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The extreme size and polarity of neurons place tremendous demands on the transport and supply systems that physically move large amounts of material from sites of synthesis in the neuronal cell body to synapses; simultaneously, the axon and dendrite must be supplied with ion channels, signaling molecules, cytoskeletal proteins, membrane proteins, organelles, and many other essential components. Deficits in this transport system are expected to lead to neurodegenerative disease. Evidence is accumulating that this transport system is defective at late stages of Alzheimer’s disease, and perhaps at earlier stages as well. Whether defects in axonal transport may be an early event in Alzheimer’s disease is being actively studied and debated.

The localization and translation of messenger RNA (mRNA) within dendrites is a specialized form of gene expression which enables neurons to locally manage information flow and provides a mechanism for the spatiotemporal regulation of structural synapse plasticity that contributes to learning and memory. The delivery of mRNA from the nucleus into dendrites involves complex formation between cis-acting elements and numerous trans-acting proteins, which display dynamic movements and play essential roles in the maintenance of several forms of synaptic plasticity in the mammalian brain. The diverse composition of these posttranscriptional operons is presented in the context of activity-regulated dendritic mRNA transport with regard to their role in synaptic plasticity and defects in their regulation that result in neuronal disease. An example of the analysis of rapid dendritic transport in living hippocampal neurons is presented that shows calcium/calmodulin-dependent protein kinase II (CaMKII)-alpha mRNA movements at rates tenfold greater than previously estimated using improved imaging techniques.

In this research project, we investigated the hypothesis that mutation of the NF1 gene might alter axonal and dendritic transport in neurons. This might provide new insights into the development of cognitive defects in patients with neurofibromatosis 1. The significant results of this pilot research project are all derived from functional studies done by sciatic nerve ligation in wild-type and Nf1-/+ mice. After nerve ligation, proximal and distal segments of the sciatic nerve were homogenized, and analyzed for content of tubulin and synaptic proteins by Western blot and immunohistochemistry. Out results support the idea that there is a quantitative decrease in the accumulation of synaptic proteins immediately proximal to the site of nerve ligature in the Nf-/+ nerve, as compared to wild-type nerves. This supports the idea that Nf1 gene mutation does indeed result in a quantitative defect in axonal transport in the peripheral nervous system. We are preparing to see if there is a similar deficiency in the rate of axonal transport in the central nervous system (CNS) as well. Once we can confirm these findings in the CNS, then we shall embark on a series of experiments that address the molecular mechanisms by which axonal transport is affected in the Nf1-/+ mice.

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP–microtubule association and FMRP–polyribosome association remains elusive. Here, we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP–microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, 3,5-dihydroxyphenylglycine-stimulated manner with similar kinetics to that of wild-type FMRP. Hence, polyribosome-free FMRP–mRNP complexes travel on microtubules and wait for activity-dependent translational derepression at the site of function. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity.

For the last several years, a considerable body of evidence has been collected indicating that MMP-9-mediated TIMP-1-regulated extracellular proteolysis is a novel mechanism contributing to synaptic plasticity. An outstanding feature of this system is that it is kept under the genetic control of a transcription factor AP-1. Both MMP-9 and TIMP-1 are AP-1 target genes in brain and their expression correlates with a spatiotemporal expression pattern of c-Fos, the major neuronal activity-driven component of AP-1. Interestingly, following the gene transcription, MMP-9 mRNA undergoes activity-driven dendritic transport, a feature that is widely accepted as one of the fundamental mechanisms of plasticity. The MMP-9/TIMP-1 system appears to be involved in a broad range of physiological and pathological phenomena in various brain regions. These include developmental reorganization of the cerebellum and visual cortex, hippocampus-dependent learning and long-term-potentiation, as well as pathogenesis of temporal lobe epilepsy. In the hippocampus, MMP-9 appears to function through a direct effect on dendritic spines. We propose an existence of a functional axis, consisting of AP-1/MMP-9/TIMP-1, together with a yet-to-be-discovered synaptic substrate(s) of MMP-9 that has a special liaison with neuronal activity. First, it is activity-driven, and second, it carries on the activity signal to produce its effects on synaptic remodeling and subsequent plasticity.

The formation of neuronal synapses is thought to depend on trans-synaptic interactions between cell adhesion molecules (CAMs) on the surface of axons and dendrites. Synapses are highly asymmetric structures. Pre- and post-synaptic domains might therefore be assembled around heterophilic CAMs which are polarized to axons vs. dendrites. We here investigated the targeting of neuroligin (NLG)-1, a heterophilic CAM, which promotes synapse formation through interaction with its receptor beta-neurexin in axons. We demonstrate that NLG-1 is highly polarized to the dendritic plasma membrane. Dendritic targeting relies on a cytoplasmic amino acid motif. By expressing chimeras of NLG-1 and CD8, an unpolarized protein, we show that the cytoplasmic domain of NLG-1 is necessary and sufficient for dendritic targeting. Furthermore, by truncation analysis we isolated a 32-amino-acid targeting motif. When appended to CD8 this cytoplasmic sequence is sufficient to direct exclusively dendritic localization of the protein. Analysis of yellow fluorescent protein-tagged NLG-1 revealed that vesicular structures containing NLG-1 are excluded from the axon indicating that polarized distribution may be achieved by direct dendritic transport. We propose that the strict polarity of NLG-1 contributes to the directional assembly of synapses during development of the central nervous system.

