Deregulation of synaptic plasticity may contribute to the pathogenesis of developmental cognitive disorders. In particular, exaggerated mGluR-dependent LTD is featured in fragile X syndrome, but the mechanisms that regulate mGluR-LTD remain incompletely understood. We report that conditional knockout of Cdh1, the key regulatory subunit of the ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC), profoundly impairs mGluR-LTD in the hippocampus. Mechanistically, we find that Cdh1-APC operates in the cytoplasm to drive mGluR-LTD. We also identify the fragile X syndrome protein FMRP as a substrate of Cdh1-APC. Endogenous Cdh1-APC forms a complex with endogenous FMRP, and knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. Knockout of FMRP suppresses, and expression of an FMRP mutant protein that fails to interact with Cdh1 phenocopies, the Cdh1 knockout phenotype of impaired mGluR-LTD. These findings define Cdh1-APC and FMRP as components of a novel ubiquitin signaling pathway that regulates mGluR-LTD in the brain.

Intellectual disability (ID) is a prevalent developmental disorder of cognition that remains incurable. Here, we report that knockdown of the X-linked ID (XLID) protein polyglutamine-binding protein 1 (PQBP1) in neurons profoundly impairs the morphogenesis of the primary cilium, including in the mouse cerebral cortex in vivo. PQBP1 is localized at the base of the neuronal cilium, and targeting its WW effector domain to the cilium stimulates ciliary morphogenesis. We also find that PQBP1 interacts with Dynamin 2 and thereby inhibits its GTPase activity. Accordingly, Dynamin 2 knockdown in neurons stimulates ciliogenesis and suppresses the PQBP1 knockdown-induced ciliary phenotype. Strikingly, a mutation of the PQBP1 WW domain that causes XLID disrupts its ability to interact with and inhibit Dynamin 2 and to induce neuronal ciliogenesis. These findings define PQBP1 and Dynamin 2 as components of a signaling pathway that orchestrates neuronal ciliary morphogenesis in the brain.

Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.

The Hedgehog (Hh) signaling pathway is a major regulator of cell differentiation and proliferation. Aberrant activation of the Hh pathway has been implicated in several types of cancer. To understand the Hedgehog pathway and fight against related diseases, it is important to inhibit Hedgehog signaling in a targeted manner. However, no tools are available for the precise inhibition of Hh signaling in a spatiotemporal manner. In this study, we synthesized and evaluated the bioactivity of a light-inducible Hh pathway inhibitor (NVOC-SANT-75). NVOC-SANT-75 inhibits transcription factor Gli1 in NIH3T3 cells and controls proliferation and differentiation of primary cultured mouse cerebellar neurons in a light-irradiation-dependent manner. The light-inducible Hedgehog signaling inhibitors may be a new candidate for light-mediated cancer treatment.

In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

Axon branching plays a critical role in establishing the accurate patterning of neuronal circuits in the brain. However, the mechanisms that control axon branching remain poorly understood. Here we report that knockdown of the brain-enriched signaling protein JNK-interacting protein 3 (JIP3) triggers exuberant axon branching and self-contact in primary granule neurons of the rat cerebellar cortex. JIP3 knockdown in cerebellar slices and in postnatal rat pups in vivo leads to the formation of ectopic branches in granule neuron parallel fiber axons in the cerebellar cortex. We also find that JIP3 restriction of axon branching is mediated by the protein kinase glycogen synthase kinase 3β (GSK3β). JIP3 knockdown induces the downregulation of GSK3β in neurons, and GSK3β knockdown phenocopies the effect of JIP3 knockdown on axon branching and self-contact. Finally, we establish doublecortin (DCX) as a novel substrate of GSK3β in the control of axon branching and self-contact. GSK3β phosphorylates DCX at the distinct site of Ser327 and thereby contributes to DCX function in the restriction of axon branching. Together, our data define a JIP3-regulated GSK3β/DCX signaling pathway that restricts axon branching in the mammalian brain. These findings may have important implications for our understanding of neuronal circuitry during development, as well as the pathogenesis of neurodevelopmental disorders of cognition.

The mTOR signaling pathway regulates protein synthesis and diverse aspects of neuronal morphology that are important for brain development and function. To identify proteins controlled translationally by mTOR signaling, we performed ribosome profiling analyses in mouse cortical neurons and embryonic stem cells upon acute mTOR inhibition. Among proteins whose translation was significantly affected by mTOR inhibition selectively in neurons, we identified the cytoskeletal regulator protein palladin, which is localized within the cell body and axons in hippocampal neurons. Knockdown of palladin eliminated supernumerary axons induced by suppression of the tuberous sclerosis complex protein TSC1 in neurons, demonstrating that palladin regulates neuronal morphogenesis downstream of mTOR signaling. Our findings provide novel insights into an mTOR-dependent mechanism that controls neuronal morphogenesis through translational regulation.SIGNIFICANCE STATEMENT This study reports the discovery of neuron-specific protein translational responses to alterations of mTOR activity. By using ribosome profiling analysis, which can reveal the location and quantity of translating ribosomes on mRNAs, multiple aspects of protein translation were quantitatively analyzed in mouse embryonic stem cells and cortical neurons upon acute mTOR inhibition. Neurons displayed distinct patterns of ribosome occupancy for each codon and ribosome stalling during translation at specific positions of mRNAs. Importantly, the cytoskeletal regulator palladin was identified as a translational target protein of mTOR signaling in neurons. Palladin operates downstream of mTOR to modulate axon morphogenesis. This study identifies a novel mechanism of neuronal morphogenesis regulated by mTOR signaling through control of translation of the key protein palladin.

