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Summary We identified ∼30-mer amyloid-β protein (Aβ) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aβ levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology., Graphical Abstract, Highlights • Proteasome inhibition increases ASPD in excitatory neurons by increasing APP and Aβ • Proteasome inhibition shifts the Aβ and ASPD distribution from axons to dendrites • ASPDs are secreted and kill surrounding ATPase α3 neurons by cell surface binding • Following apoptosis in α3 neurons, ASPD-accumulating neurons die non-apoptotically, Pathophysiology; Neuroscience; Cellular Neuroscience

SummaryMechanical stimuli on the skin of C. elegans are detected by the dendritic arbors of PVD nociceptive neurons, which provide uniform sensory coverage outside the head region across the entire animal. Through genetic screens, we isolate three mutants that display profound dendrite self-avoidance defects in PVD neurons. Studying dendrite self-avoidance in C. elegans is likely to provide new mechanistic insight into the process as the well-known self-avoidance molecule Dscam is absent from the C. elegans genome. Through whole genome sequencing, we identify the responsible mutations in the kpc-1 gene. Compared to wild-type animals, a strong kpc-1 mutant allele exhibits secondary dendrite branching defects whereas a weak kpc-1 mutant allele displays tertiary dendrite self-avoidance defects. Here, we show that the kpc-1 3’UTR is required for kpc-1’s functions in both dendrite branching and dendrite self-avoidance. The kpc-1 3’UTR facilitates kpc-1 RNA localization to branching points and contact points between sibling dendrites. Using fluorescence recovery after photoconversion, we show that the kpc-1 3’UTR promotes local protein synthesis in the distal segment of PVD dendrites. We identify an important secondary structural motif in the kpc-1 3’UTR required for tertiary dendrite self-avoidance. We demonstrate that over-expression of kpc-1 leads to greater self-avoidance without limiting initial dendrite outgrowth, supporting a direct role of kpc-1 in self-avoidance. Animals with dma-1 receptor over-expression display similar secondary dendrite branching and tertiary dendrite self-avoidance defects that are suppressed with kpc-1 over-expression, which suggests that DMA-1 is a potential KPC-1 target that is down-regulated by KPC-1. Our results support a model where KPC-1 proteins are synthesized at branching points and contact points between neighboring dendrites to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance. A recently reported Schizophrenia-associated genetic variant in the 3’UTR of the human furin gene, a homolog of kpc-1, highlights the important role of the kpc-1 (furin) 3’UTR in neuronal development, which is further demonstrated by this mechanistic study.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore. Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

In neurons, control of microtubule dynamics is required for multiple homeostatic and regulated activities. Over the past few decades, a great deal has been learned about the role of the microtubule cytoskeleton in axonal and dendritic transport, with a broad impact on neuronal health and disease. However, significantly less attention has been paid to the importance of microtubule dynamics in directly regulating synaptic function. Here, we review emerging literature demonstrating that microtubules enter synapses and control central aspects of synaptic activity, including neurotransmitter release and synaptic plasticity. The pleiotropic effects caused by a dysfunctional synaptic microtubule cytoskeleton may thus represent a key point of vulnerability for neurons and a primary driver of neurological disease.

