In this study, we isolated synaptosomes from mouse forebrains using a sucrose density gradient centrifugation procedure. Prior to characterizing the expression profile of the miRNAs, we identified the synaptosomes using several methods. Fig. 1a illustrates a representative image of synaptosomes: synaptosomes are 0.5 μm-diameter, sealed membrane vesicles that contain synaptic vesicles and occasional mitochondria (arrows). We also determined the neurotransmitter levels in the synaptosome supernatants after the depolarizing stimulus and observed expected elevations of glutamate and GABA after the stimulation (Fig. 1b). To exclude the possibility of non-specific synaptosome leakage, a LDH activity assay was employed to monitor the leakage in synaptosomes. As shown in Fig. 1c, the LDH activity of the supernatants was less than 3% that of the LDH activity in the pellets, and there was no significant difference between the control and KCl-stimulated groups. Western blotting analysis showed that the synaptic vesicular protein, synaptotagamin1 (Syt1), was enriched in the synaptosome fraction (Syn). Furthermore, the post-synaptic protein PSD-95 was enriched in the insoluble fraction (Sp) after TritonX-100 extraction (Fig. 1d). These experiments confirmed that the synaptosomes were relatively intact and enriched in our preparation and that the synaptosomes displayed active metabolic function.
Figure 1. Characterization of isolated synaptosomes. (a) Typical morphology of synaptosomes using TEM. Scale bar = 500 nm. (b) Quantitative analysis of the neurotransmitter, n = 3 or 4, *p < 0.05. (c) Lactate dehydrogenase (LDH) activities in the supernatants and pellets of the synaptosomes. Total LDH activity = LDH activity of the supernatant and pellets, n = 3 or 4. (d) Distribution of synaptosome proteins in the synaptosome and PSD preparation. Total forebrain homogenate (H) was processed to obtain a soluble cytoplasmic fraction (S1), crude pellets (P2), and a synaptosome fraction (Syn), which was then lysed using 1% TritonX-100 to yield soluble (sS) and insoluble fractions (Sp) as described in the Methods section. Equal amounts of protein were loaded and blotted against various antibodies as indicated. [Correction added after online publication 27 November 2012: n values were amended].
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Next, the miRNA expression profile of the synaptosomes was analyzed using a qRT-PCR -based microRNA microarray. The results showed that approximately 33% of the miRNAs examined (192 of 585) were highly expressed in the synaptosomes (with an average threshold criterion of CT ≤ 25.0; Table S1). Among these highly expressed miRNAs, twenty-seven were further verified by qRT-PCR using TaqMan microRNA probes (Fig. 2a). The miRNAs were normalized against the U6 control, we found 16 miRNAs (miR-9, miR-16, miR-26a, miR-26b, miR-29a, miR-29c, miR-30a, miR-99b, miR-124, miR-125a, miR-125b, miR-128, miR-134, miR-138, miR-146a, and miR-146b) were highly expressed. Four miRNAs (miR-20a, miR-27b, miR-99a, and miR-130a) were moderately expressed, and 7 miRNAs (miR-15a, mir-21, miR-25, miR-27a, miR-34a, miR-148a, and miR-154) were poorly expressed in the synaptosomes. From these results, we found that brain tissue-enriched miRNAs (miR-9, miR-124, miR-128 and miR-138) were highly expressed in the synaptosomes. The regression coefficient between the expression profiles obtained from the microarray and the qRT-PCR assay was approximately 0.7 (Fig. 2b), indicating that the profiles obtained using the microRNA microarray and qRT-PCR was quite consistent. Among the synaptosome miRNAs studied here, miR-146b exhibited the highest expression levels. This result is consistent with a previous report that found that the synaptic intensity of miR-146b was among the highest expressed miRNA group (Lugli et al. 2008).
To evaluate whether miRNAs in synaptosomes were ‘naked’ or were associated with the protein complex required for miRNA function, we performed an immunoprecipitation assay using anti-Ago2 antibodies and determined whether miRNAs were co-immunoprecipitated with Ago2. Western blotting showed a relatively high efficiency of Ago2 immunoprecipitation and a high specificity of the Ago2 antibodies (Fig. 2c, upper panel and lower panel). We checked 21 miRNAs in the synaptosome lysates (open bar, Fig. 2d), and the anti-Ago2 antibody pulled down all 21 miRNAs from the lysates in the synaptosomes (shaded bar, Fig. 2d); however, different miRNAs had a different ratio of Ago2-associated/total. These results suggest that a portion of the miRNAs in the synaptosomes were associated with Ago2 and may exhibit biologically active functions.
We further prepared crude synaptic vesicles and synaptosomes from equal-volume P2 fractions. Western blotting revealed a relatively high enrichment of Syt1 protein and poor expression of PSD-95 protein in the SV fraction. Furthermore, we examined the protein levels of Ago2 in these fractions and found that Ago2 was relatively enriched in the SV fraction compared to the P2' or Syn fractions. The LP1 fraction also exhibited high expression of Ago2 protein (slightly higher than in the SV fraction) (Fig. 2e). The LP1 pellets were PSD-enriched fraction, and the high expression level of Ago2 was consistent with a previous report (Lugli et al. 2008). Next, we quantified 8 miRNAs from synaptic vesicles or the synaptosome fraction and found that, on average, approximately 31 ± 6% miRNAs from the synaptosomes were present in synaptic vesicles (Fig. 2f, open and shaded bars). These results suggest that a group of miRNAs is associated with the Ago2 protein in the synaptic fraction, and furthermore, several had been engulfed by synaptic vesicles.