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Two papers in Cell uncover reciprocal crosstalk of local translation and mitochondria in neurons. Rangaraju et al. (2019) observe tethered compartments of stable mitochondria in dendrites that provide a local energy supply for mRNA translation at synapses. Cioni et al. (2019) report a novel association of axonal RNA granules with Rab7a-late endosomes that provides a platform for local translation supporting mitochondria., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 the authors 0270-6474/17/371685-11$15.00/0.)

There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. A few studies have revealed the presence of microRNAs (miRNAs) in axons, which may control local protein synthesis during axon development. However, so far, there has been no attempt to screen for axon-enriched miRNAs and to validate their possible localization to growth cones of developing axons from neurons of the central nervous system. In this study, the localization of miRNAs in axons and growth cones in cortical neurons was examined using a "neuron ball" culture method that is suitable to prepare axonal miRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball cultures of mouse cortical neurons were analyzed by quantitative real-time RT-PCR. Among 375 miRNAs that were analyzed, 105 miRNAs were detected in axons, and six miRNAs were significantly enriched in axonal fractions when compared with cell body fractions. Fluorescence in situ hybridization revealed that two axon-enriched miRNAs, miR-181a-1* and miR-532, localized as distinct granules in distal axons and growth cones. The association of these miRNAs with the RNA-induced silencing complex further supported their function to regulate mRNA levels or translation in the brain. These results suggest a mechanism to localize specific miRNAs to distal axons and growth cones, where they could be involved in local mRNA regulation. These findings provide new insight into the presence of axonal miRNAs and motivate further analysis of their function in local protein synthesis underlying axon guidance., (Copyright © 2013 Wiley Periodicals, Inc.)

Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1., (Copyright © 2013 Wiley Periodicals, Inc.)

Directed transport of the mRNA binding protein, zipcode binding protein1 (ZBP1), into developing axons is believed to play an important role in mRNA localization and local protein synthesis. The role of molecular motors in this process is unclear. We elucidated a role for myosin Va (MyoVa) to modulate the axonal localization and transport of ZBP1 in axons. Using cultured rat hippocampal neurons, ZBP1 colocalized with MyoVa in axons and growth cones. Interaction of MyoVa with ZBP1 was evident by coimmunoprecipitation of endogenous and overexpressed proteins. Inhibition of MyoVa function with the globular tail domain (GTD) of MyoVa protein or short hairpin RNA led to an accumulation of ZBP1 in axons. Live cell imaging of mCherryZBP1 in neurons expressing GTD showed an increase in the number of motile particles, run length, and stimulated anterograde moving ZBP1 particles, suggesting that MyoVa controls availability of ZBP1 for microtubule-dependent transport. These findings suggest a novel regulatory role for MyoVa in the transport of ZBP1 within axons.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease specifically affecting cortical and spinal motor neurons. Cytoplasmic inclusions containing hyperphosphorylated and ubiquitinated TDP-43 are a pathological hallmark of ALS, and mutations in the gene encoding TDP-43 have been directly linked to the development of the disease. TDP-43 is a ubiquitous DNA/RNA-binding protein with a nuclear role in pre-mRNA splicing. However, the selective vulnerability and axonal degeneration of motor neurons in ALS pose the question of whether TDP-43 may have an additional role in the regulation of the cytoplasmic and axonal fate of mRNAs, processes important for neuron function. To investigate this possibility, we have characterized TDP-43 localization and dynamics in primary cultured motor neurons. Using a combination of cell imaging and biochemical techniques, we demonstrate that TDP-43 is localized and actively transported in live motor neuron axons, and that it co-localizes with well-studied axonal mRNA-binding proteins. Expression of the TDP-43 C-terminal fragment led to the formation of hyperphosphorylated and ubiquitinated inclusions in motor neuron cell bodies and neurites, and these inclusions specifically sequestered the mRNA-binding protein HuD. Additionally, we showed that overexpression of full-length or mutant TDP-43 in motor neurons caused a severe impairment in axon outgrowth, which was dependent on the C-terminal protein-interacting domain of TDP-43. Taken together, our results suggest a role of TDP-43 in the regulation of axonal growth, and suggest that impairment in the post-transcriptional regulation of mRNAs in the cytoplasm of motor neurons may be a major factor in the development of ALS.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by homozygous mutations or deletions in the survival of motor neuron (SMN1) gene, encoding the ubiquitously expressed SMN protein. SMN associates with different proteins (Gemins 2-8, Unrip) to form a multimeric complex involved in the assembly of small nuclear ribonucleoprotein complexes (snRNPs). Since this activity is essential for the survival of all cell types, it still remains unclear why motor neurons are selectively vulnerable to low levels of SMN protein. Aside from its housekeeping role in the assembly of snRNPs, additional functions of SMN have been proposed. The well-documented localization of SMN in axonal transport granules and its interaction with numerous mRNA-binding proteins not involved in splicing regulation suggest a role in axonal RNA metabolism. This review will focus on the neuropathological and experimental evidence supporting a role for SMN in regulating the assembly, localization, or stability of axonal messenger ribonucleoprotein complexes (mRNPs). Furthermore, how defects in this non-canonical SMN function may contribute to the motor neuron pathology observed in SMA will be discussed. This article is part of a Special Issue entitled RNA-Binding Proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that β-actin's 3'-UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin's 3'-UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the β-actin 3'-UTR containing transgene mRNA into axons. This GFP-3'-β-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the β-actin 3'-UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo.

