Benjamin Buller

Functional MicroRNA Is Transferred between Glioma Cells

MicroRNAs (miRNA) are single-stranded 17- to 27-nucleotide RNA molecules that regulate gene expression by posttranscriptional silencing of target mRNAs. Here, we transformed rat 9L gliosarcoma cells to express cel-miR-67, a miRNA that lacks homology in rat. Coculture of these cells with cells that expressed a luciferase reporter that contained a complementary sequence to cel-miR-67 resulted in significant suppression of luciferase expression. This effect was also observed in the U87-MG human glioma cell line. Moreover, luciferase suppression was inhibited by the addition of carbenoxolone to cocultures, suggesting that gap junction communication regulates intercellular transfer of miRNA. Finally, in situ hybridization revealed the presence of cel-miR-67 in cel-miR-67-null 9L cells after coculture with cel-miR-67-expressing cells. Our data show that miRNA transcribed in glioma cells can be transferred to adjacent cells and induces targeted inhibition of protein expression in the acceptor cells. These findings reveal a novel mechanism of targeted intercellular protein regulation between brain tumor cells.

MiR-15b and miR-152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3

We tested invasion and angiogenesis related mRNA expression and miRNA profiles of glioma. Genes with mRNA expression that changed significantly were selected to predict possible miRNAs that regulate mRNA expression, and were then matched with miRNA results. NRP-2 with the matching miRNA miR-15b, and MMP-3 with the matching miRNA miR-152 were selected for further study. Luciferase activity assay confirmed that miR-15b and miR-152 attenuate expression of NRP-2 and MMP-3 protein by binding to NRP-2 and MMP-3 transcript, respectively. In vitro invasion assay data showed that miR-15b and miR-152 significantly decreased 9L cell invasiveness. In vitro tube formation assay data showed that miR-15b reduced tube formation. A preliminary pathway study indicated that miR-15b and miR-152 deactivated the MEK-ERK pathway via NRP-2 and MMP-3 in 9L cells, respectively.

Axonal Outgrowth and Dendritic Plasticity in the Cortical Peri-Infarct Area After Experimental Stroke

Background and Purpose— Axonal remodeling is critical to brain repair after stroke. The present study investigated axonal outgrowth after stroke and the signaling pathways mediating axonal outgrowth in cortical neurons. Methods— Using a rodent model of middle cerebral artery occlusion, we examined high-molecular weight neurofilament (NFH) immunoreactive axons and myelin basic protein-positive oligodendrocytes in the peri-infarct area. In vitro, using cultured cortical neurons in a microfluidic chamber challenged by oxygen-glucose deprivation (OGD), we investigated mechanisms selectively regulating axonal outgrowth after OGD. Results— NFH+ axons and MBP+ oligodendrocytes substantially increased in the peri-infarct area during stroke recovery, concomitantly with an increase in dendrites and spines identified by Golgi-Cox staining. In vitro, cortical neurons subjected to OGD exhibited significant increases in axonal outgrowth and in phosphorylated NFH protein levels, concurrently with downregulation of phosphatase tensin homolog deleted on chromosome 10, activation of Akt, and inactivation of glycogen synthase kinase-3&bgr; in regenerated axons. Blockage of phosphoinositide 3-kinase with pharmacological inhibitors suppressed Akt activation and attenuated phosphorylation of glycogen synthase kinase-3&bgr;, which resulted in suppression of phosphorylated NFH and axonal outgrowth after OGD; whereas GSK-3 inhibitors augmented axonal regeneration and elevated phosphorylated NFH levels after OGD. Conclusions— Stroke induces axonal outgrowth and myelination in rodent ischemic brain during stroke recovery, and the phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3&bgr; signaling pathway mediates axonal regeneration of cortical neurons after OGD.