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. The detailed biophysics active ion channel transport are increasingly well understood, along with the steady-state distribution of functional channels in the membrane. However we lack a quantitative understanding of how transport mechanisms give rise to stable expression patterns, and how live measurements of active transport relate to static estimates of channel density in neurites. We experimentally measured neuronal transport and expression densities of Kv4.2, a voltage-gated transient potassium channel. Kv4.2 is known to have a highly specific dendritic expression and little or no reported functional expression in axons. Surprisingly, in over 500 hours of quantitative live imaging, we found substantially higher microtubule-based transport of Kv4.2 subunits in axons compared to dendrites. We show that this paradoxical result is expected using a mass action trafficking model of intracellular transport that we calibrate to experimental measurements. Furthermore, we find qualitative differences in axonal and dendritic active transport that are captured in a stochastic model of puncta transport. This reveals that active transport is tuned to efficiently move cargo through axons while promoting mixing in dendrites. Finally, our data reveals trends in transport parameters that can explain the functional density profile of Kv4.2. Puncta velocity bias is directed distally and the magnitude of this bias increases with distance from the soma. These trends are consistent with an analytical solution of a linear transport PDE, corroborating previously unexplained distributions of Kv4.2 subunit localization and A-type current density. Together, our results provide new quantitative data on ion channel trafficking and reveal counterintuitive but mathematically consistent relationships between the distribution of cargo that is in transit and its functional expression.SIGNIFICANCEThis study of ion channel transport reveals a seemingly counterintuitive result: the majority of subunit transport occurs in axons for a cargo whose static distribution is concentrated in dendrites. This disparity is reconciled by a simple mathematical model of transport, which reveals that the local density of actively transported intracellular cargo can show an inverse relationship with its static expression density. Mass action models also reconcile the previously unexplained, highly asymmetric, increasing distribution of Kv4.2 with its measured trafficking density that resembles diffusion with minimal drift. The generality of our analysis prompts caution in how static snapshots of intracellular cargo distributions should be interpreted for any type of intracellular cargo.

Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome is caused by mutations in ATRX, which encodes a chromatin-remodeling protein. Genome-wide analyses in mouse and human cells indicate that ATRX tends to bind to G-rich sequences with a high potential to form G-quadruplexes. Here, we report that Atrx mutation induces aberrant upregulation of Xlr3b expression in the mouse brain, an outcome associated with neuronal pathogenesis displayed by ATR-X model mice. We show that ATRX normally binds to G-quadruplexes in CpG islands of the imprinted Xlr3b gene, regulating its expression by recruiting DNA methyltransferases. Xlr3b binds to dendritic mRNAs, and its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α, promoting synaptic dysfunction. Notably, treatment with 5-ALA, which is converted into G-quadruplex-binding metabolites, reduces RNA polymerase II recruitment and represses Xlr3b transcription in ATR-X model mice. 5-ALA treatment also rescues decreased synaptic plasticity and cognitive deficits seen in ATR-X model mice. Our findings suggest a potential therapeutic strategy to target G-quadruplexes and decrease cognitive impairment associated with ATR-X syndrome., 難治性疾患「ATR-X症候群」の治療に新たな光 --重度知的障がいに対する新しい治療薬候補の発見--. 京都大学プレスリリース. 2018-05-22.

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.

Microtubule motor proteins – kinesins and dyneins – play an important role in intracellular transport. Impairments to axon transport can influence neurotransmitter release and short-term presynaptic plasticity. Impairments to dendritic transport, particularly recycling of synaptic receptors, affect postsynaptic plasticity. This review seeks to follow the link between microtubule motor proteins and the mechanisms of synaptic plasticity from the point of view of their involvement in transporting proteins and organelles, where their role in the mechanisms of synaptic plasticity has been demonstrated.