N-Methyl-d-aspartate (NMDA) receptors (NMDARs), which play a key role in synaptic plasticity, are dynamically regulated by many signaling molecules and scaffolding proteins. Although actin cytoskeleton has been implicated in regulating NMDAR stability in synaptic membrane, the role of microtubules in regulating NMDAR trafficking and function is largely unclear. Here we show that microtubule-depolymerizing agents inhibited NMDA receptor-mediated ionic and synaptic currents in cortical pyramidal neurons. This effect was Ca2+-independent, required GTP, and was more prominent in the presence of high NMDA concentrations. The NR2B subunit-containing NMDA receptor was the primary target of microtubules. The effect of microtubule depolymerizers on NMDAR currents was blocked by cellular knockdown of the kinesin motor protein KIF17, which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Neuromodulators that can stabilize microtubules, such as brain-derived neurotrophic factor, significantly attenuated the microtubule depolymerizer-induced reduction of NMDAR currents. Moreover, immunocytochemical studies show that microtubule depolymerizers decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Taken together, these results suggest that interfering with microtubule assembly suppresses NMDAR function through a mechanism dependent on kinesin-based dendritic transport of NMDA receptors.

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.

Glycine receptors (GlyRs) and specific subtypes of GABAAreceptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminalsrchomology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR β subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABAAreceptor subtype.

Protein transport to and from the postsynaptic plasma membrane is thought to be of central importance for synaptic plasticity. However, the molecular details of such processes are poorly understood. One mechanism by which membrane and secretory proteins may be transported to and from postsynaptic membranes is via cargo receptors. We studied the dendritic transport of TGN38, a putative cargo receptor thought to mediate protein transport between the trans-Golgi network (TGN), endosomes, and the plasma membrane. With fluorescence time-lapse imaging of neurons expressing a TGN38-green fluorescent protein fusion protein (GFP-TGN38), we observed rapid bidirectional dynamics of the protein in dendritic shafts. In addition, the protein was present on the surface and on intracellular membranes of dendrites and dendritic spines. Finally, GFP-TGN38 was found to cycle rapidly between the plasma membrane and intracellular membranes within dendrites, including those of spines. Together, our results suggest a role for TGN38 in facilitating rapid changes in the protein composition of postsynaptic membranes.

The molecular bases underlying the pathogenesis of neurodegenerative diseases are gradually being disclosed. One problem that investigators face is distinguishing primary from secondary events. Rare, inherited mutations causing familial forms of these disorders have provided important insights into the molecular networks implicated in disease pathogenesis. Increasing evidence indicates that accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin-proteasome system dysfunction, excitotoxic insult, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis and failure of axonal and dendritic transport represent unifying events in many slowly progressive neurodegenerative disorders.

Myosin-V is a versatile motor involved in short-range axonal/dendritic transport of vesicles in the actin-rich cortex and synaptic regions of nerve cells. It binds to several different kinds of neuronal vesicles by its globular tail domain but the mechanism by which it is recruited to these vesicles is not known. In this study, we used an in vitro motility assay derived from axoplasm of the squid giant axon to study the effects of the globular tail domain on the transport of neuronal vesicles. We found that the globular tail fragment of myosin-V inhibited actin-based vesicle transport by displacing native myosin-V and binding to vesicles. The globular tail domain pulled down kinesin, a known binding partner of myosin-V, in affinity isolation experiments. These data confirmed earlier evidence that kinesin and myosin-V interact to form a hetero-motor complex. The formation of a kinesin/myosin-V hetero-motor complex on vesicles is thought to facilitate the coordination of long-range movement on microtubules and short-range movement on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated. These results are the first demonstration that the recombinant tail of myosin-V inhibits vesicle transport in an in vitro motility assay. Future experiments are designed to determine the functional significance of the interaction between myosin-V and kinesin and to identify other proteins that bind to the globular tail domain of myosin-V.

The regulation of the dendritic transport of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could precisely modulate the extent of the cerebral field subjected to catecholaminergic influence, and thus could modulate many neurovegetative and central functions. This type of regulation was particularly observed in nucleus tractus solitarius (NTS) and locus coeruleus (LC) catecholaminergic neurons, without any clues about molecular and cellular factors involved in vivo. Here, we have observed whether cerebral angiotensins are involved in this regulation, using TGR(ASrAOGEN)680 (TGR) rat strain, characterized by an inhibition of the synthesis of the only known precursor for all cerebral angiotensins, the astrocytic angiotensinogène. We have observed a sharp decrease of TH quantity within NTS and LC in these rats, specifically during the 4th postnatal week. This phenomenon is particularly characterized by a sharp accumulation of TH protein within cell bodies of NTS and LC neurons and by a blockade of TH dendritic transport, while TH transport toward axonal processes to terminal fields is maintained. The blockade of the dendritic transport of TH protein is associated to an inhibition of the phosphorylation process of MARCKS protein, which is an intracellular signalling pathway that is activated by angiotensins specific AT1 receptors and that was clearly connected in vitro to the neuritic transport of TH protein. The study of the distribution of TH protein within LC terminal fields at adulthood shows the existence of a catecholaminergic hyper-innervation of the dentate gyrus and of frontal and cingular cortices in TGR, which is most probably implicated in the alterations of the durations of sleep-wake stages and of the reactivity of rats to a novelty stress that we have observed in these rats. Thus, our work reveals that cerebral angiotensins are required during the 4th postnatal week to trigger the dendritic transport of TH protein within noradrenergic neurons, and then in the functional setting-up of the inhibitory retrocontrol exerted by the noradrenaline locally released by dendrites of these neurons, La régulation de l'adressage dendritique de la tyrosine hydroxylase (TH), l'enzyme limitante de la biosynthèse des catécholamines, permettrait de moduler avec précision l'étendue du territoire cérébral soumis à l'influence catécholaminergique et ainsi de moduler de nombreuses fonctions neurovégétatives et centrales. Ce type de régulation a notamment été observé au niveau des neurones catécholaminergiques du noyau du faisceau solitaire (NTS) et du locus coeruleus (LC), sans pour autant que les acteurs moléculaires et cellulaires impliqués n'aient été élucidés in vivo. Nous avons recherché si les angiotensines cérébrales pouvaient être potentiellement impliquées dans cette régulation, en utilisant le modèle du rat TGR(ASrAOGEN)680 (TGR), caractérisé par une inhibition de la synthèse du seul précurseur connu des angiotensines cérébrales, l'angiotensinogène astrocytaire. Nous avons mis en évidence une très forte diminution de la quantité de protéine TH dans le NTS et le LC chez ces rats, spécifiquement au cours de la 4ème semaine postnatale. Ce phénomène est notamment caractérisé par une accumulation de la protéine TH dans le corps cellulaire des neurones de ces deux structures et un blocage de son adressage dendritique, alors que le transport vers les aires de terminaisons est maintenu. Le blocage de l'adressage dendritique est associé à l'invalidation du processus de phosphorylation de la protéine MARCKS, voie de signalisation notamment induite par la stimulation du récepteur AT1 aux angiotensines qui avait été clairement impliquée in vitro dans le transport neuritique de la TH. L'étude de la répartition de la TH dans les aires de terminaison du LC à l'âge adulte a permis de montrer l'existence d'une hyper-innervation catécholaminergique du gyrus dentelé et des cortex frontal et cingulaire chez les rats TGR, vraisemblablement impliquée dans les altérations que nous avons observées de l'alternance des cycles d'éveil et de sommeil et de la réactivité à un stress de nouveauté. Ainsi, nos travaux révèlent que les angiotensines cérébrales sont impliquées à 4 semaines dans l'initiation de l'adressage dendritique de la protéine TH dans les neurones noradrénergiques, et de ce fait, dans la mise en place fonctionnelle du rétrocontrôle inhibiteur exercé par la noradrénaline libérée localement par les dendrites de ces neurones