The MicroRNA-17–92 Cluster Enhances Axonal Outgrowth in Embryonic Cortical Neurons

MicroRNAs (miRNAs) regulate dendritogenesis and plasticity. However, the biological function of miRNAs in axons has not been extensively investigated. Here, using rat primary cortical neurons cultured in a microfluidic chamber, we found that the distal axons of the neurons expressed the miR-17–92 cluster, and proteins that regulate production and activity of mature miRNAs, Dicer and Argonaute 2, respectively, were present in the distal axons. Overexpression of the miR-17–92 cluster in cortical neurons substantially increased axonal outgrowth, whereas distal axonal attenuation of endogenous miR-19a, a key miRNA of the miR-17–92 cluster, with the miRNA hairpin inhibitor suppressed axonal outgrowth. Moreover, overexpression of the miR-17–92 cluster reduced phosphatase and tensin homolog (PTEN) proteins and elevated phosphorylated mammalian target of rapamycin (mTOR) in the distal axons. In contrast, distal axonal attenuation of miR-19a increased PTEN proteins and inactivated mTOR in the axons, but did not affect these protein levels in the cell bodies. Overexpression of PTEN and attenuation of endogenous PTEN prevailed over the enhancement and inhibitory effects of the miR-19a on axonal outgrowth, respectively. Axonal application of LY294002, a phosphoinositide3-kinase inhibitor, or rapamycin, an mTOR inhibitor, abolished axonal outgrowth enhanced by overexpression of the miR-17–92 cluster. Collectively, these findings demonstrate that axonal alteration of miR-17–92 cluster expression regulates axonal outgrowth and that local modulation of PTEN protein levels by miR-19a likely contributes to the axonal outgrowth.

MicroRNA-17–92 Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats

Background and Purpose— Multipotent mesenchymal stromal cell (MSC) harvested exosomes are hypothesized as the major paracrine effectors of MSCs. In vitro, the miR-17–92 cluster promotes oligodendrogenesis, neurogenesis, and axonal outgrowth. We, therefore, investigated whether the miR-17–92 cluster–enriched exosomes harvested from MSCs transfected with an miR-17–92 cluster plasmid enhance neurological recovery compared with control MSC-derived exosomes. Methods— Rats subjected to 2 hours of transient middle cerebral artery occlusion were intravenously administered miR-17–92 cluster–enriched exosomes, control MSC exosomes, or liposomes and were euthanized 28 days post–middle cerebral artery occlusion. Histochemistry, immunohistochemistry, and Golgi–Cox staining were used to assess dendritic, axonal, synaptic, and myelin remodeling. Expression of phosphatase and tensin homolog and activation of its downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3&bgr; in the peri-infarct region were measured by means of Western blots. Results— Compared with the liposome treatment, both exosome treatment groups exhibited significant improvement of functional recovery, but miR-17–92 cluster–enriched exosome treatment had significantly more robust effects on improvement of neurological function and enhancements of oligodendrogenesis, neurogenesis, and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone (IBZ) than the control MSC exosome treatment. Moreover, miR-17–92 cluster–enriched exosome treatment substantially inhibited phosphatase and tensin homolog, a validated miR-17–92 cluster target gene, and subsequently increased the phosphorylation of phosphatase and tensin homolog downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3&bgr; compared with control MSC exosome treatment. Conclusions— Our data suggest that treatment of stroke with tailored exosomes enriched with the miR-17–92 cluster increases neural plasticity and functional recovery after stroke, possibly via targeting phosphatase and tensin homolog to activate the PI3K/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3&bgr; signaling pathway.

Niacin Treatment of Stroke Increases Synaptic Plasticity and Axon Growth in Rats

Background and Purpose— Niacin is the most effective medication in current clinical use for increasing high-density lipoprotein cholesterol. We tested the hypothesis that niacin treatment of stroke promotes synaptic plasticity and axon growth in the ischemic brain. Methods— Male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion and treated with or without Niaspan (a prolonged-release formulation of niacin, 40 mg/kg) daily for 14 days starting 24 hours after middle cerebral artery occlusion. The expression of synaptophysin, Nogo receptor, Bielschowsky silver, brain-derived neurotrophic factor, and its receptor tropomyosin-related kinase B were measured by immunohistostaining and Western blot, respectively, in the ischemic brain. Complementing in vivo studies, primary cultured neurons were used to test the effect of niacin and high-density lipoprotein on neurite outgrowth and brain-derived neurotrophic factor/tropomyosin-related kinase B expression. Results— Niaspan treatment of stroke significantly increased synaptophysin, Bielschowsky silver, brain-derived neurotrophic factor/tropomyosin-related kinase B expression, and decreased Nogo receptor expression in the ischemic brain compared with middle cerebral artery occlusion control animals (P<0.05, n=8/group). Niacin and high-density lipoprotein treatment significantly increased neurite outgrowth and brain-derived neurotrophic factor/tropomyosin-related kinase B expression in primary cultured neurons. Tropomyosin-related kinase B inhibitor attenuated niacin-induced neurite outgrowth (P<0.05, n=6/group). Conclusions— Niacin treatment of stroke promotes synaptic plasticity and axon growth, which is mediated, at least partially, by the brain-derived neurotrophic factor/tropomyosin-related kinase B pathways.

