Academic literature on the topic 'Fast axonal transport'

Axonal growth depends on axonal transport. We report the first global analysis of mitochondrial transport during axonal growth and pauses. In the proximal axon, we found that docked mitochondria attached to the cytoskeletal framework that were stationary relative to the substrate and fast axonal transport fully accounted for mitochondrial transport. In the distal axon, we found both fast mitochondrial transport and a coherent slow transport of the mitochondria docked to the axonal framework (low velocity transport [LVT]). LVT was distinct from previously described transport processes; it was coupled with stretching of the axonal framework and, surprisingly, was independent of growth cone advance. Fast mitochondrial transport decreased and LVT increased in a proximodistal gradient along the axon, but together they generated a constant mitochondrial flux. These findings suggest that the viscoelastic stretching/creep of axons caused by tension exerted by the growth cone, with or without advance, is seen as LVT that is followed by compensatory intercalated addition of new mitochondria by fast axonal transport.

When video microscopy was first used to study fast axonal transport in isolated axoplasm from squid giant axons, a torrent of membrane traffic was seen to move in both directions. Images of membrane bounded organelles (MBOs) moving along individual microtubules (MTs) in axoplasm opened the way for characterization of the microscopic properties of fast axonal transport and led to the characterization of two molecular motors involved in fast axonal transport. The pharmacology of MBO movement ruled out previously identified molecular motors and a biochemical dissection of fast axonal transport in axoplasm demonstrated the existence of a new class of molecular motors. Subsequently, the polypeptides comprising a new class of molecular motor, kinesin, were discovered initiating a new era in the study of molecular motors and intracellular motility.The effects of ATP analogues on fast axonal transport led to dicovery of kinesin. When the nonhydrolyzable ATP analogue, adenylyl 5′-imidodiphosphate (AMP-PNP), was perfused into isolated axoplasm, all MBOs moving in both anterograde and retrograde directions stopped moving and remained attached to MTs. Unlike the effects of AMP-PNP on myosin and dynein, inhibition by AMP-PNP was rapid even in the presence of equimolar ATP, but was reversed by excess ATP.

Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein pUS9 promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.

Membranous and nonmembranous cargoes are transported along axons in the fast and slow components of axonal transport, respectively. Recent observations on the movement of cytoskeletal polymers in axons suggest that slow axonal transport is generated by fast motors and that the slow rate is due to rapid movements interrupted by prolonged pauses. This supports a unified perspective for fast and slow axonal transport based on rapid movements of diverse cargo structures that differ in the proportion of the time that they spend moving. A Flash feature accompanies this Mini-Review.

ABSTRACT Following intracerebral inoculation, the DA strain of Theiler’s virus sequentially infects neurons in the gray matter and glial cells in the white matter of the spinal cord. It persists in the latter throughout the life of the animal. Several observations suggest that the virus spreads from the gray to the white matter by axonal transport. In contrast, the neurovirulent GDVII strain causes a fatal encephalitis with lytic infection of neurons. It does not infect the white matter of the spinal cord efficiently and does not persist in survivors. The inability of this virus to infect the white matter could be due to a defect in axonal transport. Using footpad inoculations, we showed that the GDVII strain is, in fact, transported in axons. Transport was prevented by sectioning the sciatic nerve. The kinetics of transport and experiments using colchicine suggested that the virus uses microtubule-associated fast axonal transport. Our results show that a cardiovirus can spread by fast axonal transport and suggest that the inability of the GDVII strain to infect the white matter is not due to a defect in axonal transport.

