Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations or deletions in the survival motoneuron 1 (SMN1) gene, resulting in deficiency of the SMN protein that is essential for motoneuron function. Smn depletion in mice disturbs axonal RNA transport and translation, thereby contributing to axon growth impairment, muscle denervation, and motoneuron degeneration. However, the mechanisms whereby Smn loss causes axonal defects remain unclear. RNA localization and translation in axons are controlled by RNA-binding proteins (RBP) and we recently observed that the neuronal RBP Ptbp2 modulates axon growth in motoneurons. Here, we identify Smn as an interactor of Ptbp2 in the cytosolic compartments of motoneurons. We show that the expression level of Ptbp2 is reduced in axons but not in the somata of Smn-depleted motoneurons. This is accompanied by reduced synthesis of the RBP hnRNP R in axons. Re-expression of Ptbp2 in axons compensates for the deficiency of Smn and rescues the defects in axon elongation and growth cone maturation observed in Smn-deficient motoneurons. Our data suggest that Ptbp2 and Smn are components of cytosolic mRNP particles, contributing to the precise spatial and temporal control of protein synthesis within axons and axon terminals.

BACKGROUND: Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity.
METHODS: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles.
RESULTS: Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles.
CONCLUSIONS: We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3\' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

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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.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here, we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.

Spatial segregation of proteins to neuronal axons arises in part from local translation of mRNAs that are first transported into axons in ribonucleoprotein particles (RNPs), complexes containing mRNAs and RNA binding proteins. Understanding the importance of local translation for a particular circuit requires not only identifying axonal RNPs and their mRNA cargoes, but also whether these RNPs are broadly conserved or restricted to only a few species. Fragile X granules (FXGs) are axonal RNPs containing the fragile X related family of RNA binding proteins along with ribosomes and specific mRNAs. FXGs were previously identified in mouse, rat, and human brains in a conserved subset of neuronal circuits but with species-dependent developmental profiles. Here, we asked whether FXGs are a broadly conserved feature of the mammalian brain and sought to better understand the species-dependent developmental expression pattern. We found FXGs in a conserved subset of neurons and circuits in the brains of every examined species that together include mammalian taxa separated by up to 160 million years of divergent evolution. A developmental analysis of rodents revealed that FXG expression in frontal cortex and olfactory bulb followed consistent patterns in all species examined. In contrast, FXGs in hippocampal mossy fibers increased in abundance across development for most species but decreased across development in guinea pigs and members of the Mus genus, animals that navigate particularly small home ranges in the wild. The widespread conservation of FXGs suggests that axonal translation is an ancient, conserved mechanism for regulating the proteome of mammalian axons.

Through the generation of humanized FUS mice expressing full-length human FUS, we identify that when expressed at near endogenous murine FUS levels, both wild-type and ALS-causing and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and to inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain of toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs. VIDEO ABSTRACT.

Local axonal protein synthesis plays a crucial role in the formation and function of neuronal circuits. Understanding the role of this mechanism in specific circuits requires identifying the protein composition and mRNA cargos of the ribonucleoprotein particles (RNPs) that form the substrate for axonal translation. FXGs (Fragile X granules) are axonal RNPs present in a stereotyped subset of mature axons in the intact brain that contain one or more of the Fragile X related (FXR) proteins (FMRP, FXR2P, and FXR1P) along with mRNA and ribosomes. Here we performed a systematic survey of the FXR protein composition and mRNA association of FXGs in the brain. We have identified four FXG types that can be categorized based on their FXR protein complement. All FXGs contain FXR2P, with FMRP and/or FXR1P present in circuit-selective subsets. Individual neuronal cell types predominantly express a single FXG type, with FMRP-containing FXGs the most prevalent in forebrain neurons. All FXG types associate with ribosomes and mRNA, but the specific mRNA cargos are a function of FXG type, brain region and neuron class. Transcripts for β-catenin and its regulator APC associate with a subset of forebrain FXGs. Moreover, both these transcripts can colocalize within individual FXGs, suggesting that the axonal translation of functionally related proteins may be coordinately regulated with high spatiotemporal resolution. Cell type-dependent expression of specific RNP types with distinct mRNA cargos, such as FXGs, presents a potential mechanism for regulating local translation and its output in a circuit-dependent manner.