Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve

Publisher's PDF.Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neurodegeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. IntroductionUniversity of Delaware. Department of Biological Sciences

Ingenuity Pathway Analysis of the differentially expressed up-regulated and down-regulated miRNA predicted targets.

<p>The listed pathways were from the category of “nervous system development and function” and sorted by <i>p</i>-value. Only rows having 3 or more molecules were shown.</p

Distinct pattern of miRNA levels in sciatic nerve in response to injury.

<p>The heatmap with a cluster dendrogram showed significant changes in levels of 63 miRNAs in rat sciatic nerve that received a crush injury and were sacrificed at 1, 4, 7, and 14 days post-injury (DPI), as compared to the sham-operated uninjured control (n = 3/group). <b>A.</b> The color scale shown on the top left denotes the relative expression level of the indicated miRNA across all time points (log₂ scale): red represents an increased change in level and green denotes a decreased level. Clustering analysis was performed using <i>Cluster 3</i>.<i>0</i> with an average linkage and Euclidean distances metric and visualized using Java <i>TreeView</i>. <b>B.</b> Each column represents different time points after injury [1, 4, 7, and 14 days post-injury (DPI)], as compared to the uninjured control (n = 3/group), and rows represents individual miRNAs. Red represents an increased change in level compared to that in the uninjured control and green denotes a decreased level. Insets on the top show three distinct clusters of miRNA changes. Dashed lines indicate miRNA levels in the uninjured control.</p

Validation of mature miRNA sequencing data by real time qPCR assay.

<p>Bar graph showed changes in levels of 8 miRNAs at 1, 4, 7, and 14 days post-injury (DPI), as compared to those of uninjured control. Top panels. miRNAs significantly up-regulated following injury. Bottom panels. miRNAs significantly down-regulated following injury. Error bars indicate standard deviation.</p

Enrichment and detection of small non-coding miRNA from sciatic nerve axoplasm.

<p><b>A.</b> Extended RT-PCR of mRNAs from sciatic nerve axoplasm using either by the conventional whole tissue lysate method or a mechanical squeezing procedure. Note that neither cell body restricted (MAP2, H1F0) nor glial (GFAP, ErbB3) mRNAs were amplified from sciatic nerve cDNAs reverse-transcribed from the squeezed axoplasmic RNAs. <b>B.</b> Size and frequency distribution of regulatory and small non-coding RNAs in uninjured sciatic nerve. <b>C.</b> A size histogram of mapped small RNAs. >40% of small RNAs were mapped to miRNAs, while 30% of small RNA sequences could not be annotated.</p

Seven miRNAs significantly up-regulated and eight miRNAs down-regulated following nerve injury.

<p>Seven miRNAs significantly up-regulated and eight miRNAs down-regulated following nerve injury.</p

Sonic hedgehog-induced histone deacetylase activation is required for cerebellar granule precursor hyperplasia in medulloblastoma.

Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP) cells. Sonic hedgehog (Shh) is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs) and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL) in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease

Additional file 1: Figure S1. of EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation

Sodium-induced post-translational modifications of tubulin is limited to acetylation. Figure S2. EGF treatment increases intracellular sodium. Figure S3. Sodium-induced acetylation of tubulin and calcium signaling. Figure S4. Average vesicle speed in nocodazole-treated cells. (PDF 360 kb

Differentiating CGP cells show a decline of HDAC activity and protein level.

<p>A, Immunoblot of CGP cells differentiating in vitro. CGP cells isolated from P5 wild type mice were plated and harvested at different days of in vitro culture (DIV). Numbers below immunoblots are the intensity normalized to undifferentiated cells. B, HDAC activity of CGP cells differentiated in vitro was measured. Assay was performed three times in triplicate and the representative one is shown. Bars correspond to mean ± S.E. of three independent experiments.</p