Abstract Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Huntington's Disease (HD) is an autosomal dominant, neurodegenerative disease that occurs when an expansion of the polyQ tract of the huntingtin gene expands to greater than ~35 residues. This mutation leads to aggregation of the Huntingtin protein (Htt) and degeneration of striatal and cortex neurons, ultimately causing motor impairment and personality changes. Neither the mechanism by which mutant Htt causes toxicity, nor the endogenous function of wild-type Htt, are well understood. To explore mechanisms of mutant Htt-induced toxicity, we generated and characterized a Drosophila model of HD by expressing a 588 amino acid N-terminal fragment of human Htt with 138 glutamines, and tagged with mRFP (Q138Htt-RFP). We used this model to conduct a screen for genes that modify cytoplasmic aggregation and/or toxicity phenotypes. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Htt expression and aggregation, and those that rescue viability independent of effects on Htt aggregation, suggesting that aggregation and toxicity can be separated. To evaluate the putative function of Htt in fast axonal transport, we characterized the co-localization of the Drosophila Htt homolog tagged with mRFP (dHtt-RFP), and the alterations in axonal transport kinetics associated with a dhtt null. We find that dHtt co-localizes with a subset of cargos including synaptic vesicles and mitochondria, and acts locally on these cargos to increase transport processivity. Finally, we evaluated the effects of Q138Htt-RFP expression on transport kinetics. We find that the majority of transport cargos bypass Q138Htt aggregates, indicating they are not complete blockages of axonal transport. We also observe reduced mitochondrial transport in the absence of aggregates, suggesting aggregate-independent transport defects. Our observations of transport in vivo support a role for wild-type Htt in mediating fast axonal transport of membrane bound organelles, and suggest that mutant Htt can cause aggregation-dependent and -independent defects in axonal transport.
by Kurt R. Weiss.
Ph.D.

De plus en plus de preuves montrent que le transport axonal rapide (FAT) joue un rôle crucial au cours des maladies neurodégénératives (NDs). La maladie de Huntington est une maladie neurodégénérative causée par une expansion anormale de polyglutamines dans la partie Nterminale de la protéine huntingtine (HTT) : une grande protéine d’échafaudage impliquée dans la régulation du transport. La présence de HTT mutante comme l’absence de la HTT induisent des défauts de transport chez les mammifères. Chez la Drosophile, la HTT mutante reproduit le phénotype observée chez les mammifères, cependant la fonction conservée de la HTT chez la Drosophile melanogaster (DmHTT) n’est pas encore clairement établie. Ici nous mettons en évidence que DmHTT s’associe aux vésicules, aux microtubules et intéragit avec la proteine dynéine. Dans les neurones corticaux de rat, DmHTT remplace partiellement la HTT de mammifère dans le transport axonal rapide, et les drosophiles invalidées pour la HTT montrent des défauts de transport axonal in vivo. Ces résultats suggèrent que la fonction de la HTT est conservée dans le modèle Drosophile.Le FAT est un processus qui requiert un apport constant d’énergie. Les mitochondries sont les principales sources de production d’ATP de la cellule. Cependant nous avons démontré que le FAT ne dépend non pas de cette source d’énergie là, contrairement à ce que l’on pensait, mais de l’ATP glycolytique produit par les vésicules. La dérégulation de GAPDH ou de PK, les deux enzymes glycolytiques productrices d’ATP, ralentit le transport vésiculaire. Néanmoins, l’invalidation de GAPDH n’affecte pas le transport mitochondrial. En outre, toutes les enzymes glycolytiques sont associées à des vésicules dynamiques et sont capables de produire leur propre ATP. Enfin nous montrons que l’ATP produit est suffisant pour assurer leur propre transport, prouvant l’autonomie énergétique des vésicules pour le transport
Growing evidence support the idea that impairments in Fast Axonal Transport (FAT) play a crucial role in Neurodegenerative Diseases (NDs). Huntington’s Disease is neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-Terminal part of huntingtin (HTT), a large scaffold protein implicated in transport regulation. Both the presence of the mutated HTT as the loss of HTT leads to transport defects in mammals. In the fruit fly overexpression of the mutant HTT recapitulates the phenotype observed in mammals. However, it is still unclear whether HTT’s function is conserved in D. melanogaster. Here, we show that D. melanogaster HTT (DmHTT) associates with vesicles, microtubules, and interacts with dynein. In rat cortical neurons, DmHTT partially replaces mammalian HTT in fast axonal transport, and DmHTT KO flies show axonal transport defects in vivo. These results suggest that HTT function in transport is conserved in D. melanogaster.FAT is a process that requires a constant supply of energy. Mitochondria are the main producers of ATP in the cell. However, we have demonstrated that FAT does not depend on this source of energy, as previously thought, but it depends on glycolytic ATP produced on vesicles. Perturbing GAPDH or PK, the two ATP generating glycolytic enzymes, slows down vesicular transport. However, knocking down GAPDH does not affect mitochondrial transport. Furthermore, all of the glycolytic enzymes are associated with dynamic vesicles, and are capable of producing their own ATP. Finally, we show that this ATP production is sufficient to sustain their own transport, demonstrating the energetical autonomy of vesicles for transport