The kinesin I family of motor proteins are crucial for axonal transport, but their roles in dendritic transport and postsynaptic function are not well-defined. Gene duplication and subsequent diversification give rise to three homologous kinesin I proteins (KIF5A, KIF5B and KIF5C) in vertebrates, but it is not clear whether and how they exhibit functional specificity. Here we show that knockdown of KIF5A or KIF5B differentially affects excitatory synapses and dendritic transport in hippocampal neurons. The functional specificities of the two kinesins are determined by their diverse carboxyl-termini, where arginine methylation occurs in KIF5B and regulates its function. KIF5B conditional knockout mice exhibit deficits in dendritic spine morphogenesis, synaptic plasticity and memory formation. Our findings provide insights into how expansion of the kinesin I family during evolution leads to diversification and specialization of motor proteins in regulating postsynaptic function., eLife digest Transporting molecules within a cell becomes a daunting task when the cell is a neuron, with fibers called axons and dendrites that can stretch as long as a meter. Neurons use many different molecules to send messages across the body and store memories in the brain. If the right molecules cannot be delivered along the length of nerve cells, connections to neighboring neurons may decay, which may impair learning and memory. Motor proteins are responsible for transporting molecules within cells. Kinesins are a type of motor protein that typically transports materials from the body of a neuron to the cell’s periphery, including the dendrites, which is where a neuron receives messages from other nerve cells. Each cell has up to 45 different kinesin motors, but it is not known whether each one performs a distinct task or if they have overlapping roles. Now, Zhao, Fok et al. have studied two similar kinesins, called KIF5A and KIF5B, in rodent neurons to determine their roles. First, it was shown that both proteins were found at dendritic spines, which are small outgrowths on dendrites where contact with other cells occurs. Next, KIF5A and KIF5B were depleted, one at a time, from neurons extracted from a brain region called the hippocampus. Removing KIF5B interfered with the formation of dendritic spines, but removing KIF5A did not have an effect. Dendritic spines are essential for learning and memory, so several behavioral tests were conducted on mice that had been genetically modified to express less KIF5B in the forebrain. These tests revealed that the mice performed poorly in tasks that tested their memory recall. This work opens a new area of research studying the specific roles of different kinesin motor proteins in nerve cells. This could have important implications because certain kinesin motor proteins such as KIF5A are known to be defective in some inherited neurodegenerative diseases.

Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3' untranslated region (UTR). Activity-regulated cytoskeleton-associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3' UTR, it was thought that arc mRNA is a natural target of nonsense-mediated mRNA decay (NMD). Here, we show that DTE in human arc 3' UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity-dependent degradation of the mRNA.

Localized translation plays a crucial role in synaptic plasticity and memory consolidation. However, it has not been possible to follow the dynamics of memory-associated mRNAs in living neurons in response to neuronal activity in real time. We have generated a novel mouse model where the endogenous Arc/Arg3.1 gene is tagged in its 3' untranslated region with stem-loops that bind a bacteriophage PP7 coat protein (PCP), allowing visualization of individual mRNAs in real time. The physiological response of the tagged gene to neuronal activity is identical to endogenous Arc and reports the true dynamics of Arc mRNA from transcription to degradation. The transcription dynamics of Arc in cultured hippocampal neurons revealed two novel results: (i) A robust transcriptional burst with prolonged ON state occurs after stimulation, and (ii) transcription cycles continue even after initial stimulation is removed. The correlation of stimulation with Arc transcription and mRNA transport in individual neurons revealed that stimulus-induced Ca2+ activity was necessary but not sufficient for triggering Arc transcription and that blocking neuronal activity did not affect the dendritic transport of newly synthesized Arc mRNAs. This mouse will provide an important reagent to investigate how individual neurons transduce activity into spatiotemporal regulation of gene expression at the synapse.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3' UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3' UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

The vast majority of the mammalian genome is transcribed, generating a wealth of transcripts that do not have protein-coding potential. These non-coding RNAs (ncRNAs) have emerged as major mediators of compartmentalized gene expression with many important regulatory functions, and are therefore at the focus of biological research in many cellular systems. The expression of ncRNAs is particularly multifaceted in neurons, as they seem to be expressed in a highly cell-type and activity-dependent manner. Specific subclasses of ncRNAs, especially microRNAs (miRNAs), were implicated in the local regulation of mRNA translation in neuronal dendrites, a process of compartmentalized gene expression that is engaged during synaptic plasticity. Recent discoveries point towards a widespread involvement of ncRNA families in local translation, including less abundant small RNAs (PIWI-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs)) and long ncRNAs (circular RNAs (circRNAs), long intergenic ncRNAs (lincRNAs)). The mechanisms underlying the dendritic transport and the regulatory function of ncRNAs in response to neuronal activity are being elucidated. The emerging picture is an intricate crosstalk between different ncRNA families, mRNAs and RNA-binding proteins (RBPs) that synergistically fine-tune the local dendritic proteome in an activity-dependent manner.

