Axonal localization of neuritin/CPG15 mRNA is limited by competition for HuD binding.

HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.

Different motif requirements for the localization zipcode element of ß-actin mRNA binding by HuD and ZBP1.

Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat ß-actin mRNA's 3' UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3' end of a 5'-ACACCC-3' motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the ß-actin zipcode, but they recognize different features of this target sequence.

Axonal amphoterin mRNA is regulated by translational control and enhances axon outgrowth.

High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3'-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3'-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3'-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body.

EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation.

BACKGROUND: Endocytosis of activated EGF receptor (EGFR) to specific endocytic compartments is required to terminate EGF signaling. Trafficking of EGFR relies on microtubule tracks that transport the cargo vesicle to their intermediate and final destinations and can be modulated through posttranslational modification of tubulin including acetylation. Na,K-ATPase maintains intracellular sodium homeostasis, functions as a signaling scaffold and interacts with EGFR. Na,K-ATPase also binds to and is regulated by acetylated tubulin but whether there is a functional link between EGFR, Na,K-ATPase and tubulin acetylation is not known. RESULTS: EGF-induced sodium influx regulates EGFR trafficking through increased microtubule acetylation. Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin. In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover. Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation. CONCLUSIONS: These studies provide a novel regulatory mechanism to attenuate EGFR signaling in which EGF modulates EGFR trafficking through intracellular sodium-mediated HDAC6 inactivation and tubulin acetylation.

Identification of precursor microRNAs within distal axons of sensory neuron.

A set of specific precursor microRNAs (pre-miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre-miRNAs are also transported into distal axons to autonomously regulate intra-axonal protein synthesis. Here, we show that a subset of pre-miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre-miRNAs (let 7c-a and pre-miRs-16, 23a, 25, 125b-1, 433, and 541) showed elevated axonal levels, while others (pre-miRs-138-2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre-miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

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 ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-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 ß-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.

Axonal transport of neural membrane protein 35 mRNA increases axon growth.

Many neuronal mRNAs are transported from cell bodies into axons and dendrites. Localized translation of the mRNAs brings autonomy to these processes that can be vast distances from the cell body. For axons, these translational responses have been linked to growth and injury signaling, but there has been little information about local function of individual axonally synthesized proteins. In the present study, we show that axonal injury increases levels of the mRNA encoding neural membrane protein 35 (NMP35) in axons, with a commensurate decrease in the cell body levels of NMP35 mRNA. The 3' untranslated region (3'UTR) of NMP35 is responsible for this localization into axons. Previous studies have shown that NMP35 protein supports cell survival by inhibiting Fas-ligand-mediated apoptosis; however, these investigations did not distinguish functions of the locally generated NMP35 protein. Using axonally targeted versus cell-body-restricted NMP35 constructs, we show that NMP35 supports axonal growth, and overexpression of an axonally targeted NMP35 mRNA is sufficient to increase axonal outgrowth.

Axonal localization of neuritin/CPG15 mRNA in neuronal populations through distinct 5' and 3' UTR elements.

Many neuronal mRNAs are actively transported into distal axons. The 3' untranslated regions (UTRs) of axonal mRNAs often contain cues for their localization. The 3' UTR of neuritin mRNA was shown to be sufficient for localization into axons of hippocampal neurons. Here, we show that neuritin mRNA localizes into axons of rat sensory neurons, but this is predominantly driven by the 5' rather than 3' UTR. Neuritin mRNA shifts from cell body to axon predominantly after nerve crush injury, suggesting that it encodes a growth-associated protein. Consistent with this, overexpression of neuritin increases axon growth but only when its mRNA localizes into the axons.

Axonally synthesized ß-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 ß-actin mRNA 3'UTR competes with endogenous ß-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 ß-actin mRNA. We took advantage of this 3'UTR competition to address the functions of axonally synthesized ß-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of ß-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of ß-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 ß-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 ß-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 ß-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of ß-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.

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 ß-actin and GAP-43 mRNAs. ß-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 ß-actin mRNA for axonal localization and decreases axon length and branching similar to the ß-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. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth.

Lysophosphatidic acid differentially regulates axonal mRNA translation through 5'UTR elements.

Sensory neurons transport a complex population of mRNAs into their axons, including many encoding ER chaperone proteins. Transport of the mRNA encoding the ER chaperone protein calreticulin is regulated through 3'UTR elements. In other cellular systems, translation of chaperone protein mRNAs can be regulated by ER stress. Here, we have asked if the translation of axonal calreticulin mRNA is regulated in a different manner than its transport into axons. Treatment with lysophosphatidic acid, which is known to trigger axon retraction and stimulate ER Ca(2+) release, caused a translation-dependent increase in axonal calreticulin protein levels. RNA sequences in the 5'UTR of calreticulin confer this translational control through a mechanism that requires an inactivating phosphorylation of eIF2α. In contrast to calreticulin, these signaling events do not activate axonal translation through ß-actin's 5'UTR. Together, these data indicate that stimulation of ER stress can regulate specificity of localized mRNA translation through 5'UTR elements.

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.

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.

MicroRNAs 21 and 199a-3p Regulate Axon Growth Potential through Modulation of Pten and mTor mRNAs.

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA levels. Conversely, miR-199a-3p directly bound to mTor mRNA and miR-199a-3p overexpression decreased mTor mRNA levels. Overexpressing miR-21 increased both overall and intra-axonal protein synthesis in cultured DRGs, while miR-199a-3p overexpression decreased this protein synthesis. The axon growth phenotypes seen with miR-21 and miR-199a-3p overexpression were reversed by co-transfecting PTEN and mTOR cDNA expression constructs with the predicted 3' untranslated region (UTR) miR target sequences deleted. Taken together, these studies indicate that injury-induced alterations in miR-21 and miR-199a-3p expression can alter axon growth capacity by changing overall and intra-axonal protein synthesis through regulation of the PTEN/mTOR pathway.

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, TNFalpha, 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.

Differential regulation of brain-derived neurotrophic factor (BDNF) expression in sensory neuron axons by miRNA-206.

Distinct subcellular localization and subsequent translational control of 3' UTR variants of mRNA encoding brain-derived neurotrophic factor (BDNF) are critical for the development and plasticity of neurons. Although the processes that lead to preferential localization of BDNF have been well studied, it is still not clear how neurons ensure differential BDNF production in a spatial-specific manner. Here, we identified that microRNA (miRNA)-206 has the potential to specifically regulate BDNF with a long 3' UTR without affecting its short 3' UTR counterpart. Overexpression of miRNA-206 in sensory neurons resulted in a 30% and 45% reduction of BDNF protein expression in the cell bodies and axons, respectively. The work described in the present study indicates that miRNAs can differentially and specifically regulate the expression of transcript variants with different localization patterns.

Analysis of piRNA-Like Small Non-coding RNAs Present in Axons of Adult Sensory Neurons.

Small non-coding RNAs (sncRNAs) have been shown to play pivotal roles in spatiotemporal-specific gene regulation that is linked to many different biological functions. PIWI-interacting RNAs (piRNAs), typically 25-34-nucleotide long, are originally identified and thought to be restricted in germline cells. However, recent studies suggest that piRNAs associate with neuronal PIWI proteins, contributing to neuronal development and function. Here, we identify a cohort of piRNA-like sncRNAs (piLRNAs) in rat sciatic nerve axoplasm and directly contrast temporal changes of piLRNA levels in the nerve following injury, as compared with those in an uninjured nerve using deep sequencing. We find that 32 of a total of 53 annotated piLRNAs show significant changes in their levels in the regenerating nerve, suggesting that individual axonal piLRNAs may play important regulatory roles in local messenger RNA (mRNA) translation during regeneration. Bioinformatics and biochemical analyses show that these piLRNAs carry characteristic features of mammalian piRNAs, including sizes, a sequence bias for uracil at the 5'-end and a 2'-O-methylation at the 3'-end. Their axonal expression is directly visualized by fluorescence in situ hybridization in cultured dorsal root ganglion neurons as well as immunoprecipitation with MIWI. Further, depletion of MIWI protein using RNAi from cultured sensory neurons increases axon growth rates, decreases axon retraction after injury, and increases axon regrowth after injury. All these data suggest more general roles for MIWI/piLRNA pathway that could confer a unique advantage for coordinately altering the population of proteins generated in growth cones and axons of neurons by targeting mRNA cohorts.

Murine Oviductosomes (OVS) microRNA profiling during the estrous cycle: Delivery of OVS-borne microRNAs to sperm where miR-34c-5p localizes at the centrosome.

Oviductosomes (OVS) are nano-sized extracellular vesicles secreted in the oviductal luminal fluid by oviductal epithelial cells and known to be involved in sperm capacitation and fertility. Although they have been shown to transfer encapsulated proteins to sperm, cargo constituents other than proteins have not been identified. Using next-generation sequencing, we demonstrate that OVS are carriers of microRNAs (miRNAs), with 272 detected throughout the estrous cycle. Of the 50 most abundant, 6 (12%) and 2 (4%) were expressed at significantly higher levels (P < 0.05) at metestrus/diestrus and proestrus/estrus. RT-qPCR showed that selected miRNAs are present in oviductal epithelial cells in significantly (P < 0.05) lower abundance than in OVS, indicating selective miRNA packaging. The majority (64%) of the top 25 OVS miRNAs are present in sperm. These miRNAs' potential target list is enriched with transcription factors, transcription regulators, and protein kinases and there are several embryonic developmentally-related genes. Importantly, OVS can deliver to sperm miRNAs, including miR-34c-5p which is essential for the first cleavage and is solely sperm-derived in the zygote. Z-stack of confocal images of sperm co-incubated with OVS loaded with labeled miRNAs showed the intracellular location of the delivered miRNAs. Interestingly, individual miRNAs were predominantly localized in specific head compartments, with miR-34c-5p being highly concentrated at the centrosome where it is known to function. These results, for the first time, demonstrate OVS' ability to contribute to the sperm's miRNA repertoire (an important role for solely sperm-derived zygotic miRNAs) and the physiological relevance of an OVS-borne miRNA that is delivered to sperm.

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.