Regulation of serum response factor by miRNA-200 and miRNA-9 modulates oligodendrocyte progenitor cell differentiation.

Serum response factor (SRF) is a transcription factor that transactivates actin‐associated genes and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in OL differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the micro‐RNAs (miRNAs) miR‐9 and the miR‐200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG‐1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Overexpression of miR‐9 and miR‐200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co‐expression of miR‐9 and miR‐200 attenuated activity of a luciferase reporter assay containing the Srf 3′ untranslated region. Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation.

MiR-145 reduces ADAM17 expression and inhibits in vitro migration and invasion of glioma cells

MicroRNAs are important regulators of gene expression and have been suggested to play a key role in tumorigenesis. In this study, we show that miR-145 is significantly downregulated in glioma cell lines compared to normal brain tissue and negatively regulates tumorigenesis. Restoration of miR-145 in glioma cells significantly reduced in vitro proliferation, migration and invasion. Also, overexpression of miR-145 reduced ADAM17 and EGFR expression. In addition, we tested the hypothesis that the miR-145-mediated suppression of cell proliferation, migration and invasion is, at least in part, due to silencing of ADAM17 and EGFR gene expression. Using luciferase reporters carrying the 3′-untranslated region of ADAM17 combined with western blotting, we identified ADAM17 as a direct target of miR-145. Collectively, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor proliferation, but also cell migration and invasion, and warrants further investigation.

TGF-β1 promotes motility and invasiveness of glioma cells through activation of ADAM17.

The transforming growth factor β1 (TGF-β1) belongs to a family of structurally related polypeptide factors. TGF-beta plays an important role in the pathobiology of invasion of malignant gliomas. The objective of the present study was to investigate the impact of TNF-α converting enzyme (TACE/ADAM17) signaling on the process of TGF-β1-stimulated migration and invasion of T98G glioma cells. We found that TGF-β1 increased migration and invasiveness in glioma cells. Addition of the TGF-β1 receptor inhibitor, SB431542, reduced the TGF-β1-stimulated migration and invasiveness of glioma cells. In addition, TGF-β1-induced migration and invasiveness were also blocked by exposure to an ADAM17 inhibitor, TAPI-2. Furthermore, ADAM17 mRNA and protein expression were up-regulated by TGF-β1. Treatment with SB431542 and TAPI-2 blocked TGF-β1-induced ADAM17 protein expression. In summary, these results indicate that TGF-β1 promotes cell migration and invasiveness of glioma cells through stimulation of ADAM17.

Mesenchymal Stem Cell-Derived Exosomes Provide Neuroprotection and Improve Long-Term Neurologic Outcomes in a Swine Model of Traumatic Brain Injury and Hemorrhagic Shock

Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) remains a leading cause of preventable death worldwide. Mesenchymal stem cell-derived exosomes have demonstrated promise in small animal models of neurologic injury. To investigate the effects of exosome treatment in a clinically realistic large animal model, Yorkshire swine underwent TBI and HS. Animals were maintained in shock for 2 h before resuscitation with normal saline (NS). Animals were then resuscitated either with NS (3 × volume of shed blood) or with the same volume of NS with delayed exosome administration (1 × 1013 particles/4 mL) (n = 5/cohort). Exosomes were administered 9 h post-injury, and on post-injury days (PID) 1, 5, 9, and 13. Neurologic severity scores (NSS) were assessed for 30 days, and neurocognitive functions were objectively measured. Exosome-treated animals had significantly lower NSS (p < 0.05) during the first five days of recovery. Exosome-treated animals also had a significantly shorter time to complete neurologic recovery (NSS = 0) compared with animals given NS alone (days to recovery: NS = 16.8 ± 10.6; NS + exosomes = 5.6 ± 2.8; p = 0.03). Animals treated with exosomes initiated neurocognitive testing earlier (days to initiation: NS = 9.6 ± 0.5 vs. NS + exosomes = 4.2 ± 0.8; p = 0.008); however, no difference was seen in time to mastery of tasks. In conclusion, treatment with exosomes attenuates the severity of neurologic injury and allows for faster neurologic recovery in a clinically realistic large animal model of TBI and HS.