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity, allowing for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in C. elegans neurons and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for proper microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron however, use an additional pathway to nucleate microtubules from the tip of the dendrite, at the base of the sensory cilium in ciliated neurons. Since, inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule organizing center at the dendrite tip ensures proper dendritic cargo transport.

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra-axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time-dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO-exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs) is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer's disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer's disease, chronic stress and epilepsy.

Local protein synthesis occurs in axons and dendrites of neurons, enabling fast and spatially restricted responses to a dynamically changing extracellular environment. Prior to local translation, mRNA that is to be translated is packed into ribonucleoprotein particles (RNPs) where RNA binding proteins ensure mRNA silencing and provide a link to molecular motors. ZBP1 is a component of RNP transport particles and is known for its role in the local translation of β-actin mRNA. Its binding to mRNA is regulated by tyrosine 396 phosphorylation, and this particular modification was shown to be vital for axonal growth and dendritic branching. Recently, additional phosphorylation of ZBP1 at serine 181 (Ser181) was described in non-neuronal cells. In the present study, we found that ZBP1 is also phosphorylated at Ser181 in neurons in a mammalian/mechanistic target of rapamycin complex 2-, Src kinase-, and mRNA binding-dependent manner. Furthermore, Ser181 ZBP1 phosphorylation was essential for the proper dendritic branching of hippocampal neurons that were cultured in vitro and for the proper ZBP1 dendritic distribution and motility.

Ca2+-dependent RNA–protein interactions enable activity-inducible RNA transport in dendrites., A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell’s activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca2+ levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca2+ through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca2+-dependent RNA–protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs., This work was funded by grants from the Ministry of Economy and Competitiveness of Spain (BFU2014-52591-R CG) and the European Union (FEDER) to C.G.

Summary Translation of mRNAs in dendrites mediates synaptic plasticity, the probable cellular basis of learning and memory. Coordination of translational inhibitory and stimulatory mechanisms, as well as dendritic transport of mRNA, is necessary to ensure proper control of this local translation. Here, we find that the deadenylase CNOT7 dynamically regulates dendritic mRNA translation and transport, as well as synaptic plasticity and higher cognitive function. In cultured hippocampal neurons, synaptic stimulation induces a rapid decrease in CNOT7, which, in the short-term, results in poly(A) tail lengthening of target mRNAs. However, at later times following stimulation, decreased poly(A) and dendritic localization of mRNA take place, similar to what is observed when CNOT7 is depleted over several days. In mice, CNOT7 is essential for hippocampal-dependent learning and memory. This study identifies CNOT7 as an important regulator of RNA transport and translation in dendrites, as well as higher cognitive function.

BC1 RNA is a small brain specific non-protein coding RNA. It is transported from the cell body into dendrites where it is involved in the fine-tuning translational control. Due to its compactness and established secondary structure, BC1 RNA is an ideal model for investigating the motifs necessary for dendritic localization. Previously, microinjection of in vitro transcribed BC1 RNA mutants into the soma of cultured primary neurons suggested the importance of RNA motifs for dendritic targeting. These ex vivo experiments identified a single bulged nucleotide (U22) and a putative K-turn (GA motif) structure required for dendritic localization or distal transport, respectively. We generated six transgenic mouse lines (three founders each) containing neuronally expressing BC1 RNA variants on a BC1 RNA knockout mouse background. In contrast to ex vivo data, we did not find indications of reduction or abolition of dendritic BC1 RNA localization in the mutants devoid of the GA motif or the bulged nucleotide. We confirmed the ex vivo data, which showed that the triloop terminal sequence had no consequence on dendritic transport. Interestingly, changing the triloop supporting structure completely abolished dendritic localization of BC1 RNA. We propose a novel RNA motif important for dendritic transport in vivo.

Neuralized, an E3 ubiquitin ligase, interacts with and positively modulates the Notch pathway by promoting ubiquitination and subsequent endocytosis of its ligands. NEURL1 mRNA is dendritically localised in the dentate gyrus of an adult rat brain, implying that it may be locally translated, but its transport mechanisms remain unstudied. Here, we report the presence of a previously unknown, shorter splice-variant of rat NEURL1 3′UTR (1477 bp in length), and identify it as a potential target of nonsense-mediated decay. We show that endogenous NEURL1 mRNAs with both longer and shorter 3′UTRs are enriched in the neurites of cultured rat primary hippocampal neurons. Both NEURL1 3′UTRs can mediate transport of reporter mRNAs into dendrites in primary hippocampal neurons. By analysing the dendritic trafficking capacity of reporter mRNAs linked to various regions of longer or shorter NEURL1 3′UTR, we localise the dendritic targeting element (DTE) of spliced version of NEURL1 3′UTR to its first half, corresponding to the nucleotides 1-148 and 416-914 of the full-length 3′UTR. In contrast, the dendritic targeting capacity of the full-length NEURL1 3′UTR is abolished by splitting its 3′UTR in two halves (nt 1-914 and nt 915-1744), suggesting that slightly different DTE might mediate dendritic transport of the two transcripts.

Local, synaptic synthesis of new proteins in response to neuronal stimulation plays a key role in the regulation of synaptic morphogenesis. Recent studies indicate that matrix metalloproteinase-9 (MMP-9), an endopeptidase that regulates the pericellular environment through cleavage of its protein components, plays a critical role in regulation of spine morphology and synaptic plasticity. Here, we sought to determine whether MMP-9 mRNA is transported to dendrites for local translation and protein release. First, dendritic transport of MMP-9 mRNA was seen in primary hippocampal neuronal cultures treated with glutamate and in dentate gyrus granule cells in adult anesthetized rats after induction of long-term potentiation. Second, rapid, activity-dependent polyadenylation of MMP-9 mRNA; association of the mRNA with actively translating polysomes; andde novoMMP-9 protein synthesis were obtained in synaptoneurosomes isolated from rat hippocampus. Third, glutamate stimulation of cultured hippocampal neurons evoked a rapid (in minutes) increase in MMP-9 activity, as measured by cleavage of its native substrate, β-dystroglycan. This activity was reduced by the polyadenylation inhibitor, thus linking MMP-9 translation with protein function. In aggregate, our findings show that MMP-9 mRNA is transported to dendrites and locally translated and that the protein is released in an activity-dependent manner. Acting in concert with other dendritically synthesized proteins, locally secreted MMP-9 may contribute to the structural and functional plasticity of the activated synapses.

SummaryCellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3′ UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.PaperClip

Polarized kinesin-driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons. Seven kinesins translocated preferentially to axons and five translocated into both axons and dendrites. None translocated selectively to dendrites. Highly homologous members of the same subfamily displayed distinctly different translocation preferences and were differentially regulated during development. By expressing chimeric kinesins, we identified two microtubule-binding elements within the motor domain that are important for selective translocation. We also discovered elements in the dimerization domain of kinesin-2 motors that contribute to their selective translocation. These observations indicate that selective interactions between kinesin motor domains and microtubules can account for polarized transport to the axon, but not for selective dendritic transport.

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions.

