A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery.

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Astrocytes have an important role in synaptic formation and function but how astrocytic processes become associated with synaptic structures during development is not well understood. Here we analyzed the pattern of growth of the processes extending off the main Bergmann glial (BG) shafts during synaptogenesis in the cerebellum. We found that during this period BG process outgrowth was correlated with increased ensheathment of dendritic spines. In addition, two-photon time-lapse imaging revealed that BG processes were highly dynamic, and processes became more stable as the period of spine ensheathment progressed. While process motility was dependent on actin polymerization, activity of cytoskeletal regulators Rac1 and RhoG did not play a role in glial process dynamics or density, but was critical for maintaining process length. We extended this finding to probe the relationship between process morphology and ensheathment, finding that shortened processes result in decreased coverage of the spine. Furthermore, we found that areas in which BG expressed dn-Rac1, and therefore had a lower level of synaptic ensheathment, showed an overall increase in synapse number. These analyses reveal how BG processes grow to surround synaptic structures, elucidate the importance of BG process structure for proper development of synaptic ensheathment, and reveal a role for ensheathment in synapse formation.

The pattern of growth of Purkinje cell dendrites has been analyzed and related to their interactions with Bergmann glial radial processes. In cerebellar slice cultures from mice expressing green fluorescent protein (GFP) under the glial fibrillary acidic protein (GFAP) promoter, Purkinje cells were transfected and imaged with two-photon microscopy over 2 days. We report that while the Purkinje cell dendritic tree grows, individual dendrites increase or decrease in length. Importantly, we demonstrate that vertical growth of Purkinje cell dendrites occurs primarily in alignment with radial glial processes. These findings suggest that radial glial processes provide a structural substrate for the directional growth of Purkinje cell dendrites, thus influencing the shape of the dendritic tree.

BackgroundMicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response.MethodsIntravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO.ResultsIn the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-β and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls.ConclusionsBased on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0753-x) contains supplementary material, which is available to authorized users.

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In the cerebellum, lamellar Bergmann glial (BG) appendages wrap tightly around almost every Purkinje cell dendritic spine. The function of this glial ensheathment of spines is not entirely understood. The development of ensheathment begins near the onset of synaptogenesis, when motility of both BG processes and dendritic spines are high. By the end of the synaptogenic period, ensheathment is complete and motility of the BG processes decreases, correlating with the decreased motility of dendritic spines. We therefore have hypothesized that ensheathment is intimately involved in capping synaptogenesis, possibly by stabilizing synapses. To test this hypothesis, we misexpressed GluR2 in an adenoviral vector in BG towards the end of the synaptogenic period, rendering the BG α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) Ca2+-impermeable and causing glial sheath retraction. We then measured the resulting spine motility, spine density and synapse number. Although we found that decreasing ensheathment at this time does not alter spine motility, we did find a significant increase in both synaptic pucta and dendritic spine density. These results indicate that consistent spine coverage by BG in the cerebellum is not necessary for stabilization of spine dynamics, but is very important in the regulation of synapse number.

Short chain fatty acids, produced as gut microbiome metabolites but also present in the diet, exert broad effects in host physiology. Propionic acid (PPA), along with butyrate and acetate, plays a growing role in health, but also in neurological conditions. Increased PPA exposure in humans, animal models and cell lines elicit diverse behavioural and biochemical changes consistent with organic acidurias, mitochondrial disorders and autism spectrum disorders (ASD). ASD is considered a disorder of synaptic dysfunction and cell signalling, but also neuroinflammatory and neurometabolic components. We examined behaviour (Morris water and radial arm mazes) and the ultrastructure of the hippocampus and medial prefrontal cortex (electron microscopy) following a single intraperitoneal (i.p.) injection of PPA (175 mg/kg) in male adolescent rats. PPA treatment showed altered social and locomotor behaviour without changes in learning and memory. Both transient and enduring ultrastructural alterations in synapses, astro‐ and microglia were detected in the CA1 hippocampal area. Electron microscopic analysis showed the PPA treatment significantly decreased the total number of synaptic vesicles, presynaptic mitochondria and synapses with a symmetric active zone. Thus, brief systemic administration of this dietary and enteric short chain fatty acid produced behavioural and dynamic brain ultrastructural changes, providing further validation of the PPA model of ASD.