Background: Lafora disease is a progressive myoclonus epilepsy with polyglucosan accumulations and a peculiar neurodegeneration with generalised organellar disintegration. It causes severe seizures, leading to dementia and eventually death in early adulthood. Methods: One Lafora disease gene, EPM2A , has been identified on chromosome 6q24. Locus heterogeneity led us to search for a second gene using a genome wide linkage scan in French-Canadian families. Results: We mapped a second Lafora disease locus, EPM2B , to a 2.2 Mb region at 6p22, a region known to code for several proteins, including kinesins. Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport. Conclusion: Analysis of the present locus in other non- EPM2A families will reveal whether there is further locus heterogeneity. Identification of the disease gene will be of major importance towards our understanding of the pathogenesis of Lafora disease.

Caldendrin is a neuronal Ca(2+)-sensor protein (NCS), which represents the closest homologue of calmodulin (CaM) in nerve cells. It is tightly associated with the somato-dendritic cytoskeleton of neurons and highly enriched in the postsynaptic cytomatrix. Here, we report that caldendrin specifically associates with the microtubule cytoskeleton via an interaction with light chain 3 (LC3), a microtubule component with sequence homology to the GABAA receptor-associated protein (GABARAP), which is, like LC3, probably involved in cellular transport processes. Interestingly, two binding sites exist in LC3 for caldendrin from which only one exhibits a strict Ca(2+)-dependency for the interaction to take place but both require the presence of the first two EF-hands of caldendrin. CaM, however, is not capable of binding to LC3 at both sites despite its high degree of primary structure similarity with caldendrin. Computer modelling suggests that this might be explained by an altered distribution of surface charges at the first two EF-hands rendering each molecule, in principle, specific for a discrete set of binding partners. These findings provide molecular evidence that NCS can transduce signals to a specific target interaction irrespective of Ca(2+)-concentrations and CaM-levels.

Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT2A) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT2A receptor distribution. MAP1A co-localized mainly with 5-HT2A receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input. © 2004 Elsevier B.V. All rights reserved.

In neurons, the proteins derived from mRNAs localized in dendrites have been implicated in synaptic plasticity. The cytoplasmic polyadenylation element (CPE), a cis element in the 3′-UTRs of specific dendritic mRNAs, promotes cytoplasmic polyadenylation-induced translation in response to synaptic stimulation. Here, we demonstrate that the CPE and its binding protein CPEB facilitate mRNA transport to dendrites. In rat hippocampal neurons infected with recombinant viruses, the CPE is sufficient to direct a reporter RNA into dendrites. CPEB-GFP protein forms RNA-containing particles that are transported into dendrites in a microtubule-dependent fashion at an average velocity of 4–8 μm/min. Such particles also contain maskin, a CPEB-associated factor that mediates cap-dependent translational repression of CPE-containing mRNA, and the molecular motors dynein and kinesin. Overexpression of CPEB in neurons promotes the transport of CPE-containing endogenous MAP2 mRNA to dendrites, whereas overexpression of a mutant CPEB that is defective for interaction with molecular motors inhibits this transport. In neurons derived from CPEB knockout mice, the dendritic transport of a CPE-containing reporter RNA is reduced. These results suggest a mechanism whereby CPE-containing mRNAs can be transported to dendrites in a translationally dormant form, but activated at synapses in response to NMDA receptor stimulation.

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. In the mouse, several kinesin motors have been characterized and are suggested to play roles in axonal and/or dendritic transport. One such kinesin is KifC2. Sequence and secondary structure analysis revealed that KifC2 is a member of the C-terminal motor family. Northern and Western blot analyses indicated that KifC2 is specifically expressed in both the central and peripheral nervous systems. The cellular locations of the KifC2 proteins were found to be mainly in neural cell bodies and dendrites but also in axons. To understand the in vivo function of the KifC2 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC2 gene. Homozygous KifC2 mutants were viable and reproduced normally, and their development was apparently normal. These results suggest that KifC2 is dispensable for normal neural development and behavior in the mouse.