We report that the transacting factor CArG Box binding Factor A (CBF-A) binds the RNA trafficking sequences found in activity-regulated cytoskeleton-associated protein, calmodulin-dependent protein kinase II, and brain-derived neurotrophic factor mRNAs in an activity-dependent manner and accompanies the transcripts from gene to dendrites. CBF-A gene silencing impaired dendritic mRNA localization. We propose that CBF-A is important for trafficking of RNA trafficking sequence–containing neuronal mRNAs., In neurons certain mRNA transcripts are transported to synapses through mechanisms that are not fully understood. Here we report that the heterogeneous nuclear ribonucleoprotein CBF-A (CArG Box binding Factor A) facilitates dendritic transport and localization of activity-regulated cytoskeleton-associated protein (Arc), brain-derived neurotrophic factor (BDNF), and calmodulin-dependent protein kinase II (CaMKIIα) mRNAs. We discovered that, in the adult mouse brain, CBF-A has a broad distribution. In the nucleus, CBF-A was found at active transcription sites and interchromosomal spaces and close to nuclear pores. In the cytoplasm, CBF-A localized to dendrites as well as pre- and postsynaptic sites. CBF-A was found in synaptosomal fractions, associated with Arc, BDNF, and CaMKIIα mRNAs. Electrophoretic mobility shift assays demonstrated a direct interaction mediated via their hnRNP A2 response element (A2RE)/RNA trafficking sequence (RTS) elements located in the 3′ untranslated regions. In situ hybridization and microscopy on live hippocampal neurons showed that CBF-A is in dynamic granules containing Arc, BDNF, and CaMKIIα mRNAs. N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) postsynaptic receptor stimulation led to CBF-A accumulation in dendrites; increased Arc, BDNF, and CaMKIIα mRNA levels; and increased amounts of transcripts coprecipitating with CBF-A. Finally, CBF-A gene knockdown led to decreased mRNA levels. We propose that CBF-A cotranscriptionally binds RTSs in Arc, BDNF, and CaMKIIα mRNAs and follows the transcripts from genes to dendrites, promoting activity-dependent nuclear sorting of transport-competent mRNAs.

Although the dendritic localization and translation of a subset of mRNAs plays a pivotal role in synaptic plasticity, the dendritic mRNAs and their functions have been only minimally characterized thus far. In this study, we isolated mRNAs from Staufen2-containing ribonucleoprotein complexes, which function as modules for the transport of mRNA to the dendrites, and then constructed a cDNA library. Apolipoprotein E gene (APOE) mRNA was isolated from the dendritic mRNA-specific cDNA library. The specific localization of APOE mRNA was evaluated via in situ hybridization. The specific regions involved in the dendritic transport of APOE mRNA were determined using a visualization system employing green fluorescent protein-tagged bacteriophage MS2 RNA-binding protein. As a result, the proximal N-terminal or C-terminal regions of the ApoE-coding sequences were determined to be sufficient for dendritic transport. The level of dendritic APOE mRNA was significantly increased by depolarization-induced neuronal activity, but was reduced in the cell body regions. We assessed the functions of neuronal ApoE. The reduction of ganglioside GM1 by cholesterol depletion was completely blocked by ApoE over-expression. In addition, ApoE over-expression increased the immunoreactivity of the post-synaptic density 95 kDa antibody in the dendrites. These findings indicate that neuronal ApoE may be relevant to lipid rafts or synaptic structural plasticity.

Dysregulation of mitochondrial structure and function has emerged as a central factor in the pathogenesis of Parkinson's disease and related parkinsonian disorders (PD). Toxic and environmental injuries and risk factors perturb mitochondrial complex I function, and gene products linked to familial PD often affect mitochondrial biology. Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) cause an L-DOPA responsive parkinsonian syndrome, stimulating extensive interest in the normal neuroprotective and mitoprotective functions of PINK1. Recent data from mammalian and invertebrate model systems converge upon interactions between PINK1 and parkin, as well as DJ-1, α-synuclein and leucine rich repeat kinase 2 (LRRK2). While all studies to date support a neuroprotective role for wild type, but not mutant PINK1, there is less agreement on subcellular compartmentalization of PINK1 kinase function and whether PINK1 promotes mitochondrial fission or fusion. These controversies are reviewed in the context of the dynamic mitochondrial lifecycle, in which mitochondrial structure and function are continuously modulated not only by the fission–fusion machinery, but also by regulation of biogenesis, axonal/dendritic transport and autophagy. A working model is proposed, in which PINK1 loss-of-function results in mitochondrial reactive oxygen species (ROS), cristae/respiratory dysfunction and destabilization of calcium homeostasis, which trigger compensatory fission, autophagy and biosynthetic repair pathways that dramatically alter mitochondrial structure. Concurrent strategies to identify pathways that mediate normal PINK1 function and to identify factors that facilitate appropriate compensatory responses to its loss are both needed to halt the aging-related penetrance and incidence of familial and sporadic PD.

Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. Here we show that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity.

The dendritic localization of mRNAs and their subsequent translation at stimulated synapses contributes to the experience-dependent remodeling of synapses and thereby to the establishment of long-term memory. Localized mRNAs are transported in a translationally silent manner to distal dendrites in specific ribonucleoprotein particles (RNPs), termed transport RNPs. A recent study suggested that processing bodies (P-bodies), which have recently been identified as sites of RNA degradation and translational control in eukaryotic cells, may participate in the translational control of dendritically localized mRNAs inDrosophilaneurons. This study raised the interesting question of whether dendritic transport RNPs are distinct from P-bodies or whether those structures share significant overlap in their molecular composition in mammalian neurons. Here, we show that P-body and transport RNP markers do not colocalize and are not transported together in the same particles in dendrites of mammalian neurons. Detailed time-lapse videomicroscopy analyses reveal, however, that both P-bodies and transport RNPs can interact in a dynamic manner via docking. Docking is a frequent event involving as much as 50% of all dendritic P-bodies. Chemically induced neuronal activity results in a 60% decrease in the number of P-bodies in dendrites, suggesting that P-bodies disassemble after synaptic stimulation. Our data lend support to the exciting hypothesis that dendritically localized mRNAs might be stored in P-bodies and be released and possibly translated when synapses become activated.

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.

The phenomenon of dendritic transport and local translation of mRNA is considered to be one of the most fundamental mechanisms underlying long-term synaptic plasticity. Matrix metalloproteinase 9 (gelatinase B) (MMP-9) is a matrix metalloproteinase implicated in synaptic long-term potentiation and hippocampus-dependent memory. It was recently shown to be prominently up-regulated in the hippocampal dentate gyrus (DG) upon kainate-mediated seizures. Here, using a high resolution nonradioactive in situ hybridization at the light- and electron-microscopic levels, as well as subcellular fractionation, we provide evidence that in the rat hippocampus, MMP-9 mRNA is associated with dendrites and dendritic spines bearing asymmetric (excitatory) synapses. Moreover we observe that after kainate treatment the number of dendrites and synapses containing MMP-9 mRNA increases markedly. Our results indicate that we are observing the phenomenon of dendritic transport of seizure-induced MMP-9 mRNA.

The mRNA for the immediate early geneArc/Arg3.1is induced by strong synaptic activation and is rapidly transported into dendrites, where it localizes at active synaptic sites. NMDA receptor activation is critical for mRNA localization at active synapses, but downstream events that mediate localization are not known. The patterns of synaptic activity that induce mRNA localization also trigger a dramatic polymerization of actin in the activated dendritic lamina and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) throughout the postsynaptic cytoplasm. The local polymerization of actin in the activated dendritic lamina is of particular interest because it occurs in the same dendritic domains in which newly synthesized Arc/Arg3.1 mRNA localizes. Here, we explore the role of activity-induced alterations in the actin network and mitogen-activated protein (MAP) kinase activation in Arc/Arg3.1 mRNA localization. We show that actin polymerization induced by high-frequency stimulation is blocked by local inhibition of Rho kinase, and Arc/Arg3.1 mRNA localization is abrogated in the region of Rho kinase blockade. Local application of latrunculin B, which binds to actin monomers and inhibits actin polymerization, also blocked the targeting of Arc/Arg3.1 mRNA to activated synaptic sites. Local application of the MAP kinase kinase inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene) blocked ERK phosphorylation, and also blocked Arc/Arg3.1 mRNA localization. Our results indicate that the reorganization of the actin cytoskeletal network in conjunction with MAP kinase activation is required for targeting newly synthesized Arc/Arg3.1 mRNA to activated synaptic sites.

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca 2+ /calmodulin-dependent protein kinase II a-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogenactivated protein kinase pathway.