Extracellular Vesicles Derived from Young Neural Cultures Attenuate Astrocytic Reactivity In Vitro

Abstract

Extracellular vesicles (EVs) play an important role in intercellular communication and are involved in both physiological and pathological processes. In the central nervous system (CNS), EVs secreted from different brain cell types exert a sundry of functions, from modulation of astrocytic proliferation and microglial activation to neuronal protection and regeneration. However, the effect of aging on the biological functions of neural EVs is poorly understood. In this work, we studied the biological effects of small EVs (sEVs) isolated from neural cells maintained for 14 or 21 days in vitro (DIV). We found that EVs isolated from 14 DIV cultures reduced the extracellular levels of lactate dehydrogenase (LDH), the expression levels of the astrocytic protein GFAP, and the complexity of astrocyte architecture suggesting a role in lowering the reactivity of astrocytes, while EVs produced by 21 DIV cells did not show any of the above effects. These results in an in vitro model pave the way to evaluate whether similar results occur in vivo and through what mechanisms.

Keywords: aging; astrocytes; extracellular vesicles (EVs); glial fibrillary acidic protein (GFAP); neural cultures.

Figure 1

Characterization of extracellular vesicles (EVs) isolated from 14 and 21 DIV rat cortical neural cultures. EVs samples from neuronal culture media were processed by serial centrifugation, as described in “Materials and Methods”. (A) Plot showing the size distribution of EVs secreted by 14 and 21 DIV rat cortical neural cultures and quantified in the post-10,000 g supernatant through Nanoparticle tracking analysis with the help of a NanoSight NS500 instrument. (B) Representative Transmission Electron Microscopy (TEM) images of EVs isolated from the media of 14 and 21 DIV neural cultures using the protocol shown in “Materials and Methods”. Scale bars on both images represent 100 nm. (C) Plot comparing the total number of EVs present in the media of 14 and 21 DIV neural cultures, determined from the area under the curve (AUC) of size distribution profiles like the ones shown in panel A. Data are expressed as vesicles/mL, and the bars represent mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t-test (*** p < 0.001, n = 6). (D) Plot comparing the size of EVs present in the media of 14 and 21 DIV neural cultures, determined from size distribution profiles as the ones shown in panel A. Data are expressed as the mean size of vesicles, and the bars represent mean ± SEM. Statistical significance was analyzed by a two-tailed unpaired t-test (ns = non-significant, n = 6).

Figure 2

Confocal analysis of Bodipy-cholesterol-labeled EVs uptake by neurons and astrocytes. (A) Schematics of the procedure followed to label EVs using Bodipy-cholesterol. EVs in PBS are incubated with 1 µM Bodipy-cholesterol diluted for 1 h at 37 °C. Subsequently, the unincorporated Bodipy-cholesterol was removed using Invitrogen™ spin columns (MW 3000, Thermo Fisher). Bodipy-cholesterol-labeled EVs were recovered in the eluted fraction. (B) Representative Transmission Electron Microscopy (TEM) images of unlabeled EVs or Bodipy-cholesterol labeled EVs derived from the media of 14 DIV neural cultures after elusion from the Invitrogen™ spin columns. (C) Plot comparing the fluorescence (Excitation 495 nm/Emission 510 nm) of unlabeled EVs (autofluorescence) or Bodipy-cholesterol-labeled EVs using the protocol schematized in panel A. The graph shows the mean fluorescence ± SEM. Statistical significance was analyzed by a two-tailed unpaired t-test (* p < 0.05, n = 3). (D) Representative confocal images of 14 DIV neural cultures treated for 24 h with Bodipy-cholesterol-labeled EVs isolated from the media of 14 DIV neural cultures. Both neurons (stained with the neuronal marker MAP2, in white) and astrocytes (stained with the astrocytic marker GFAP, in red) show the capacity to take up Bodipy-cholesterol-labeled EVs (Bodipy, in green). Nuclei are stained with DAPI. Scale bars represent 10 µm in all the images.

Figure 3

Biological effect of 14 and 21 DIV isolated EVs on 14 DIV rat cortical neural culture for a 24 h treatment period. (A) Western blot analysis of total tau protein in 14 DIV neural culture lysates untreated (cnt) or treated for 24 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. Actin was used as loading control. The plot shows the levels for total tau protein normalized by actin and quantified from Western blot experiments as the ones shown in this panel. Graph shows the mean protein levels relative to the untreated (control) condition ± SEM (B) Same analysis as in panel A, but for tau phosphorylated at S396/T404 (PHF-1 epitope). (C) Plots comparing the Aβ40 levels (upper graph) or the Aβ42 levels (lower graph) in the media of rat cortical neural cultures untreated (control) or treated for 24 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. Graph shows the Aβ concentration (pM) ± SEM. (D) Same analysis as in panel A was performed for the postsynaptic protein PSD95, (E) the presynaptic protein synaptophysin, (F) the phosphorylated form of p38, (G) the receptor for neurotrophins TrkB, and (H) the phosphorylated form of AKT at S473. (I) The plot compares the relative levels of lactate dehydrogenase (LDH) measured in the media of 14 DIV neural cultures untreated (control) or treated for 24 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. All graphs show the mean LDH levels relative to the control condition ± SEM. Statistical significance was analyzed by one-way ANOVA. Post hoc analysis was analyzed by Tukey’s Multiple Comparison Test (* p < 0.05; ns = non-significant, n = 3 independent experiments). Note: the bands for actin are the same for panels A and H since the same Western blot was used to analyze the expression levels of total tau and p-AKT.