RNA transport in dendrites, and subsequent local translation in postsynaptic microdomains, have increasingly been recognized as potentially powerful tools for neurons in long-term modulations of individual synapses (see reviews by Steward 1997; Kuhl and Skehel 1998; Tiedge et al. 1999). While analogous mechanisms have also been reported to be employed by other cell types (reviewed by Bassell et al. 1999), neurons face a particular challenge in that thousands of synapses per dendritic arborization (many of which at considerable distances from the soma) have to be regulated in an independent and input-specific manner. It now appears plausible that such regulation is achieved by two mechanisms: (1) targeted delivery of specific proteins to dendritic sites of demand, and (2) dendritic transport of select RNAs, followed by local postsynaptic translation when and where needed.

Neural BC1 RNA is distributed in neuronal dendrites as RNA-protein complexes (BC1 RNPs) containing Translin. In this study, we demonstrated that the single-stranded DNA- and RNA-binding protein pur alpha and its isoform, pur beta, which have been implicated in control of DNA replication and transcription, linked BC1 RNA to microtubules (MTs). The binding site was within the 5' proximal region of BC1 RNA containing putative dendrite-targeting RNA motifs rich in G and U residues, suggesting that in the cytoplasm of neurons, these nuclear factors are involved in the BC1 RNA transport along dendritic MTs. The pur proteins were not components of BC1 RNP but appeared to associate with MTs in brain cells. Therefore, it is suggested that they may transiently interact with the RNP during transport. In this respect, the interaction of pur proteins with BC1 RNA could be regulated by the Translin present within the RNP, because the binding mode of these two classes of proteins (pur proteins and Translin) to the dendrite-targeting RNA motifs was mutually exclusive. As the motifs are well conserved in microtubule-associated protein 2a/b mRNA as well, the pur proteins may also play a role(s) in the dendritic transport of a subset of mRNAs.

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Though the Rab8b-knockout mice did not display an overt phenotype, the Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than the Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisations of basolateral/dendritic markers of the double-knockout mice were apparently normal. The morphology and the lengths of various primary/motile cilia and the frequency of ciliated cells appeared to be identical between the control and double-knockout mice. However, an additional knockdown of Rab10 in the double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral/dendritic transport. In the meantime, the simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, while the additional loss of Rab10 greatly affects ciliogenesis. (203 words)

Previous studies have demonstrated that neurotensin is internalized and retrogradely transported in neurons of the substantia nigra following its intracerebral injection in the neostriatum. The aim of the present study was to compare the intracellular distribution of retrogradely transported material with that observed following internalization of the peptide at the somatodendritic level and to confirm that the internalization was confined to dopamine neurons. To document somatodendritic internalization, slices (350 μm) from the rat ventral midbrain were incubated in vitro with 20 nM fluoresceinylated neurotensin, a fluorescent derivative of neurotensin, and immunostained 5–60 min later for tyrosine hydroxylase. To document retrograde transport, rats were injected with the same compound into the neostriatum and the brains processed for tyrosine hydroxylase immunohistochemistry 4.5 and 8 h later. Confocal laser microscopic examination of superfused slices revealed that fluoresceinylated neurotensin was internalized at the level of the perikarya and processes of neurons in the substantia nigra, ventral tegmental area and interfascicular nucleus. At short time intervals, the label was detected in the form of small, intensely fluorescent particles distributed within the cytoplasm of both perikarya and dendrites. At longer time intervals, these fluorescent particles were larger, less numerous and confined to the perikarya where they eventually clustered against the nucleus. Following intrastriatal injection of fluoresceinylated neurotensin, retrogradely labeled cells were apparent throughout the substantia nigra, pars compacta, as well as in the lateral part of the ventral tegmental area. Here again, the label took the form of small fluorescent particles, comparable in size, shape and distribution to those detected following superfusion of midbrain slices. In both labeling conditions, fluoresceinylated neurotensin was almost exclusively confined to tyrosine hydroxylase-immunoreactive cells. These results indicate that neurotensin is internalized throughout the terminal and dendritic arborizations of mesostriatal dopamine cells and that the internalized peptide is transported centripetally from both locations to the soma of the cells. The clustering of fluorescent particles in the perinuclear region of the cells further suggests that the internalized process may play a role in long term transcellular signalling.

mRNA transport and subsequent local translation are key mechanisms for several cellular processes during development. In neurons, local translation in dendrites plays important roles in use-dependent synaptic modification and in higher-order brain functions. RNA granules that consist of clusters of ribosomes and mRNAs play central roles in the mRNA transport and local translational control in neuronal dendrites. We identified RNG105 (RNA granule protein 105) as a novel RNA-binding protein that localizes to RNA granules in neurons. We further identified RNG105-associated mRNAs, which included mRNAs encoding Na+/K+ ATPase (NKA) subunit isoforms, i.e., α3, FXYD1, FXYD6, and FXYD7. We generated RNG105-knockout mice and found that the mice died soon after birth because of respiratory failure, suggesting that their brainstem function was impaired. In Rng105–/– neurons, dendritic localization of the NKA mRNAs was reduced, which was accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, Knockout of RNG105 impaired the formation of synapses and neuronal networks, which was mimicked by inhibition and knockdown of NKA in neurons. We propose that RNG105 participates in the dendritic transport of the mRNAs to regulate the development and maintenance of functional networks.

Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin- dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha- Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta- subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co- localization of translational machinery and high concentrations of CaM- KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.

[ 3 H]Glycine was applied to the acoustic cortex of anaesthetized cats during continuous click stimulation for 1 and 9 h, respectively. In other experiments (4 h) stimulation periods of 5 min alternated with 20 min rest. The underlying cortical areas were excised and processed for light and electron microscopic autoradiography. After 1 h of [ 3 H]glycine application labelled proteins appeared in the perikaryon and proximal segments of dendrites. In the case of 4 h application more distant dendritic regions and axon terminals also became labelled. After 9 h glycine application the number of pre- and postsynaptically labelled terminals showed a considerable increase. During periodic stimulation dendritic labelling became intermittent. Distances between groups of silver grains allowed assessment of dendritic transport rate.

Introduction The neuronal cytoskeJeton controls a myriad of functions in the adult and developing central nervous system including cell division, growth, motility, cellular shape, axonal and dendritic transport, neurosecretion, etc (1). The assembled fibrillar components of the neuronal cytoskeleton may be divided into microtubules (approx. 25 nm diameter)~ neurofilaments (approx. 10 nm diameter), and micro- filaments (approx. 6 nm diameter). Although microfilaments and mlcrotubules

Both the slow bulk flow (1 mm/day) of cytoplasm and the rapid transport of specific small molecules, as well as macromolecules (at rates of up to 410 mm/day), are known to occur in the neuronal cell body, axon, and dendrites. The basic phenomena as well as mechanisms of these transport phenomena and their implication for a variety of pathophysiological processes in the central and peripheral nervous system are reviewed. Brought to bear on a variety of neurosurgical problems, ranging from herniated discs to central pain, head injury, ablative surgery, and recovery from cerebrovascular insults, the principles of axoplasmic and dendritic transport suggest opportunities for neurosurgical research that should lead not only to improved understanding of pathophysiological mechanisms but also to the further elucidation of these important neuronal phenomena.

Colchicine and certain other agents which disrupt microtubules and interfere with axonal and dendritic transport are highly toxic to certain CNS neurons. The present chapter summarizes our knowledge about this selective neurotoxicity. Injections of colchicine into several bran regions lead to the death of selected populations of neurons within those regions. Intra-hippocampal injections selectively destroy granule cells of the dentate gyrus; hippocampal pyramidal cells are essentially unaffected. Injections into the cerebellum, olfactory bulb, and caudate nucleus also destroy resident neurons. In these areas several cell types are vulnerable. Neurons of the cerebral cortex appear to be much less affected by colchicine, although some neurons of paleocortical regions are vulnerable. Colchicine does not appear to be an excitotoxin like kainic acid. The neurotoxicity of colchicine appears to be related to the destruction of microtubules, since other agents which disrupt microtubules have similar toxic effects, and since analogs of colchicine which do not disrupt microtubules are non-toxic. Colchicine may induce an autotoxic response which leads to neuronal death in certain populations due to the accumulation of some toxic cellular product which is normally transported by a microtubule-dependent process. The selective vulnerability of neurons to the neurotoxic effects of colchicine may be a model for system degenerations of the central nervous system in which certain subpopulations of neurons are selectively vulnerable to abnormal accumulations of metabolic products.

Cerebellar Purkinje cells contain intracellular membrane systems that are highly enriched in concanavalin A (Con A)-binding sites and which may be involved in axonal and dendritic transport. We have attempted to characterize these Con A-binding sites in fixed slices of cerebellum by their susceptibility to endoglycosidase H (Endo H) digestion. Tissue slices labeled with Con A-peroxidase without prior Endo H digestion had moderately dense label in the molecular and granular layers with the heaviest label occurring in Purkinje cell somata. Endo H digestion of tissue slices produced little change in the pattern of Con A label in the molecular and granular layers, but the Con A label in Purkinje cell somata was removed except for small discrete patches. Electron microscopic examination of Purkinje cells from tissue slices not digested with Endo H showed Con A label in cisternal elements of the endoplasmic reticulum (ER), nuclear envelope, hypolemmal cisternae, and the Golgi complex. Presynaptic terminal smooth membrane cisternae and dendritic hypolemmal cisternae of Purkinje cells were also labeled with Con A-peroxidase. Endo H digestion removed Con A-binding sites in cisternae of the ER, somal and dendritic hypolemmal membrane system, and presynaptic terminal smooth membrane profiles. Con A-binding sites in the nuclear envelope and Golgi complexes of Purkinje cells were not removed by Endo H digestion. These results present ultrastructural evidence in support of biochemical studies which report the rough ER as the site of polymannose oligosaccharide addition to nascent polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate of tritiated fucose in rat cerebellum has been followed using autoradiographic techniques for a period up to one week after intraventricular injection. At intervals of up to three hours after injection, Purkinje and granule cell bodies were found to be most heavily labelled. Subsequently this labelling declined and the highest grain density was observed over the molecular layer. The data is interpreted in terms of fucose incorporation into glycoproteins in nerve cell bodies followed by rapid dendritic transport in the Purkinje cell and fast transport in granule cells along ascending axons and parallel fibres.

The synthesis and transport of newly synthesized proteins in dendrites of rat hippocampal pyramid cells were investigated. Labelled leucine was injected into the left lateral ventricle and the hippocampal region was processed for light and electron microscopic autoradiography. To differentiate between the silver grains originating from ‘sedentary’ or ‘migratory’ proteins, the radioactivity in dendritic areas free of ribosomes and rich in ribosomes was determined separately. Several conclusions were reached. (1) Protein synthesis in dendrites takes place mainly in the proximal parts although a slight synthetic activity can be observed along the whole dendritic tree as well. (2) Newly synthesized proteins are transported toward the distal dendritic region; the data obtained suggest that cisterns of the smooth endoplasmic reticulum and the microtubular system may be involved in this transport. (3) Two phases of dendritic transport may be distinguished; a fast phase with a rate of 100–200 mm/day and a slow phase with a rate of 2.8–10 mm/day. It also seems probable that the majority of the proteins newly synthesized in dendrites are transported by the slow phase.

Intracellular application of tritiated amino acids was performed by microelectrophoresis of single lumbar motoneurones in the cat. Incorporation and transport of the radiochemicals were studied in autoradiographs. Simultaneous recording of the electrophysiological activity permitted direct correlation of biochemical, morphological and physiological parameters in the same individual neurone. This technique was used to test the effect of colchicine. The drug produced severe morphological changes but did not significantly influence amino acid incorporation. But transport of proteins, especially in the dendrites, was impaired. This suggest that dendritic transport depends on the integrity of the neurotubular system. Despite severe morphological changes the neurones retained normal resting potentials and generated spikes when stimulated antidromically. Yet, the majority of the neurones do not respond to orthodromic stimulation. This suggests colchicine-induced structural changes of the presynaptic side.

The immediate early gene Arc is emerging as a versatile, Wnely tuned system capable of coupling changes in neuronal activity patterns to synaptic plasticity, thereby optimizing information storage in the nervous system. Here, we attempt to overview the Arc system spanning from transcriptional regulation of the Arc gene, to dendritic transport, metabolism, and translation of Arc mRNA, to post-translational modiWcation, localization, and degradation of Arc protein. Within this framework we discuss the function of Arc in regulation of actin cytoskeletal dynamics underlying consolidation of long-term potentiation (LTP) and regulation of AMPA-type glutamate receptor endocytosis underlying long-term depression (LTD) and homeostatic plasticity. Behaviorally, Arc has a key role in consolidation of explicit and implicit forms of memory, with recent work implicating Arc in adaptation to stress as well as maladaptive plasticity connected to drug addiction. Arc holds considerable promise as a “master regulator” of protein synthesis-dependent forms of synaptic plasticity, but the mechanisms that modulate and switch Arc function are only beginning to be elucidated. publishedVersion

Abnormal accumulation of β-secretase (BACE1) in dystrophic neurites and presynaptic β-amyloid (Aβ) production contribute to Alzheimer's disease pathogenesis. Little, however, is known about BACE1 dynamic transport in neurons. We investigated BACE1 trafficking in hippocampal neurons using live-cell imaging and selective labeling. We report that transport vesicles containing internalized BACE1 in dendrites undergo exclusive retrograde transport, whereas they undergo bidirectional transport in axons. Unidirectional dendritic transport requires Eps15 homology domain-containing (EHD) 1 and 3 protein function. Furthermore, loss of EHD function compromises axonal sorting and dynamic axonal transport of BACE1. EHD1/3 colocalize with BACE1 and APP β-C-terminal fragments in hippocampal mossy fiber terminals, and their depletion in neurons significantly attenuates Aβ levels. These results represent the first demonstration of unidirectional endocytic transport of any cargo in dendrites. Moreover, they reveal a novel role for EHD proteins in neuronal BACE1 transcytosis and Aβ production, processes that are highly relevant for Alzheimer's disease.

Typical neurons of the central nervous system (CNS) elaborate tens of thousands of membrane specializations at sites of synaptic contacts on their dendrites. To construct, maintain, and modify these specializations, neurons must produce and deliver the appropriate molecular constituents to particular synaptic sites. Previous studies have revealed that polyribosomes are selectively positioned beneath postsynaptic sites, suggesting that in neurons, as in other cell types, protein synthetic machinery is located at or near the sites where particular proteins are needed. The mechanisms that deliver ribosomes and messenger RNA to their specific destinations in cells are therefore of considerable interest. Here we describe a system for RNA transport in dendrites that could provide a mechanism for the delivery of ribosomes and mRNA to synaptic sites in dendrites. Hippocampal neurons grown in culture incorporate 3H-uridine in the nucleus, then selectively transport the newly synthesized RNA into dendrites at a rate of about 0.5 mm day-1. The transport is inhibited by metabolic poisons, suggesting that it is an active, energy-dependent process. The RNA may be transported in association with the cytoskeleton.

Injection of colchicine into the lateral cerebral ventricle of the rat was found to induce a paradoxical translocation of two lysosomal enzymes, dipeptidyl peptidase II (Dpp II) and acid phosphatase, from the soma of neurons to the dendrites. Following a single injection of colchicine, neuronal somata, which normally contain the bulk of these lysosomal enzymatic activities, become depleted of these enzymes, whereas dendrites become abnormally enriched. All neurons which contained these enzymes, except those of the mesencephalic nucleus of the trigeminal nerve, displayed this phenomenon. Lysosomal enzyme translocation into dendrites was observed in the mitral cell layer within 1 hr after a colchicine injection and could be induced in most neuronal populations by injections of colchicine as low as 25 micrograms. Five days after a 100-micrograms colchicine injection, a normal pattern of enzyme distribution was observed, indicating that the effect of colchicine was reversible. Enzyme translocation was not accompanied by gross changes in cell morphology, nor did it result in the specific loss of neuronal cell bodies which contained these enzymes. The results indicate that colchicine, under conditions known to inhibit axoplasmic transport, stimulates the transport of lysosomal enzymes from the cell body to the dendrites.

We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, approximately 75 microns from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and (+)-ends directed toward the cell body. At distances within 15 micron of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.

