Soonmoon Yoo

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Axonal Degeneration Is Mediated by the Mitochondrial Permeability Transition Pore

Axonal degeneration is an active process that has been associated with neurodegenerative conditions triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. This degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. Several signaling pathways are implicated in axonal degeneration, but identification of an integrative mechanism for this self-destructive process has remained elusive. Here, we show that rapid axonal degeneration triggered by distinct mechanical and toxic insults is dependent on the activation of the mitochondrial permeability transition pore (mPTP). Both pharmacological and genetic targeting of cyclophilin D, a functional component of the mPTP, protects severed axons and vincristine-treated neurons from axonal degeneration in ex vivo and in vitro mouse and rat model systems. These effects were observed in axons from both the peripheral and central nervous system. Our results suggest that the mPTP is a key effector of axonal degeneration, upon which several independent signaling pathways converge. Since axonal and synapse degeneration are increasingly considered early pathological events in neurodegeneration, our work identifies a potential target for therapeutic intervention in a wide variety of conditions that lead to loss of axons and subsequent functional impairment.

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity

Subcellular localization of mRNAs is regulated by RNA–protein interactions. Here, we show that introduction of a reporter mRNA with the 3′UTR of β‐actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β‐actin mRNA, and introducing GFP with the 3′UTR of β‐actin mRNA depletes axons of endogenous β‐actin and GAP‐43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β‐actin and GAP‐43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

Axonally Synthesized β-Actin and GAP-43 Proteins Support Distinct Modes of Axonal Growth

Increasing evidence points to the importance of local protein synthesis for axonal growth and responses to axotomy, yet there is little insight into the functions of individual locally synthesized proteins. We recently showed that expression of a reporter mRNA with the axonally localizing β-actin mRNA 3′UTR competes with endogenous β-actin and GAP-43 mRNAs for binding to ZBP1 and axonal localization in adult sensory neurons (Donnelly et al., 2011). Here, we show that the 3′UTR of GAP-43 mRNA can deplete axons of endogenous β-actin mRNA. We took advantage of this 3′UTR competition to address the functions of axonally synthesized β-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of β-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of β-actin protein while increasing axonal synthesis of GAP-43 protein resulted in long axons with few branches. siRNA-mediated depletion of overall GAP-43 mRNA from dorsal root ganglia (DRGs) decreased the length of axons, while overall depletion of β-actin mRNA from DRGs decreased the number of axon branches. These deficits in axon growth could be rescued by transfecting with siRNA-resistant constructs encoding β-actin or GAP-43 proteins, but only if the mRNAs were targeted for axonal transport. Finally, in ovo electroporation of axonally targeted GAP-43 mRNA increased length and axonally targeted β-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of β-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.

Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration.

Locally generating new proteins in subcellular regions provide means to spatially and temporally modify protein content in polarized cells. Recent years have seen resurgence of the concept that axonal processes of neurons can locally synthesize proteins. Experiments from a number of groups have now shown that axonal protein synthesis helps to initiate growth, provides a means to respond to guidance cues, and generates retrograde signaling complexes. Additionally, there is increasing evidence that locally synthesized proteins provide functions beyond injury responses and growth in the mature peripheral nervous system. A key regulatory event in this translational regulation is moving the mRNA templates into the axonal compartment. Transport of mRNAs into axons is a highly regulated and specific process that requires interaction of RNA binding proteins with specific cis-elements or structures within the mRNAs. mRNAs are transported in ribonucleoprotein particles that interact with microtubule motor proteins for long-range axonal transport and likely use microfilaments for short-range movement in the axons. The mature axon is able to recruit mRNAs into translation with injury and possibly other stimuli, suggesting that mRNAs can be stored in a dormant state in the distal axon until needed. Axotomy triggers a shift in the populations of mRNAs localized to axons, indicating a dynamic regulation of the specificity of the axonal transport machinery. In this review, we discuss how axonal mRNA transport and localization are regulated to achieve specific changes in axonal RNA content in response to axonal stimuli.