Figure 4

Biological effect of 14 and 21 DIV isolated EVs on 14 DIV rat cortical neural culture for a 48 h treatment period. (A) Western blot analysis of total tau protein in 14 DIV neural culture lysates untreated (cnt) or treated for 48 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. Actin was used as loading control. The plot shows the levels for total tau protein normalized by actin and quantified from Western blot experiments as the ones shown in this panel. Graph shows the mean protein levels relative to the untreated (control) condition ± SEM (B) Same analysis as in panel A, but for tau phosphorylated at S396/T404 (PHF-1 epitope). (C) Plots comparing the Aβ40 levels (upper graph) or the Aβ42 levels (lower graph) in the media of rat cortical neural cultures untreated (control) or treated for 48 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. Graph shows the Aβ concentration (pM) ± SEM. (D) Same analysis as in panel A was performed for the postsynaptic protein PSD95, (E) the presynaptic protein synaptophysin, (F) the phosphorylated form of p38, (G) the receptor for neurotrophins TrkB, and (H) the phosphorylated form of AKT at S473. (I) The plot compares the relative levels of lactate dehydrogenase (LDH) measured in the media of 14 DIV neural cultures untreated (control) or treated for 48 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. All graphs show the mean LDH levels relative to the control condition ± SEM. Statistical significance was analyzed by one-way ANOVA. Post hoc analysis was analyzed by Tukey’s Multiple Comparison Test (* p < 0.05; ns = non-significant, n = 3 independent experiments). Note: the bands for actin are the same for panels D and F since the same Western blot was used to analyze the expression levels of total PSD95 and p-p38.

Figure 5

Effect of 14 and 21 DIV isolated EVs on neuronal branching. (A) Representative confocal images of neuronal processes, detected with an antibody against the dendritic marker MAP2, of 14 DIV neural culture untreated (control) or treated for 24 h with EVs isolated from the media of 14 DIV (EVs 14 DIV) or 21 DIV (EVs 21 DIV) neural cultures. A scheme highlighting the primary processes, primary branches, and secondary branches of neurons is shown on the left upper quadrant of this panel. 14 DIV neural cultures untreated (control) or treated for 24 h with EVs 14 DIV or EVs 21 DIV were analyzed for the number of primary and secondary branches per neuron (bar = 100 µm) (B,C) number of primary processes. All graphs show mean values ± SEM. Statistical significance was analyzed by one-way ANOVA. Tuckey’s Multiple Comparison Test was used for post hoc analysis (ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3 independent experiments).

Figure 6

The levels of the astrocytic protein GFAP decrease after treatment with 14 DIV EVs. (A) Western blot analysis of microglial marker Iba1 and the astrocytic marker GFAP in 14 DIV neural culture lysates untreated (cnt) or treated for 24 h with EVs isolated from the media of 14 DIV or 21 DIV neural cultures. Actin was used as a loading control. (B) Plot comparing the levels of Iba1 quantified from Western blot experiments like the one shown in panel A (n = 4 independent experiments). (C) Plot comparing the levels of GFAP, quantified from Western blot experiments like the one shown in panel A (n = 5 independent experiments). All graphs show the mean protein levels relative to the untreated (control) condition ± SEM. Statistical significance was analyzed by one-way ANOVA. Tuckey’s Multiple Comparison Test was used for post hoc analysis (** p < 0.01, *** p < 0.001, ns = non-significant).

Figure 7

Inflammatory effect of 14 and 21 DIV isolated EVs on 14 DIV neural cultures. (A) Representative confocal images of astrocytic processes, detected with an antibody against GFAP, of 14 DIV neural cultures untreated (control) or treated for 24 h with EVs isolated from the media of 14 DIV (EVs 14 DIV) or 21 DIV (EVs 21 DIV) neural cultures. A scheme highlighting the primary processes, primary branches, and secondary branches of astrocytes is shown on the left upper quadrant of this panel. 14 DIV neural cultures untreated (control) or treated for 24 h with EVs 14 DIV or EVs 21 DIV, were analyzed for the number of primary and secondary branches per astrocyte (bar = 100 µm) (B), the length of primary astrocytic processes (µm) (C), the maximum length of the primary astrocytic processes (µm) (D) and the density of the branches on the main processes (number of primary branches normalized to the length of the primary astrocytic processes (num/µm); panel (E). All graphs show mean values ± SEM. Statistical significance was analyzed by one-way ANOVA. Tuckey’s Multiple Comparison Test was used for post hoc analysis (* p < 0.05, ns = non-significant; n = 3 independent experiments).