Recent studies have shown that protein synthetic machinery consisting of polyribosomes and associated membranous cisterns is selectively localized beneath synaptic sites on neurons. In the present paper, the role of this machinery in neuronal function will be considered. We will: 1. Summarize the studies that characterize the polyribosomes and define their associations with membranous cisterns. Taken together, these observations suggest the existence of a system for the synthesis and posttranslational processing of proteins at individual synaptic sites; 2. Review the evidence that the protein synthetic machinery is particularly prominent during the initial formation of synaptic contacts (during early development), and during lesion-induced synaptogenesis in mature animals. These observations have led to the hypothesis that the polyribosomes produce proteins that play a role in the formation of the synaptic junction; 3. Review evidence that supports the hypothesis that there is a local synthesis of protein within dendrites, as well as local glycosylation; 4. Describe the evidence suggesting that at least some of the protein constituents of the synaptic junction itself are synthesized locally; and 5. Describe our studies that reveal a mechanism for selective dendritic transport of RNA; this transport mechanism permits the delivery of RNA to postsynaptic sites throughout the dendritic arbor. We will advance the hypothesis that neurons position protein synthetic machinery together with the mRNA's that are appropriate for particular synapses beneath synaptic contact regions. At the synaptic site, this machinery could then direct the synthesis of particular proteins that are critical for synapse formation or maintenance. The positioning of protein synthetic machinery at postsynaptic sites permits a rapid local regulation of the production of key proteins by events at individual synapses.

Recent studies have shown that protein synthetic machinery consisting of polyribosomes and associated membranous cisterns is selectively localized beneath synaptic sites on neurons. In the present paper, the role of this machinery in neuronal function will be considered. We will: 1. Summarize the studies that characterize the polyribosomes and define their associations with membranous cisterns. Taken together, these observations suggest the existence of a system for the synthesis and posttranslational processing of proteins at individual synaptic sites; 2. Review the evidence that the protein synthetic machinery is particularly prominent during the initial formation of synaptic contacts (during early development), and during lesion-induced synaptogenesis in mature animals. These observations have led to the hypothesis that the polyribosomes produce proteins that play a role in the formation of the synaptic junction; 3. Review evidence that supports the hypothesis that there is a local synthesis of protein within dendrites, as well as local glycosylation; 4. Describe the evidence suggesting that at least some of the protein constituents of the synaptic junction itself are synthesized locally; and 5. Descibe our studies that reveal a mechanism for selective dendritic transport of RNA; this transport mechanism permits the delivery of RNA to postsynaptic sites throughout the dendritic arbor.

The effect of colchicine injections on the ultrastructural localization of dipeptidyl peptidase II (Dpp II) was studied in the mitral cells of the rat olfactory bulb. In control animals, electron-dense reaction product representing Dpp II activity was observed in lysosomes, lipofuscin granules, short cisternae located close to the granular endoplasmic reticulum, and dense granules. Lysosomes and lipofuscin granules were the most intensely stained organelles. Dpp II-containing organelles were localized mainly to the cell body and were randomly distributed in the perikaryal cytoplasm. Twenty-four hours after a 100- micrograms intracerebroventricular colchicine injection, the distribution of Dpp II-containing organelles was drastically altered. Short cisternae and dense granules containing Dpp II reaction product were noticeably absent in these preparations. Lysosomes and lipofuscin granules were depleted from the perikaryal cytoplasm and were concentrated in dendrites. Lysosomes were observed to extend for considerable distances in dendrites where they acquired elongated and dumbbell shapes. The shapes of some of these labeled lysosomes gave the impression that they were actively being “pulled” into the dendrites. These results indicate that microtubules sequester lysosomes to the perikaryal cytoplasm and suggest the presence of a novel transport system responsible for the movement of lysosomes from the cell body to the dendrites.

Neuroaxonal Dystrophy in Man: Character and Natural History.- Neuropathological Conditions Related to Neuroaxonal Dystrophy.- Infantile Neuroaxonal Dystrophy or Seitelberger's Disease: II. Peripheral Nerve Involvement. Electron Microscopy Study in one Case.- How Long Can Degenerating Axons in the Central Nervous System Produce Reactive Changes? An Electron Microscopic Investigation.- Fine Structural Changes of Neurites in Alzheimer's Disease.- Mitochondrial Changes in Axonal Dystrophy Produced by Vitamin E Difficiency.- Permeability of Blood Vessels and Connective Tissue Sheaths in the Peripheral Nervous System to Exogenous Proteins.- Changes in Axonal Flow During Regeneration of Mammalian Motor Nerves.- Nuclear, Cytoplasmic and Axoplasmic RNA in Experimental Neuroaxonal Dystrophy.- The Dependence of Fast Transport in Mammalien Nerve Fibers on Metabolism.- Slow and Rapid Transport of Protein to Nerve Endings in Mouse Brain.- Transport of S-100 Protein in Mammalian Nerve Fibers and Transneuronal Signals.- Dynamic Condition of Protein in Axons and Axon Terminales.- Effect of Nerve Section on Protein Metabolism of Ganglion Cells and Preganglionic Nerve Endings.- Some Observations on the Experimental Production of Acute Neuroaxonal and Synaptosomal Dystrophy.- Independence of the Rapid Axonal Transport of Protein from the Flow of Free Amino Acids.- Acetylcholinesterase in Mammalian Peripheral Nerves and Characteristics of its Migration.- Role of Slow Axonal Transport in Nerve Regeneration.- Axoplasmic Streaming and Proteins in the Retino-Tectal Neurons of the Pigeon.- Different Modes of Substance Flow in the Optic Tract.- A Symmetrical Double-Label Method for Studying the Rapid Axonal Transport of Radioactivity from Labelled d-Glucosamine in the Gold Fish Visual System.- Single Cell Isotope Injection Technique, a Tool for Studying Axonal and Dendritic Transport.- Neuronal Organelles in Neuroplasmic ("Axonal") Flow, I. Mitochondria.- Neuronal Organelles in Neuroplasmic ("Axonal") Flow, II. Neurotubules.- Axonal Transport of Proteins in the Optic Nerve and Tract of the Rabbit.- Changes in Microtubules and Neurofilaments in Constricted, Hypoplastic Nerve Fibers.- Effects of Vinblastine and Colchicine on Monoamine Containing Neurons of the Rat, with Special Regard to the Axoplasmic Transport of Amine Granules.- The Importance of Axoplasmic Transport of Amine Granules for the Functions of Adrenergic Neurons.- Axonal Transport of Proteins in the Hypothalamo-Neurohypophysial System of the Rat.- Axonal Transport in the Goldfish Visual System.

