Kristy Welshhans

Netrin-1-Induced Local β-Actin Synthesis and Growth Cone Guidance Requires Zipcode Binding Protein 1

Local β-actin synthesis in growth cones of developing axons plays an important role in growth cone steering; however, the mRNA binding proteins required for this process are unknown. Here we used Zbp1/Imp1−/− mice to test the hypothesis that zipcode binding protein 1 (ZBP1) is required for the regulation of β-actin mRNA transport and local translation underlying growth cone guidance. To address the biological function of ZBP1, we developed a novel in vitro turning assay with primary cortical neuron balls having axons >1 mm in length and demonstrate that growth cones of mammalian neurons exhibit protein synthesis-dependent attraction to either netrin-1 or brain-derived neurotrophic factor (BDNF). Interestingly, this attraction is lost in Zbp1-deficient neurons. Furthermore, BDNF-stimulated β-actin mRNA localization was attenuated in Zbp1-deficient neurons, which impaired enrichment of β-actin protein in the growth cone. Finally, using a photoconvertible translation reporter, we found that ZBP1 is necessary for netrin-1 stimulated local translation of β-actin mRNA in axonal growth cones. Together, these results suggest that netrin-1- and BDNF-induced growth cone attraction required ZBP1-mediated local translation of β-actin mRNA, and therefore ZBP1 regulates protein synthesis-dependent axon guidance. Thus, mRNA binding proteins regulating local translation can control spatiotemporal protein expression in response to guidance cues and directional cell motility.

Phosphorylation of Zipcode Binding Protein 1 Is Required for Brain-Derived Neurotrophic Factor Signaling of Local β-Actin Synthesis and Growth Cone Turning

The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the β-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for β-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phosphorylation site, and immunofluorescence revealed BDNF-induced phosphorylation of ZBP1 within growth cones. The BDNF-induced increase in fluorescent signal of a green fluorescent protein translation reporter with the 3′ untranslated region of β-actin was attenuated with the Src family kinase-specific inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Furthermore, a nonphosphorylatable mutant, ZBP1 Y396F, suppressed the BDNF-induced and protein synthesis-dependent increase in β-actin localization in growth cones. Last, the ZBP1 Y396F mutant blocked BDNF-induced attractive growth cone turning. These results indicate that phosphorylation of ZBP1 at Tyr396 within growth cones has a critical role to regulate local protein synthesis and growth cone turning. Our findings provide new insight into how the regulated phosphorylation of mRNA-binding proteins influences local translation underlying growth cone motility and axon guidance.

The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of rats

5-Hydroxytryptophan (5-HTP), which is the rate-limiting precursor in serotonin (5-hydroxytryptamine (5-HT)) biosynthesis, is used as an oral supplement to enhance serotonin levels in humans. To evaluate its effects on serotonin levels and localization, 5-hydroxytryptophan was administered to Sprague-Dawley rats either orally or via intraperitoneal injection. 5-Hydroxytryptophan-immunoreactivity was co-localized with serotonin-immunoreactivity in the serotonergic dorsal raphe nucleus of control animals and this was not changed in animals given 5-hydroxytryptophan. Oral 5-HTP administration increased the intensity of both 5-HTP and serotonin immunoreactivity in raphe neurons. However, 5-HTP treatment also caused ectopic 5-hydroxytryptophan-immunoreactivity and serotonin-immunoreactivity in normally dopaminergic neurons of the substantia nigra par compacta. Serotonin-immunoreactivity was confined to neurons that also displayed amino acid decarboxylase immunoreactivity, but in a small percentage of substantia nigra neurons, serotonin immunoreactivity was not co-localized with tyrosine hydroxylase-immunoreactivity. The intensity of the immunoreactivity to serotonin and 5-hydroxytryptophan in the substantia nigra was maximal within 2h of 5-hydroxytryptophan administration and returned to control levels by 24h. This time course mirrored changes in HPLC measurements of 5-hydroxytryptophan, serotonin, and the metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the urine. 5-Hydroxytryptophan administration did not cause ectopic appearance of either serotonin or 5-hydroxytryptophan in the noradrenergic locus coeruleus. These results suggest that a single oral dose of 5-HTP increases the 5-HTP and serotonin content of serotonergic neurons and causes the transient ectopic appearance of serotonin in some normally non-serotonergic neurons.

Local activation of the nitric oxide/cyclic guanosine monophosphate pathway in growth cones regulates filopodial length via protein kinase G, cyclic ADP ribose and intracellular Ca2+ release

Nitric oxide (NO) is a gaseous messenger that has been shown to affect growth cone motility and neurite outgrowth in several model systems, but how NO brings about its effects is not understood. We have previously demonstrated that global and long‐term application of NO to Helisoma trivolvis B5 neurons results in a transient increase in filopodial length, decrease in filopodial number and decrease in neurite outgrowth, all of which are mediated via soluble guanylyl cyclase (sGC) and involve an increase in the intracellular Ca2+ concentration [S. Van Wagenen & V. Rehder (1999)Journal of Neurobiology, 39, 168–185; K.R. Trimm & V. Rehder (2004)European Journal of Neuroscience, 19, 809–818]. The goal of the current study was twofold: to investigate the effects of short‐term NO exposure on individual growth cones and to further elucidate the downstream pathway through which NO exerts its effects. Local application of the NO donor NOC‐7 for 10–20 ms via puffer micropipette resulted in a transient increase in filopodial length and a small decrease in filopodial number. We show evidence that these effects of NO are mediated via sGC, protein kinase G and cyclic ADP ribose, resulting in the release of Ca2+ from intracellular stores, probably of the ryanodine‐sensitive type. These results suggest that growth cones expressing sGC are highly sensitive to local and short‐term exposure to NO, which they may experience during pathfinding, and that the stereotyped response of transient filopodial elongation seen in B5 neurons in response to NO requires intracellular Ca2+ release.

RACK1 Is a Ribosome Scaffold Protein for β-actin mRNA/ZBP1 Complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.

Nitric oxide regulates growth cone filopodial dynamics via ryanodine receptor‐mediated calcium release

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC‐7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca2+]i). Moreover, these NO‐induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca2+]i that results from local NO application is mediated via release from ryanodine receptor (RyR)‐sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO‐induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO‐induced calcium increase; however, this calcium influx is secondary to the RyR‐induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.

Sonic Hedgehog guides axons via Zipcode Binding Protein 1-mediated local translation

Sonic hedgehog (Shh) attracts spinal cord commissural axons toward the floorplate. How Shh elicits changes in the growth cone cytoskeleton that drive growth cone turning is unknown. We find that the turning of rat commissural axons up a Shh gradient requires protein synthesis. In particular, Shh stimulation increases β-actin protein at the growth cone even when the cell bodies have been removed. Therefore, Shh induces the local translation of β-actin at the growth cone. We hypothesized that this requires zipcode binding protein 1 (ZBP1), an mRNA-binding protein that transports β-actin mRNA and releases it for local translation upon phosphorylation. We found that Shh stimulation increases phospho-ZBP1 levels in the growth cone. Disruption of ZBP1 phosphorylation in vitro abolished the turning of commissural axons toward a Shh gradient. Disruption of ZBP1 function in vivo in mouse and chick resulted in commissural axon guidance errors. Therefore, ZBP1 is required for Shh to guide commissural axons. This identifies ZBP1 as a new mediator of noncanonical Shh signaling in axon guidance. SIGNIFICANCE STATEMENT Sonic hedgehog (Shh) guides axons via a noncanonical signaling pathway that is distinct from the canonical Hedgehog signaling pathway that specifies cell fate and morphogenesis. Axon guidance is driven by changes in the growth cone in response to gradients of guidance molecules. Little is known about the molecular mechanism of how Shh orchestrates changes in the growth cone cytoskeleton that are required for growth cone turning. Here, we show that the guidance of axons by Shh requires protein synthesis. Zipcode binding protein 1 (ZBP1) is an mRNA-binding protein that regulates the local translation of proteins, including actin, in the growth cone. We demonstrate that ZBP1 is required for Shh-mediated axon guidance, identifying a new member of the noncanonical Shh signaling pathway.

