Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1

Abstract

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.

FIGURE 1.

Secreted neuronal exosomes contain miR-124a. Representative electron microscopy images of neuronal (A) and astroglial (B) exosome preparation. Scale bar = 200 nm in neuronal exosomes and 50 nm in astroglial exosomes. C, detection of exosome markers Alix and Tsg101 in neuronal and astroglial exosomes. D, bioanalyzer pico analysis of neuronal exosome RNA. smRNA, small RNA. E, relative miR-124a levels in astrocytes, neurons, and neuronal exosomes. n = 3–4. ***, p < 0.001 from one-way ANOVA with Bonferroni post-test. F, MiR-124a levels in neuronal exosomes following tetrodotoxin or KCL treatment. A.U., arbitrary unit. n = 4. *, p < 0.05 from Student's t test with control separately.

FIGURE 2.

Internalization of neuronal exosomes into astrocytes. A, time-lapse images of cultured astrocytes treated with labeled neuronal exosomes. Scale bar = 50 μm. B, neuronal exosome treatment increases miR-124a levels in cultured astrocytes. n = 3. ***, p < 0.001 from Student's t test.

FIGURE 3.

Neuronal miR-124a up-regulates astroglial GLT1 protein expression levels. A, expression of GLT1 protein levels in astrocyte cultures following transfection of miR-124a. B, quantification of GLT1 expression levels following miR-124a transfection. n = 4–6. *, p < 0.05 from one-way ANOVA with Bonferroni post-test. C, expression of GLAST protein levels in astrocyte cultures following transfection of miR-124a. D, quantification of GLAST expression levels following miR-124a transfection. n = 5–6. The three band sizes represent trimers, dimers, and monomers of GLT1 or GLAST. E, GLT1 mRNA expression levels in cultured astrocytes following miR-124a transfection. n = 3–5. F, activity of the GLT1 mRNA 3′UTR luciferase reporter following miR-124a or mixture of miR-124a/miR-124a-A/S transfection. n = 5/experiment for three independent experiments

FIGURE 4.

MiR-124a mediates neuronal exosome-dependent up-regulation of GLT1 expression in astrocytes. A, neuronal exosome treatment increases GLT1 protein levels in cultured astrocytes. B, GLT1 protein expression levels in miR-124a-A/S-transfected astrocytes following treatment with neuronal exosomes. Neuronal exosomes collected from one 10-cm dish were added into four wells of astrocyte cultures in a 6-well plate. C, quantification of the GLT1 protein levels in astrocytes following treatment with neuronal exosomes/pre-transfection of miR-124a-A/S. n = 4–6. *, p < 0.05 from one-way ANOVA with Bonferroni post-test

FIGURE 5.

Intrastriatal injection of miR-124a antisense (miR-124a-A/S) significantly reduces GLT1 protein expression levels in striatum. A, GLT1 expression levels in striatum following intrastriatal injection of miR-124a-A/S. B, quantification of the GLT1 expression levels from immunoblot analysis. n = 5–7. *, p < 0.05 from one-way ANOVA with Bonferroni test. C, GLT1 mRNA levels following miR-124a-A/S injection. D, expression changes of other glutamate transporters following miR-124a-A/S injection. Quantification of the GLAST (E) and EAAC1 (F) expression levels from immunoblot analysis. n = 3–5. G, measurement of functional glutamate uptake in striatum tissue following miR-124a-A/S injection. n = 3–4. ***, p < 0.001, one-way ANOVA with Bonferroni post-test. N.S., not significant.

FIGURE 6.

Astroglial ephrinA3 is not the downstream target of miR-124a in astrocytes in GLT1 expression regulation. A, increased GLT1 protein levels in cultured ephrinA3^−/− astrocytes. B, quantitative analysis of the GLT1 expression from the ephrinA3^−/− astrocytes. n = 6. **, p < 0.01, Student's t test. C, luciferase activity of the ephrinA3 mRNA 3′UTR luciferase reporter in astrocytes following miR-124a transfection. n = 10 from two independent experiments. D, representative immunoblot analysis of ephrinA3 expression in astrocytes following miR-124a transfection. E, quantification of ephrinA3 expression in astrocytes following miR-124a transfection. n = 6. N.S., not significant.

FIGURE 7.

Stereotaxically injected miR-124a increases GLT1 protein expression in SOD1G93A mice. A, down-regulation of miR-124a in the spinal cord of end-stage SOD1G93A mice. n = 5–6. *, p < 0.05, Student's t test. B, high abundance of injected miR-124a in astrocytes of cervical spinal cord of SOD1G93A mice. Scale bar = 50 μm. Astrocytes with abundant miR-124a are highlighted with yellow arrows. C, GLT1 immunostaining in cervical cord following miR-124a injection. Scale bar = 50 μm. GLT1 immunoreactivity from individual astrocyte (circled in a dashed line from the GFAP staining) was quantified. D, quantitative analysis of GLT1 protein levels following miR-124a injection. GLT1 immunoreactivity was measured in miR-124a⁺GFAP⁺ cells in the cervical cord and normalized with the GLT1 immunoreactivity in the lumbar cord of the same SOD1 G93A mouse. n = 78–114 cells from three SOD1 G93A mice. **, p < 0.01 from Student's t test.

FIGURE 8.

Exosome-mediated miR-124a transfer from neurons to astrocytes and its regulation of GLT1 protein expression.