) APP, BACE1, and Aβ42 were detected by Western blot assay. GAPDH was used as the internal control. The experiments were performed in triplicate. (d) Relative quantification of APP, BACE1, and Aβ42 immunoblot bands was performed by densitometry normalized to GAPDH after the cells were transfected with miR inhibitors and its negative control (NC). *, p < 0.05, **, p < 0.01. (e) Relative quantification of APP, BACE1, and Aβ42 immunoblot bands was performed by densitometry after the cells were transfected with miR mimics and the negative control (NC). *, p < 0.05. (f) A sequence alignment was presented for miR-342-5p with predicted sequence from wild-type Bace1 and its mutant (mut) sequence. (g) Relative luciferase activities of wild-type and mutant Bace1 reporter were measured in HEK293T cells overexpressing miR-342-5p mimics or the negative control (NC). ***, p < 0.001.

Figure 3

SEVs from APP transgenic mice distinctly promoted Aβ formation in mouse hippocampal HT-22 cells. SEVs were purified from APP mice (a–d) and C57BL/6 control mice (e–h). The sEVs were subjected to nanoparticle tracking analysis with a ZetaView instrument utilizing the properties of light scattering and Brownian motion to evaluate the exosome size distribution. In (a,e), the horizontal axis shows the particle diameters (nm) in the samples, and the vertical axis displays the concentrations (particles/mL) at a certain size. In (b,f), the horizontal axis shows the particle diameters (nm), and the vertical axis displays the particle volumes (×10⁶ nm³) at a certain diameter size. In (c,g), NTA video visualized light scattering sEVs, revealing the presence of vesicles of different sizes. In (d,h), the morphology of sEVs was visualized by transmission electron microscope (TEM). The scale bar is 200 nm. In (i), exosome markers CD9 and CD63 were detected by Western blot assay. GAPDH was used as a negative control. In (j), the RT-PCR assay quantified the relative levels of miR-342-5p in sEVs from APP and C57BL/6 mice. **, p < 0.01. In (k), RT-PCR quantified the relative levels of BACE1 in APP and C57BL/6 sEVs. **, p < 0.01. In (l), a Nikon fluorescent microscopy was used to visualize the internalization of Dil-labeled sEVs into HT22 cells. Cell nuclei were detected with the fluorescent dye DAPI. Scale bars represent 50 μm. In (m), an RT-PCR was applied to detect the relative levels of BACE1 in HT22 cells incubated with the sEVs from APP and C57BL/6 mice, respectively. ***, p < 0.001. In (n), a Western blot assay was used to detect APP, BACE1, and Aβ42 proteins in HT22 cells incubated with the sEVs from APP and C57BL/6 mice, respectively. The experiments were performed in triplicate. In (o), the intensities of the immunobands for APP, BACE1, and Aβ42, normalized to the inner control GAPDH were quantified by densitometry in HT22 cells incubated with sEVs from APP and C57BL/6 mice, respectively. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 4

Enrichment of miR-342-5p ameliorated Aβ pathology evoked by Exo-APP in mouse hippocampal HT-22 neurons. (a) The internalization of Dil-labeled sEVs into HT22 cells was visualized using a Nikon fluorescent microscope. DAPI staining was used to detect cell nuclei. Scale bars represent 50 μm. (b) Exo-APP were transfected with miR-342-5p mimics (EXO-miR-mimics) or its negative control (EXO-miR-NC), respectively. The levels of miR-342-5p in the sEVs were evaluated using qPCR. **, p < 0.01. (c) HT22 cells were treated with the sEVs enriched with miR-342-5p mimics (EXO-miR-mimics) or its negative control (EXO-miR-NC). APP, BACE1, and Aβ42 proteins in HT22 cells incubated with EXO-miR-mimics or EXO-miR-NC were detected using Western blot assay. The experiments were performed in triplicate. (d) Relative levels of APP, BACE1, and Aβ42 in HT22 cells were quantified by densitometry. GAPDH was used as an inner control. *, p < 0.05, **, p < 0.01.