Increased growth factor expression and cell proliferation after contusive spinal cord injury

The damage caused by traumatic central nervous system (CNS) injury can be divided into two phases: primary and secondary. The initial injury destroys many of the local neurons and glia and triggers secondary mechanisms that result in further cell loss. Approximately 50% of the astrocytes and oligodendrocytes in the spared white matter of the epicenter die by 24 h after spinal cord injury (SCI), but their densities return to normal levels by 6 weeks. This repopulation is largely due to the proliferation of local progenitors that divide in response of CNS injury. Previous studies indicate that the secondary events that cause cell death after SCI also increase the local levels of several growth factors that stimulate the proliferation of these endogenous progenitors. We compared the spatial pattern of the post-injury up-regulation of the pro-mitotic growth factors with that of 5-bromodeoxyuridine (BrdU) incorporation to determine if each could play a role in proliferation. Three days after a standard contusive SCI or laminectomy, animals received intraperitoneal BrdU injections to label dividing cells and were perfused 2 h after the last injection. Immunohistochemistry for BrdU and basic fibroblast growth factor (FGF2) and in situ hybridization for ciliary neurotrophic factor (CNTF) and glial growth factor (GGF2) mRNA were used to compare the number of dividing cells with growth factor levels in sections 2 and 4 mm from the epicenter. All three growth factors are significantly up-regulated 3 days after SCI, when cell proliferation is maximal. The increase in GGF2 and FGF2 levels is highest in sections 2 mm rostral to the epicenter, mimicking BrdU incorporation. Addition of rhGGF2 to cultured cells isolated from the spinal cord 3 days after SCI increased the number of NG2+ glial progenitors. These data suggest that FGF2 and GGF2 may contribute to the spontaneous recovery observed after SCI by stimulating the proliferation of local progenitors that help repopulate the injured cord.

MicroRNAs and Potential Targets in Osteosarcoma: Review

Osteosarcoma is the most common bone cancer in children and young adults. Surgery and multi-agent chemotherapy are the standard treatment regimens for this disease. New therapies are being investigated to improve overall survival in patients. Molecular targets that actively modulate cell processes, such as cell-cycle control, cell proliferation, metabolism, and apoptosis, have been studied, but it remains a challenge to develop novel, effective-targeted therapies to treat this heterogeneous and complex disease. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating cell processes including growth, development, and disease. miRNAs function as oncogenes or tumor suppressors to regulate gene and protein expression. Several studies have demonstrated the involvement of miRNAs in the pathogenesis of osteosarcoma with the potential for development in disease diagnostics and therapeutics. In this review, we discuss the current knowledge on the role of miRNAs and their target genes and evaluate their potential use as therapeutic agents in osteosarcoma. We also summarize the efficacy of inhibition of oncogenic miRNAs or expression of tumor suppressor miRNAs in preclinical models of osteosarcoma. Recent progress on systemic delivery as well as current applications for miRNAs as therapeutic agents has seen the advancement of miR-34a in clinical trials for adult patients with non-resectable primary liver cancer or metastatic cancer with liver involvement. We suggest a global approach to the understanding of the pathogenesis of osteosarcoma may identify candidate miRNAs as promising biomarkers for this rare disease.

A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.

Conserved 3′-Untranslated Region Sequences Direct Subcellular Localization of Chaperone Protein mRNAs in Neurons

mRNA localization provides polarized cells with a locally renewable source of proteins. In neurons, mRNA translation can occur at millimeters to centimeters from the cell body, giving the dendritic and axonal processes a means to autonomously respond to their environment. Despite that hundreds of mRNAs have been detected in neuronal processes, there are no reliable means to predict mRNA localization elements. Here, we have asked what RNA elements are needed for localization of transcripts encoding endoplasmic reticulum chaperone proteins in neurons. The 3′-untranslated regions (UTRs) of calreticulin and Grp78/BiP mRNAs show no homology to one another, but each shows extensive regions of high sequence identity to their 3′UTRs in mammalian orthologs. These conserved regions are sufficient for subcellular localization of reporter mRNAs in neurons. The 3′UTR of calreticulin has two conserved regions, and either of these is sufficient for axonal and dendritic targeting. However, only nucleotides 1315–1412 show ligand responsiveness to neurotrophin 3 (NT3) and myelin-associated glycoprotein (MAG). This NT3- and MAG-dependent axonal mRNA transport requires activation of JNK, both for calreticulin mRNA and for other mRNAs whose axonal levels are commonly regulated by NT3 and MAG.

Interaction of NG2+ glial progenitors and microglia/macrophages from the injured spinal cord

Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2+ progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell–cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2+ progenitors and OX42+ microglia/macrophages from contused rat spinal cord. Purified NG2+ cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42+ cells did not form spheroids and significantly reduced sphere growth by NG2+ cells in co‐cultures. Conditioned medium from these OX42+ cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2+ cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42+ cells for a panel of six genes for secreted factors showed expression of several that could contribute to inhibition of NG2+ cells. Further, the pattern of expression of four of these, TNFα, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42+ cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2+ cells in vivo after SCI. Thus, the nonreplacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion.