Alexander K. Murashov

Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain.

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.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

RNAi pathway is functional in peripheral nerve axons

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 β‐tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal β‐tubulin III mRNA and corresponding protein, as well as a signifi‐cant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is func‐tional 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.—Murashov, A. K., Chintalgattu, V., Islamov, R. R., Lever, T. E., Pak, E. S., Sierpinski, P. L., Katwa, L. C., Van Scott, M. R. RNAi pathway is functional in peripheral nerve axons. FASEB J. 21, 656–670 (2007)

17β-Estradiol stimulates regeneration of sciatic nerve in female mice

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.

Peripheral myelin protein 22 is regulated post-transcriptionally by miRNA-29a†

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.

Directed differentiation of embryonic stem cells into dorsal interneurons

During neural development caudalization and dorsoventral patterning of the neural tube is directed by several inductive factors including retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt signaling. The purpose of the current study was to investigate whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals. Here we show that specific combination of developmental signaling molecules including all trans‐retinoic acid, Shh, bone morphogenetic protein 2 (BMP2), and Wnt3A can direct differentiation of ES cells into dorsal interneurons possessing appropriate neuronal markers, synaptic proteins and functional neurotransmitter machineries. We introduce a concept that Wnt3A morphogenic action relies on crosstalk with both Shh and BMP2 signaling pathways.

17β‐Estradiol enhances neuronal differentiation of mouse embryonic stem cells

Existing protocols show a variety in the percentage of neurons that can be generated from mouse embryonic stem (ES) cells. In the current study, we compared effects of various differentiating conditions, including gelatin and poly‐l‐ornithine/fibronectin coatings, and NGF and 17β‐estradiol treatments on the total yield of neurons, as well as, neurite growth and branching. Here, we show that combination of fibronectin coating with 17β‐estradiol increased number of generated neurons over 50%. Poly‐l‐ornithine/fibronectin increased the percent of neurons in all cultures, suggesting its direct influence on neurogenesis. Addition of 17β‐estradiol reduced mean neurite length in culture, but significantly increased branching. Our results indicate a substrate‐dependent regulation of estrogen‐induced ES cells differentiation into neuronal cells.

MicroRNA machinery responds to peripheral nerve lesion in an injury-regulated pattern

Recently, functional and potent RNA interference (RNAi) has been reported in peripheral nerve axons transfected with short-interfering RNA (siRNA). In addition, components of RNA-induced silencing complex (RISC) have been identified in axotomized sciatic nerve fibers as well as in regenerating dorsal root ganglia (DRG) neurons in vitro. Based on these observations, and on the fact that siRNA and microRNA (miRNA) share the same effector enzymes, we hypothesized that the endogenous miRNA biosynthetic pathway would respond to peripheral nerve injury. To answer this question, we investigated changes in the expression of miRNA biosynthetic enzymes following peripheral nerve crush injury in mice. Here, we show that several pivotal miRNA biosynthetic enzymes are expressed in an injury-regulated pattern in sciatic nerve in vivo, and in DRG axons in vitro. Moreover, the sciatic nerve lesion induced expression of mRNA-processing bodies (P-bodies), which are the local foci of mRNA degradation in DRG axons. In addition, a group of injury-regulated miRNAs was identified by miRNA microarray and validated by real-time quantitative PCR (qPCR) and in situ hybridization analyses. Taken together, our data support the hypothesis that the peripheral nerve regeneration processes may be regulated by miRNA pathway.

Dicer-microRNA pathway is critical for peripheral nerve regeneration and functional recovery in vivo and regenerative axonogenesis in vitro

Both central and peripheral axons contain pivotal microRNA (miRNA) proteins. While recent observations 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:Dicer(fl/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.

MicroRNA-431 regulates axon regeneration in mature sensory neurons by targeting the Wnt antagonist Kremen1

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.