Netrin-1 induces local translation of down syndrome cell adhesion molecule in axonal growth cones.

Down syndrome cell adhesion molecule (DSCAM) plays an important role in many neurodevelopmental processes such as axon guidance, dendrite arborization, and synapse formation. DSCAM is located in the Down syndrome trisomic region of human chromosome 21 and may contribute to the Down syndrome brain phenotype, which includes a reduction in the formation of long‐distance connectivity. The local translation of a select group of mRNA transcripts within growth cones is necessary for the formation of appropriate neuronal connectivity. Interestingly, we have found that Dscam mRNA is localized to growth cones of mouse hippocampal neurons, and is dynamically regulated in response to the axon guidance molecule, netrin‐1. Furthermore, netrin‐1 stimulation results in an increase in locally translated DSCAM protein in growth cones. Deleted in colorectal cancer (DCC), a netrin‐1 receptor, is required for the netrin‐1‐induced increase in Dscam mRNA local translation. We also find that two RNA‐binding proteins—fragile X mental retardation protein (FMRP) and cytoplasmic polyadenylation element binding protein (CPEB)—colocalize with Dscam mRNA in growth cones, suggesting their regulation of Dscam mRNA localization and translation. Finally, overexpression of DSCAM in mouse cortical neurons results in a severe stunting of axon outgrowth and branching, suggesting that an increase in DSCAM protein results in a structural change having functional consequences. Taken together, these results suggest that netrin‐1‐induced local translation of Dscam mRNA during embryonic development may be an important mechanism to regulate axon growth and guidance in the developing nervous system.

Local translation of cell adhesion molecules in axons

During development and regeneration, axonal growth depends on a rapid response to extracellular growth and guidance molecules. One mechanism underlying this rapid response is local protein synthesis (Jung et al., 2012). Local protein synthesis is a highly tuned, multi-step process by which mRNA binding proteins bind to specific sequences within the untranslated regions of particular mRNAs in the soma; mRNAs are then transported to axons and growth cones and translated at the appropriate spatial location in response to specific guidance cues. Many studies have now demonstrated that stimulus-induced local translation is critical to appropriate axon growth, guidance and regeneration. Multiple types of proteins are locally translated within axons, including those regulating the cytoskeleton, retrograde signaling, regeneration and cellular survival.

RACK1 regulates neural development

Receptor for activated C kinase 1 (RACK1) is an evolutionarily conserved scaffolding protein within the tryptophan-aspartate (WD) repeat family of proteins. RACK1 can bind multiple signaling molecules concurrently, as well as stabilize and anchor proteins. RACK1 also plays an important role at focal adhesions, where it acts to regulate cell migration. In addition, RACK1 is a ribosomal binding protein and thus, regulates translation. Despite these numerous functions, little is known about how RACK1 regulates nervous system development. Here, we review three studies that examine the role of RACK1 in neural development. In brief, these papers demonstrate that (1) RACK-1, the C. elegans homolog of mammalian RACK1, is required for axon guidance; (2) RACK1 is required for neurite extension of neuronally differentiated rat PC12 cells; and (3) RACK1 is required for axon outgrowth of primary mouse cortical neurons. Thus, it is evident that RACK1 is critical for appropriate neural development in a wide range of species, and future discoveries could reveal whether RACK1 and its signaling partners are potential targets for treatment of neurodevelopmental disorders or a therapeutic approach for axonal regeneration.