Dendritic Transport Research Articles

The kpc-1 3'UTR facilitates dendritic transport and translation efficiency of mRNAs for dendrite arborization of a mechanosensory neuron important for male courtship.

A recently reported Schizophrenia-associated genetic variant in the 3'UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3'UTR in neuronal development. We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3'UTR participates in dendrite branching and self-avoidance. The kpc-1 3'UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes translation efficiency. A predicted secondary structural motif in the kpc-1 3'UTR is required for dendrite self-avoidance. Animals with over-expression of DMA-1, a PVD dendrite receptor, exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship.

Chemical LTP induces confinement of BDNF mRNA under dendritic spines and BDNF protein accumulation inside the spines.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays a key role in neuronal development and synaptic plasticity. The discovery that BDNF mRNA can be transported in neuronal dendrites in an activity-dependent manner has suggested that its local translation may support synapse maturation and plasticity. However, a clear demonstration that BDNF mRNA is locally transported and translated at activated synapses in response to long-term potentiation (LTP) is still lacking. Here, we study the dynamics of BDNF mRNA dendritic trafficking following the induction of chemical LTP (cLTP). Dendritic transport of BDNF transcripts was analyzed using the MS2 system for mRNA visualization, and chimeric BDNF-GFP constructs were used to monitor protein synthesis in living neurons. We found that within 15 min from cLTP induction, most BDNF mRNA granules become stationary and transiently accumulate in the dendritic shaft at the base of the dendritic spines, while at 30 min they accumulate inside the spine, similar to the control CamkIIα mRNA which also increased inside the spines at 60 min post-cLTP. At 60 min but not at 15 min from cLTP induction, we observed an increase in BDNF protein levels within the spines. Taken together, these findings suggest that BDNF mRNA trafficking is arrested in the early phase of cLTP, providing a local source of mRNA for BDNF translation at the base of the spine followed by translocation of both the BDNF mRNA and protein within the spine head in the late phase of LTP.

TauSTED super-resolution imaging of labile iron in primary hippocampal neurons.

Iron dyshomeostasis is involved in many neurological disorders, particularly neurodegenerative diseases where iron accumulates in various brain regions. Identifying mechanisms of iron transport in the brain is crucial for understanding the role of iron in healthy and pathological states. In neurons, it has been suggested that iron can be transported by the axon to different brain regions in the form of labile iron; a pool of reactive and exchangeable intracellular iron. Here we report a novel approach to imaging labile ferrous iron, Fe(II), in live primary hippocampal neurons using confocal and TauSTED (stimulated emission depletion) microscopy. TauSTED is based on super-resolution STED nanoscopy, which combines high spatial resolution imaging (<40nm) with fluorescence lifetime information, thus reducing background noise and improving image quality. We applied TauSTED imaging utilizing biotracker FerroFarRed Fe(II) and found that labile iron was present as submicrometric puncta in dendrites and axons. Some of these iron-rich structures are mobile and move along neuritic pathways, arguing for a labile iron transport mechanism in neurons. This super-resolution imaging approach offers a new perspective for studying the dynamic mechanisms of axonal and dendritic transport of iron at high spatial resolution in living neurons. In addition, this methodology could be transposed to the imaging of other fluorescent metal sensors.

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics.

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.

A small excitation window allows long-duration single-molecule imaging, with reduced background autofluorescence, in C. elegans neurons

Single-particle imaging using laser-illuminated widefield epi-fluorescence microscopy is a powerful tool to investigate molecular processes in vivo. Performing high-quality single-molecule imaging in such biological systems, however, remains a challenge due to difficulties in controlling the number of fluorescing molecules, photobleaching, and the autofluorescence background. Here, we show that by exciting only a small, 5-15 μm wide region in chemosensory neurons in live C. elegans, we can significantly improve the duration and quality of single-molecule imaging. Small-window illumination microscopy (SWIM) allows long-duration single-particle imaging since fluorescently labelled proteins are only excited upon entering the small excited area, limiting their photobleaching. Remarkably, we also find that using a small excitation window significantly improves the signal-to-background ratio of individual particles. With the help of theoretical calculations, we explain that the improved signal-to-background ratio is due to reduced background, mostly caused by out-of-focus autofluorescence. We demonstrate the potential of this approach by studying the dendritic transport of a ciliary calcium channel protein, OCR-2, in the chemosensory neurons of C. elegans. We reveal that OCR-2-associated vesicles are continuously transported back and forth along the length of the dendrite and can switch between directed and diffusive states. Furthermore, we perform single-particle tracking of OCR-2-associated vesicles to quantitatively characterize the transport dynamics. SWIM can be readily applied to other in vivo systems where intracellular transport or cytoskeletal dynamics occur in elongated protrusions, such as axons, dendrites, cilia, microvilli and extensions of fibroblasts.

