Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3′untranslated region (3′UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3′UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively-proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs.
Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin-resistant phenotype. Although diet-induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long-term voluntary wheel-running in C57BL/6J male mice on their offspring's predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel-running for 12 wk produced offspring that were more susceptible to the adverse effects of a high-fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long-term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro-RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA-induced modification of sperm.
MicroRNAs (miRNAs) are small, non-coding RNAs that function as key post-transcriptional regulators in neural development, brain function, and neurological diseases. Growing evidence indicates that miRNAs are also important mediators of nerve regeneration, however, the affected signaling mechanisms are not clearly understood. In the present study, we show that nerve injury-induced miR-431 stimulates regenerative axon growth by silencing Kremen1, an antagonist of Wnt/beta-catenin signaling. Both the gain-of-function of miR-431 and knockdown of Kremen1 significantly enhance axon outgrowth in murine dorsal root ganglion neuronal cultures. Using cross-linking with AGO-2 immunoprecipitation, and 3′-untranslated region (UTR) luciferase reporter assay we demonstrate miR-431 direct interaction on the 3′-UTR of Kremen1 mRNA. Together, our results identify miR-431 as an important regulator of axonal regeneration and a promising therapeutic target.
Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.
Embryonic stem (ES) cells have been investigated in various animal models of neurodegenerative disease; however, few studies have examined the ability of ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to examine the ability of pre-differentiated murine ES cells (neuronal and glial precursors) to improve functional outcome. Rats were prepared with a unilateral controlled cortical impact injury or sham and then transplanted 7 days later with 100K ES cells (WW6G) (~30% neurons) or media. Two days following transplantation rats were tested on a battery of behavioral tests. It was found that transplantation of ES cells improved behavioral outcome by reducing the initial magnitude of the deficit on the bilateral tactile removal and locomotor placing tests. ES cells also induced almost complete recovery on the vibrissae --> forelimb placing test, whereas, media-transplanted rats failed to show recovery. Acquisition of a reference memory task in the Morris water maze was not improved by transplantation of ES cells. Histological analysis revealed a large number of surviving ES cells in the lesion cavity and showed migration of ES cells into subcortical structures. It was found that transplantation of ES cells prevented the occurrence of multiple small necrotic cavities that were seen in the cortex adjacent to the lesion cavity in media transplanted rats. Additionally, ES cells transplants also significantly reduced lesion size. Results of this study suggest that ES cells that have been pre-differentiated into neuronal precursors prior to transplantation have therapeutic potential.
Relatively little is known about the underlying neuropathology of dysphagia in amyotrophic lateral sclerosis (ALS); thus, effective treatments remain elusive. Tremendous progress toward understanding and treating dysphagia in ALS may be possible through the use of an animal model of dysphagia in ALS research; however, no such animal model currently exists. The most logical candidate to consider is the SOD1-G93A transgenic mouse, the most widely investigated animal model of ALS. To investigate whether this animal model develops dysphagia, oral behaviors (lick and mastication rates) of SOD1-G93A transgenic mice (n = 30) were evaluated at three time points based on hind limb motor function: asymptomatic (60 days), disease onset (approximately 110 days), and disease end-stage (approximately 140 days). Age-matched nontransgenic littermates (n = 30) served as controls. At each time point, lick and mastication rates were significantly lower (p< 0.05) for transgenic mice compared with controls. Histologic analysis of the brainstem showed marked neurodegeneration (vacuolation) of the trigeminal and hypoglossal nuclei, two key motor components involved in mastication and licking behaviors. These results demonstrate a clinicopathologic correlation of oral dysfunction in SOD1-G93A transgenic mice, thereby establishing the SOD1-G93A transgenic mouse as a bona fide animal model of oral dysphagia in ALS.
Both central and peripheral axons contain pivotal microRNA (miRNA) proteins. While recent observation demonstrated that miRNA biosynthetic machinery responds to peripheral nerve lesion in an injury-regulated pattern, the physiological significance of this phenomenon remains to be elucidated. In the current paper we hypothesized that deletion of Dicer would disrupt production of Dicer-dependent miRNAs and would negatively impact regenerative axon growth. Taking advantage of tamoxifen-inducible CAG-CreERt:Dicerfl/fl knockout (Dicer KO), we investigated the results of Dicer deletion on sciatic nerve regeneration in vivo and regenerative axon growth in vitro. Here we show that the sciatic functional index, an indicator of functional recovery, was significantly lower in Dicer KO mice in comparison to wild-type animals. Restoration of mechanical sensitivity recorded in the von Frey test was also markedly impaired in Dicer mutants. Further, Dicer deletion impeded the recovery of nerve conduction velocity and amplitude of evoked compound action potentials in vitro. Histologically, both total number of regenerating nerve fibers and mean axonal area were notably smaller in the Dicer KO mice. In addition, Dicer-deficient neurons failed to regenerate axons in dissociated dorsal root ganglia (DRG) cultures. Taken together, our results demonstrate that knockout of Dicer clearly impedes regenerative axon growth as well as anatomical, physiological and functional recovery. Our data suggest that the intact Dicer-dependent miRNA pathway is critical for the successful peripheral nerve regeneration after injury.
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