We have previously reported that recently synthesized RNA is selectively transported into the dendrites of hippocampal neurons grown in culture (Davis et al., 1987). The present study provides further details about this transport process, focusing especially on the velocity of transport, by comparing the velocity of dendritic transport of RNA in neurons of different ages and in the branched and unbranched dendrites of individual neurons. In our previous study, we recognized that calculations of transport velocity could be compromised because transport was being evaluated in a population of dendrites of varying lengths. The present study uses a mathematical modeling approach to determine how the morphology of the population of dendrites would affect the analysis of transport velocity. Focusing first on a simple model, we compared the distribution of transported material at various times when all dendrites were of the same length and when the population included dendrites of different lengths. We found that the distance of labeling increased linearly over time when all dendrites were of the same length, but increased with a negatively accelerating curve when dendrites were of different lengths. We then determined the actual distribution of dendritic lengths in cultured hippocampal neurons, based on immunostaining with an antibody directed against the selective dendritic marker, microtubule-associated protein 2 (MAP2). Using a computer model, we calculated the mean distance of transport as a function of time in this population of dendrites, assuming different velocities of transport. The velocity that best fit the measured distances of RNA transport in both 7- and 15-d-old neurons was 11 microns/hr (0.26 mm/d). However, for the dendrites exhibiting the longest distance of labeling, the best-fitting curve assumed a velocity of 21 microns/hr in both 7- and 15-d-old neurons (0.50 mm/d). Comparisons of transport in branched and unbranched dendrites revealed that the distance of labeling over branched dendrites was consistently longer than over unbranched dendrites of individual neurons. However, neurons with a larger proportion of branched dendrites did not exhibit a greater mean distance of transport. The density of silver grains was higher over branched than over unbranched dendrites, suggesting that a greater amount of recently synthesized RNA may be transported into branched dendrites. Taken together, these results suggest that RNA transport into dendrites is regulated differentially in the dendrites of individual neurons.

Previous studies established that the kinesin adaptor proteins, TRAK1 and TRAK2, play an important role in mitochondrial transport in neurons. They link mitochondria to kinesin motor proteins via a TRAK acceptor protein in the mitochondrial outer membrane, the Rho GTPase, Miro. TRAKs also associate with enzyme, O-linked N-acetylglucosamine transferase (OGT), to form a quaternary, mitochondrial trafficking complex. A recent report suggested that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons whereas TRAK2 controls mitochondrial transport in dendrites. However, it is not clear whether the function of any of these proteins is exclusive to axons or dendrites and if their mechanisms of action are conserved between different neuronal populations and also, during maturation. Here, a comparative study was carried out into TRAK-mediated mitochondrial mobility in axons and dendrites of hippocampal and cortical neurons during maturation in vitro using a shRNA gene knockdown approach. It was found that in mature hippocampal and cortical neurons, TRAK1 predominantly mediates axonal mitochondrial transport whereas dendritic transport is mediated via TRAK2. In young, maturing neurons, TRAK1 and TRAK2 contribute similarly in mitochondrial transport in both axons and dendrites in both neuronal types. These findings demonstrate maturation regulation of mitochondrial transport which is conserved between at least two distinct neuronal subtypes.

The envelope glycoproteins of Semliki Forest virus (SFV), Vesicular Stomatitis virus (VSV), and Influenza Fowl Plague virus (FPV) are vectorially targeted in neurons to the plasma membrane of dendrites (SFV and VSV) and axons (FPV). To gain insight into the mechanisms responsible for such polarized delivery we have examined the effects on neurons of nocodazole and brefeldin A (BFA), which are known to cause microtubule depolymerization and disassembly of the Golgi apparatus, respectively. Nocodazole treatment blocked transport of all viral glycoproteins to both axons and dendrites. BFA treatment induced disruption of the Golgi complex, including the trans-Golgi network (TGN), and tubulation of endosomes. However, the delivery of the SFV and FPV glycoproteins to the cell surface was not affected significantly by BFA, although processing and sorting were altered, as revealed by surface biotinylation and immunofluorescence microscopy of fixed nonpermeabilized cells. These results demonstrate the involvement of microtubules in axonal and dendritic transport of integral membrane glycoproteins, and the existence of a BFA-sensitive component in the sorting but not in the transport machinery.

Within mouse forebrain, a subset of microRNAs are significantly enriched in synaptoneurosomes (a synaptic fraction containing pinched-off dendritic spines) and a subset are significantly depleted relative to total forebrain homogenate. Here I show that, as a group, the pre-miR hairpin precursors of synaptically enriched microRNAs exhibit significantly different structural features than those that are non-enriched or depleted. Precursors of synaptically enriched microRNAs tend to have a) shorter uninterrupted double-stranded stem segments, and b) more symmetrical bulges containing a single nucleotide on each side. These structural differences may provide a basis for the differential binding of proteins that mediate dendritic transport of pre-miRs, or that prevent pre-miRs from being prematurely processed into mature miRNAs during the transport process. Reviewers This article was reviewed by I. King Jordan and Jerzy Jurka.