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: A new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. To test if the dynein adapter RAB-interacting lysosomal protein (RILP) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents previously validated in non-neuronal cells. Striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of staining. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. It would be interesting to identify the actual target for this neuronal Golgi phenotype. Cell type-specific off-target phenotypes therefore likely occur in neurons, necessitating revalidation of reagents that were previously validated in other cell types.

Lattice light-sheet microscopy and evaluation of dendritic transport in cultured hippocampal tissue reveal high variability in mobility of the KIF1A motor domain and entry into dendritic spines

The unique morphology of neurons consists of a long axon and a highly variable arbour of dendritic processes, which assort neuronal cells into the main classes. The dendritic tree serves as the main domain for receiving synaptic input. Therefore, to maintain the structure and to be able to plastically change according to the incoming stimuli, molecules and organelles need to be readily available. This is achieved mainly via bi-directional transport of cargo along the microtubule lattices. Analysis of dendritic transport is lagging behind the investigation of axonal transport. Moreover, addressing transport mechanisms in tissue environment is very challenging and, therefore, rare. We employed high-speed volumetric lattice light-sheet microscopy and single particle tracking of truncated KIF1A motor protein lacking the cargo-binding domain. We focused our analysis on dendritic processes of CA1 pyramidal neurons in cultured hippocampal tissue. Analysis of individual trajectories revealed detailed information about stalling and high variability in movement and speed, and biased directionality of KIF1A. Furthermore, we could also observe KIF1A shortly entering into dendritic spines. We provide a workflow to analyse variations in the speed and direction of motor protein movement in dendrites that are either intrinsic properties of the motor domain or depend on the structure and modification of the microtubule trails.

Expected and unexpected roles for dynein regulation of dendritic late endosomes.

Dendrites differ from axons in multiple ways, including the presence of minus-end out microtubules intermixed with the more conventional plus-end out microtubules. The mixed microtubule polarity makes regulation of directional transport in dendrites a challenge. Dynein can in principle be a retrograde and anterograde motor in dendrites. We show in our recent paper that dynein supports bi-directional transport of late endosomes in dendrites. We also show that overexpression of the RAB7 effector RILP which recruits dynein to late endosomes imparts retrograde bias onto late endosomes. Inhibition of dynein leads to a decrease in bi-directional motility of late endosomes, an expected result. Unexpectedly, inhibition of dynein also impairs endosome maturation as evidenced by increased association of GTP-RAB7 with late endosomes. Ultimately, dynein inhibition causes degradation defects of short-lived dendritic receptors and stunted dendrite morphologies. Much more work is required to fully understand how endosomal pathways are regulated in time and space in dendrites. Given the prevalence of neurological disorders where endosome-lysosome functions are impaired, this is a topic of great translational relevance.

Autoimmune RNA dysregulation and seizures: therapeutic prospects in neuropsychiatric lupus.

Lupus autoimmunity frequently presents with neuropsychiatric manifestations, but underlying etiology remains poorly understood. Human brain cytoplasmic 200 RNA (BC200 RNA) is a translational regulator in neuronal synapto-dendritic domains. Here, we show that a BC200 guanosine-adenosine dendritic transport motif is recognized by autoantibodies from a subset of neuropsychiatric lupus patients. These autoantibodies impact BC200 functionality by quasi irreversibly displacing two RNA transport factors from the guanosine-adenosine transport motif. Such anti-BC autoantibodies, which can gain access to brains of neuropsychiatric lupus patients, give rise to clinical manifestations including seizures. To establish causality, naive mice with a permeabilized blood-brain barrier were injected with anti-BC autoantibodies from lupus patients with seizures. Animals so injected developed seizure susceptibility with high mortality. Seizure activity was entirely precluded when animals were injected with lupus anti-BC autoantibodies together with BC200 decoy autoantigen. Seizures are a common clinical manifestation in neuropsychiatric lupus, and our work identifies anti-BC autoantibody activity as a mechanistic cause. The results demonstrate potential utility of BC200 decoys for autoantibody-specific therapeutic interventions in neuropsychiatric lupus.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

The synaptic life of microtubules.

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.

Paradoxical relationships between active transport and global protein distributions in neurons

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. Many kinds of ion channels are trafficked over long distances to specific cellular compartments. This raises the question of whether cargo is directed with high specificity during transit or whether cargo is distributed widely and sequestered at specific sites. We addressed this question by experimentally measuring transport and expression densities of Kv4.2, a voltage-gated transient potassium channel that exhibits a specific dendritic expression that increases with distance from the soma and little or no functional expression in axons. In over 500 h of quantitative live imaging, we found substantially higher densities of actively transported Kv4.2 subunits in axons as opposed to dendrites. This paradoxical relationship between functional expression and traffic density supports a model—commonly known as the sushi belt model—in which trafficking specificity is relatively low and active sequestration occurs in compartments where cargo is expressed. In further support of this model, we find that kinetics of active transport differs qualitatively between axons and dendrites, with axons exhibiting strong superdiffusivity, whereas dendritic transport resembles a weakly directed random walk, promoting mixing and opportunity for sequestration. Finally, we use our data to constrain a compartmental reaction-diffusion model that can recapitulate the known Kv4.2 density profile. Together, our results show how nontrivial expression patterns can be maintained over long distances with a relatively simple trafficking mechanism and how the hallmarks of a global trafficking mechanism can be revealed in the kinetics and density of cargo.

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal.

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 electrophysiologicalapproaches. 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.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Neuronal trafficking dynamics and regulators

AbstractBackgroundNeurons are highly polarized cells continuously relying on intracellular transport, through which they communicate with the external world. Among the somatodendritic and axonal compartments many proteins with different functions are sorted to reach long distances. The relevance of neuronal transport in ensuring cell development and homeostasis maintenance raises a big interest on this topic. Moreover, several neurodegenerative diseases, such as Alzheimer’s or Amyotrophic Lateral Sclerosis, exhibit significant axonal pathology, where transport impairment seems to have a crucial influence. However, the role of this process in neurodegeneration, remains poorly understood. In this study we aim to characterize neuronal transport dynamics of specific proteins, and the molecular machinery regulating this process. Furthermore, we aim to explore transport from the pathological point of view, evaluating the effect that mutant proteins (e.g. TDP43‐related) may have on it.MethodHuman Neural Stem Cells derived neurons were differentiated for 30 DIV. Plasmids encoding for different GFP‐coupled proteins were designed to express those who are involved in main neuronal functions (e.g. synaptic activity) and transfected in neurons. Time‐lapse movies of cargoes were acquired to detect changes in movement. Data analysis was performed using object segmentation and tracking algorithms. Raw data were processed to describe the main motion parameters: average velocity, directionality, track length, and others. Post‐imaging Immunocytochemistry was performed to assess cargoes localization and discriminate between axonal and dendritic transport.ResultAmyloid Precursor Protein (APP) cargoes with higher velocity in anterograde direction were observed compared with the retrograde one; meanwhile for Synaptophysin, a major population of stationary particles was found. Moreover, sorted data for axonal and dendritic transport description shown faster APP particles in axonal anterograde movement. Relying on the movement dynamics description, we decided to screen for protein‐protein interactions, using GFP‐trap and CoIP approaches followed by Mass Spectrometry analysis, which can shed light on possible partners involved in the transport regulation.ConclusionOur work describes an in vitro model for neuronal transport study and highlights the different behavior of proteins with various physiological functions. A complete view will elucidate details about transport regulation and will open a way to understand its role in neuropathological processes.