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.

Spinal muscular atrophy (SMA), an inherited disease of motor neuron dysfunction, results from insufficient levels of the survival motor neuron (SMN) protein. Movement of the SMN protein as granules within cultured axons suggests that the pathogenesis of SMA may involve defects in neuronal transport, yet the nature of axon transport vesicles remains enigmatic. Here we show that SMN directly binds to the α-subunit of the coat protein I (COPI) vesicle coat protein. The α-COP protein co-immunoprecipitates with SMN, small nuclear ribonucleoprotein-associated assembly factors and β-actin mRNA. Although typically Golgi associated, in neuronal cells α-COP localizes to lamellipodia and growth cones and moves within the axon, with a subset of these granules traveling together with SMN. Depletion of α-COP resulted in mislocalization of SMN and actin at the leading edge at the lamellipodia. We propose that neurons utilize the Golgi-associated COPI vesicle to deliver cargoes necessary for motor neuron integrity and function.

Spinal muscular atrophy (SMA) results from reduced levels of the survival of motor neuron (SMN) protein, which has a well characterized function in spliceosomal small nuclear ribonucleoprotein assembly. Currently, it is not understood how deficiency of a housekeeping protein leads to the selective degeneration of spinal cord motor neurons. Numerous studies have shown that SMN is present in neuronal processes and has many interaction partners, including mRNA-binding proteins, suggesting a potential noncanonical role in axonal mRNA metabolism. In this study, we have established a novel technological approach using bimolecular fluorescence complementation (BiFC) and quantitative image analysis to characterize SMN-protein interactions in primary motor neurons. Consistent with biochemical studies on the SMN complex, BiFC analysis revealed that SMN dimerizes and interacts with Gemin2 in nuclear gems and axonal granules. In addition, using pull down assays, immunofluorescence, cell transfection, and BiFC, we characterized a novel interaction between SMN and the neuronal mRNA-binding protein HuD, which was dependent on the Tudor domain of SMN. A missense mutation in the SMN Tudor domain, which is known to cause SMA, impaired the interaction with HuD, but did not affect SMN axonal localization or self-association. Furthermore, time-lapse microscopy revealed SMN cotransport with HuD in live motor neurons. Importantly, SMN knockdown in primary motor neurons resulted in a specific reduction of both HuD protein and poly(A) mRNA levels in the axonal compartment. These findings reveal a noncanonical role for SMN whereby its interaction with mRNA-binding proteins may facilitate the localization of associated poly(A) mRNAs into axons.

Spinal muscular atrophy (SMA) is a neurodegenerative disease that results from loss of function of the SMN1 gene, encoding the ubiquitously expressed survival of motor neuron (SMN) protein, a protein best known for its housekeeping role in the SMN-Gemin multiprotein complex involved in spliceosomal small nuclear ribonucleoprotein (snRNP) assembly. However, numerous studies reveal that SMN has many interaction partners, including mRNA binding proteins and actin regulators, suggesting its diverse role as a molecular chaperone involved in mRNA metabolism. This review focuses on studies suggesting an important role of SMN in regulating the assembly, localization, or stability of axonal messenger ribonucleoprotein (mRNP) complexes. Various animal models for SMA are discussed, and phenotypes described that indicate a predominant function for SMN in neuronal development and synapse formation. These models have begun to be used to test different therapeutic strategies that have the potential to restore SMN function. Further work to elucidate SMN mechanisms within motor neurons and other cell types involved in neuromuscular circuitry hold promise for the potential treatment of SMA.

Homozygous deletion of the survival motor neuron 1 gene (SMN1) causes spinal muscular atrophy (SMA), the most frequent genetic cause of early childhood lethality. In rare instances, however, individuals are asymptomatic despite carrying the same SMN1 mutations as their affected siblings, thereby suggesting the influence of modifier genes. We discovered that unaffected SMN1-deleted females exhibit significantly higher expression of plastin 3 (PLS3) than their SMA-affected counterparts. We demonstrated that PLS3 is important for axonogenesis through increasing the F-actin level. Overexpression of PLS3 rescued the axon length and outgrowth defects associated with SMN down-regulation in motor neurons of SMA mouse embryos and in zebrafish. Our study suggests that defects in axonogenesis are the major cause of SMA, thereby opening new therapeutic options for SMA and similar neuromuscular diseases.