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

A unique signature of neurons is the high expression of the longest genes in the genome. These genes have essential neuronal functions, and disruption of their expression has been implicated in neurological disorders. DNA topoisomerases resolve DNA topological constraints and facilitate neuronal long gene expression. Conversely, the Rett syndrome protein, methyl-CpG-binding protein 2 (MeCP2), can transcriptionally repress long genes. How these factors regulate long genes is not well understood, and whether they interact is not known. Here, we identify and map a functional interaction between MeCP2 and topoisomerase IIβ (TOP2β) in mouse neurons. We profile neuronal TOP2β activity genome wide, detecting enrichment at regulatory regions and gene bodies of long genes, including MeCP2-regulated genes. We show that loss and overexpression of MeCP2 alter TOP2β activity at MeCP2-regulated genes. These findings uncover a mechanism of TOP2β inhibition by MeCP2 in neurons and implicate TOP2β dysregulation in disorders caused by MeCP2 disruption.

Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

Lysidine (2-lysyl cytidine) is a lysine-containing cytidine derivative commonly found at the wobble position of bacterial AUA codon-specific tRNA(Ile). This modification determines both codon and amino acid specificities of tRNA(Ile). We previously identified tRNA(Ile)-lysidine synthetase (tilS) that synthesizes lysidine, for which it utilizes ATP and lysine as substrates. Here, we show that lysidine synthesis consists of two consecutive reactions that involve an adenylated tRNA intermediate. A mutation study revealed that Escherichia coli TilS discriminates tRNA(Ile) from the structurally similar tRNA(Met) having the same anticodon loop by recognizing the anticodon loop, the anticodon stem, and the acceptor stem. TilS was shown to bind to the anticodon region and 3' side of the acceptor stem, which cover the recognition sites. These findings reveal a dedicated mechanism embedded in tRNA(Ile) that controls its recognition and discrimination by TilS, and indicate the significance of this enzyme in the proper deciphering of genetic information.

Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4.

We developed a compartmentalized culture system of single embryoid bodies (EBs) utilizing a through-hole on a membrane to induce spatially patterned differentiation. An EB derived from mouse pluripotent stem cells was immobilized on the through-hole. By introducing a stem cell maintenance medium and a differentiation medium into upper and lower culture compartments, respectively, a localized differentiated state was achieved only in the lower part of EB, which is exposed to the medium in the lower compartment. This system may enable us to reconstruct complex tissues and to recapitulate developmental processes using EBs.

Modified uridine containing taurine, 5-taurinomethyluridine (τm5U), is found at the anticodon first position of mitochondrial (mt-)transfer RNAs (tRNAs). Previously, we reported that τm5U is absent in mt-tRNAs with pathogenic mutations associated with mitochondrial diseases. However, biogenesis and physiological role of τm5U remained elusive. Here, we elucidated τm5U biogenesis by confirming that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for τm5U formation catalyzed by MTO1 and GTPBP3. GTPBP3-knockout cells exhibited respiratory defects and reduced mitochondrial translation. Very little τm5U34 was detected in patient's cells with the GTPBP3 mutation, demonstrating that lack of τm5U results in pathological consequences. Taurine starvation resulted in downregulation of τm5U frequency in cultured cells and animal tissues (cat liver and flatfish). Strikingly, 5-carboxymethylaminomethyluridine (cmnm5U), in which the taurine moiety of τm5U is replaced with glycine, was detected in mt-tRNAs from taurine-depleted cells. These results indicate that tRNA modifications are dynamically regulated via sensing of intracellular metabolites under physiological condition.

During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and β isoforms in the brain. Although CaMKIIα functions have been elucidated, the isoform-specific catalytic functions of CaMKIIβ have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKIIβ to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20-anaphase promoting complex) as a centrosomal substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKIIβ that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. We report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. We also find that the centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity.

Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

Melanocytes are surrounded by diverse cells, including sensory neurons in our skin, but their interaction and functional importance have been poorly investigated. In this study, we find that melanocytes and nociceptive neurons contact more in human skin color patch tissue than control. Co-culture with human iPSC-derived sensory neurons significantly induces morphogenesis and pigmentation of human melanocytes. To reveal melanocyte-stimulating factors secreted from neurons, we perform proteomic analyses and identify RGMB in the sensory neuron-conditioned medium. RGMB protein induces morphogenesis and melanin production of melanocytes, demonstrating that RGMB is a melanocyte-stimulating factor released from sensory neurons. Transcriptome analysis suggests that the melanosome transport machinery can be controlled by RGMB, leading us to identify the vesicle production response of melanocytes upon RGMB treatment. This study discovers a role of sensory neurons in modulating multiple aspects of human melanocytes through secretion of a key factor: RGMB.