Abstract A78: IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma

Abstract Peripheral tissues adopt mechanisms of immune suppression that counteract protective immunity to prevent immunopathology and are co-opted by tumors for immune escape. In tumors, cytotoxic, antigen-specific T cells produce IFNg and activate multiple mechanisms of adaptive immune resistance, including upregulation of the T-cell inhibitory molecule programed death ligand 1 (PD-L1). Tumor cell PD-L1 expression does not predict response to anti-PD-L1 immunotherapy, indicating, however, that other sources of PD-L1 in tumor microenvironments may contribute to PD-L1-dependent immune suppression. Lymphatic vessels facilitate antigen and dendritic cell transport to draining lymph nodes and are required for antitumor immunity. Interestingly, lymphatic endothelial cells in lymph nodes are tolerogenic at steady-state and constitutively express PD-L1 to delete self-reactive CD8+ T cells. However, whether lymphatic endothelial cells in tumor microenvironments are also directly immunosuppressive remains unclear. Here we test the hypothesis that peripheral, tumor-associated lymphatic vessels acquire immunosuppressive mechanisms, e.g., PD-L1 expression, that limit effector CD8+ T-cell accumulation and function in melanoma. We demonstrate that host nonhematopoietic, nontumor, PD-L1 limits CD8+ T-cell accumulation in murine melanoma. Further, we show that tumor-associated lymphatic endothelial cells upregulate PD-L1 in response to IFNg produced by tumor-infiltrating, antigen-specific CD8+ T cells. As such, loss of IFNgR by lymphatic endothelial cells improves CD8+ T-cell accumulation in murine melanomas, thereby enhancing tumor control and improved overall survival. Importantly, this robust lymphatic vessel-dependent tumor control was revealed in the context of murine melanomas with significant UVB-induced somatic mutation burden, consistent with a cytotoxic T cell-mediated negative feedback mechanism. Consequently, we present IFNg-mediated activation of tumor-associated lymphatic vessels as a new component of adaptive immune resistance, suggesting that context-dependent lymphatic vessel function may provide novel, targetable mechanisms of immune control in melanoma. Citation Format: Ryan S. Lane, Alec P. Breazeale, Amanda W. Lund. IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A78.

Tracking Down the Fast and Superprocessive KIF1A with Gold Scattering Microscopy

The kinesin-3 family member KIF1A is a neuronal kinesin that performs long-distance anterograde vesicle transport in axons and dendrites. Single molecule studies observe KIF1A velocities > 1 μm/s and average run lengths > 5 μm, making KIF1A one of the fastest and most processive members of the kinesin superfamily; however, the mechanistic basis of these high speeds and long run lengths is unknown. One prevailing model for superprocessivity holds that the positively-charged “K-loop” in the KIF1A motor domain diffusively tethers the motor to the negatively-charged microtubule, which prevents complete dissociation of the motor and effectively links together short runs. However, this model does not account for how KIF1A reaches such high speeds, or what role the K-loop plays in the ATP-driven stepping mechanism. To address these questions, we labeled the C-terminus of a truncated KIF1A dimer with a 30-nm gold nanoparticle and used interferometric scattering microscopy (iSCAT) to observe the KIF1A stepping cycle at sub-millisecond time scales and nanometer-level precision. Our preliminary data reveal that KIF1A motility is characterized by periods of directed stepping interspersed by periods where the motor is statically bound to the microtubule, rather than diffusively tethered, as predicted by the K-loop model. An alternate explanation for superprocessivity, that could also account for the stepping speed, is that during each step the K-loop ensures the tethered head binds to the microtubule hundreds of times faster than the bound head detaches from the microtubule. Our ongoing investigations employ a KIF1A construct with a nanoparticle-functionalized motor domain to characterize the nature of these paused states, and the duration of the one-head bound state in the chemomechanical cycle.

Specific depletion of the motor protein KIF5B leads to deficits in dendritic transport, synaptic plasticity and memory.

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.

Функциональная роль гипоталамо- гипофизарной нейросекреторной системы в миграциях и нересте рыб

The results of the ecological-histophysiological study of the hypothalamo-hypophysial neurosecretory system (HHNS) by use of light- and electron-microscopical morphometric methods and comparative analysis of the results are presented. At the beginning of migrations a state of “HHNS mobilization” has been established. It is expressed in the form of the strong synthesis activation of neurosecretary products in neurosecretory cells (NSC) and their transport in dendrites and axons to neurohypophysis (NH), where, their mass accumulation, however, occurs. This completion of moderate “normal” level of excretion of nonapeptide neurohormones (NP-NH) into the bloodstream disrupts the body’s water-salt homeostasis. The synchronized transventricular direction of the NP-NH excretion from dendrites and axons into the brain liquor of III ventricle provides their neurotropic effect in the behavioral centers of Central Nervous System (CNS). As a result, HHNS has a decisive double synchronous effect, which disrupts the long-adapted “pasture” type of osmoregulation (hyper- or hypoosmotic), which is probably the main physiological stimulus of habitat change and, at the same time, it includes the behavioral centers of the CNS, which causes a dominant state, originally defined as a “migration impulse.”The interactions of nonapeptide- and luliberinergic centers of the hypothalamus in the navigational mechanisms of homing and imprinting are discussed. At the beginning of spawning, regardless of its season a strong activation of HHNS, followed by a decrease in its functional activity, was discovered in fish of continuous spawning. The detected two-phase reaction of the HHNS seems to be a reflection of its participation in the body’s protective and adaptive responses to physiological stress. The key role of the HHNS in integrating migration and spawning processes is discussed.

Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

Postsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.