Spinal muscular atrophy (SMA) is a motor neuron degenerative disease caused by low levels of the survival motor neuron (SMN) protein and is linked to mutations or loss of SMN1 and retention of SMN2. How low levels of SMN cause SMA is unclear. SMN functions in small nuclear ribonucleoprotein (snRNP) biogenesis, but recent studies indicate that SMN may also function in axons. We showed previously that decreasing Smn levels in zebrafish using morpholinos (MO) results in motor axon defects. To determine how Smn functions in motor axon outgrowth, we coinjected smn MO with various human SMN RNAs and assayed the effect on motor axons. Wild-type SMN rescues motor axon defects caused by Smn reduction in zebrafish. Consistent with these defects playing a role in SMA, SMN lacking exon 7, the predominant form from the SMN2 gene, and human SMA mutations do not rescue defective motor axons. Moreover, the severity of the motor axon defects correlates with decreased longevity. We also show that a conserved region in SMN exon 7, QNQKE, is critical for motor axon outgrowth. To address the function of SMN important for motor axon outgrowth, we determined the ability of different SMN forms to oligomerization and bind Sm protein, functions required for snRNP biogenesis. We identified mutations that failed to rescue motor axon defects but retained snRNP function. Thus, we have dissociated the snRNP function of SMN from its function in motor axons. These data indicate that SMN has a novel function in motor axons that is relevant to SMA and is independent of snRNP biosynthesis.

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF-/-) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF-/-grafts was markedly less than WTgrafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that traininginduced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP+ neurons (SLICK) were cut and repaired with BDNF-/- and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF-/- grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves. [ABSTRACT FROM AUTHOR]

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 b-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 bactin 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. [ABSTRACT FROM AUTHOR]

Neurons are exquisitely polarized cells that extend intricate axonal and dendritic arbors. Developmental cues guide axons and dendrites into circuits by inducing rapid changes in local protein expression and cytoskeletal structure. Neurons can transduce these signals through local mRNA regulation. Here, we review the latest insights regarding post-transcriptional control of gene expression through mRNA transport and local protein synthesis in developing neurons. We focus on local mRNA regulation during axon growth and guidance, dendrite morphogenesis, and synapse formation and refinement. Dysregulated mRNA transport and translation in neurological disorders are also discussed. The collection of molecules and mechanisms reviewed includes sequence-specific RNA binding proteins, microtubule motors and adaptors, microRNAs, translation initiation factors, and the receptor-mediated signaling that modulates these molecules. [ABSTRACT FROM AUTHOR]

Local β-actin synthesis in growth cones of developing axons plays an important role in growth cone steering; however, them RNA 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. [ABSTRACT FROM AUTHOR]

The article highlights the European Molecular Biology Organization Workshop on RNA Control of Neuronal Function in Kfar Blum, Israel in May 22 and 26, 2005. The article gives importance on the recent reports on mechanisms, regulation and function of mRNA transport and local translation in both dendrites and axons.

Abstract: Genetic deficiency of the mRNA binding protein FMRP results in the most common inherited form of mental retardation, Fragile X syndrome. We investigated the localization and function of FMRP during development of hippocampal neurons in culture. FMRP was distributed within granules that extended into developing axons and growth cones, detectable at distances over 300 μm from the cell body. In mature cultures, FMRP granules were present in both axons and dendrites, with pockets of higher concentrations appearing intermittently, along distal axon segments and near synapses. MAP1b mRNA, a known FMRP target, was also localized to axon growth cones. Morphometric analysis of growth cones from the FMR1 KO revealed both excess filopodia and reduced motility. At later stages during synapse formation, FMR1 KO neurons exhibited excessive filopodia and long spines along dendrites, yet there was a marked decrease in the density of spine-like protrusions juxtaposed to presynaptic terminals. In contrast, there was no difference in the density of shaft synapses between FMR1 KO and WT. Brief depolarization of WT neurons resulted in increased numbers of filopodia and spine synapses, whereas no additional morphologic changes were observable in dendrites of FMR1 KO neurons that already had increased density of filopodia–spines. These findings suggest that alterations in the regulation of axonal growth and innervation in FMR1 KO neurons may contribute to the dendritic and spine pathology in Fragile X syndrome. This work has broader implications for understanding the role of mRNA binding proteins in developmental and protein-synthesis-dependent plasticity. [Copyright &y& Elsevier]