碩士
國立清華大學
分子與細胞生物研究所
104
Various neuronal intermediate filament defects are associated with neurological disorders, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). To study how human diseases develop, scientists frequently employ model organisms, such as zebrafish, fruit flies, and worms. However, whether a neurofilament homolog exists in the nematode Caenorhabditis elegans remains unclear. In this study, we investigated a candidate gene, temporarily assigned gene-63 (tag-63), which we hypothesized to be a homolog to human neurofilament heavy polypeptide (nefh) according to WormBase BLASTP. We established a stable transgenic worm line expressing the tag-63 transcriptional reporter and found that tag-63 expresses in some neurons showing partial co-occurrence with the pan-neuronal reporter UNC-104. Additionally, we sequenced and outcrossed the deletion mutant strain VC275 tag-63(ok471) and separately crossed it with the translational reporters UNC-104/KIF1A (kinesin-3 motors) and SNB-1/VAMP1 (synaptobrevin-1 synaptic vesicles). We verified the crossing through genotyping PCR and Western blotting and examined anterograde motor UNC-104 and corresponding cargo SNB-1 fast axonal transport dynamics in the mechanosensory neuron ALM by using time-lapse imaging in live worms. Intriguingly, we found that worms carrying the TAG-63 mutation significantly activate the retrograde machinery and deactivate the anterograde machinery of UNC-104 and SNB-1 by acting on differential transport parameters. Moreover, the TAG-63 mutation causes similar motility schemes between UNC-104 and SNB-1 in anterograde but not retrograde transport directions. Based on these results, we propose a novel model that neurofilament homolog TAG-63 affecting fast axonal transport in C. elegans via transient interactions between TAG-63 and the motor-cargo complex.

博士
國立清華大學
分子與細胞生物研究所
106
Abstract I Newly synthesized synaptic proteins in the cell body are transported down the axonal processes to their respective destinations by microtubule-dependent motors. UNC-104, a homologue of KIF1A in C. elegans is required for the transport of synaptic vesicle precursors. Its C-terminal PH domain can specifically and directly bind to acidic phospholipids (PI (4,5)P2 ) located on the vesicular membrane and associate the motor to the cargo. However, we hypothesize that the classical linkage of UNC-104 to the cargo via its PH domain is weak owing to protein-lipid interaction and may not be sufficient for motor-cargo binding and recognition. Interestingly, it has been shown that RIM/UNC-10 reveals a binding site for Liprin-α/SYD-2 at the C-terminus, while its N-terminus binds to Rab3/RAB-3. As it has been shown that SYD-2 is a functional adaptor for UNC-104 and RAB-3 is known to be a synaptic-vesicle specific, membrane-bound protein, we propose the presence of an additional linker RAB-3/UNC-10/SYD-2 that could enhance the motor-cargo connectivity. To prove the existence of this proposed linker, we performed real-time PCR analysis in the first place indicating an increase of SYD-2 expression in unc-10 mutants while its expression in rab-3 mutants is significantly reduced. Further, from Co-IP assays, we identified that SYD-2’s association to UNC-104 was strongly reduced in both unc-10 and rab-3 mutants. Also, the UNC-104 cluster area in sublateral as well as ALM neurons was severely affected in these (unc-10 and rab-3) mutants. SYD-2 and UNC-104 colocalize with each other in C. elegans neurons. However, the colocalization between SYD-2 and UNC-104 largely diminished in unc-10 and rab-3 mutants. Similarly, the distribution patterns of bimolecular fluorescence complementation assay (BiFC) signals revealing UNC-104 and SYD-2 interactions were also reduced in unc-10 and rab-3 mutants respectively. Alongside, UNC-104 motor motility was 5 significantly affected in all the three active zone protein (syd-2, unc-10 and rab-3) mutants. In addition, we also observed the motility pattern of RAB-3-containing vesicles demonstrating that SYD-2/UNC-10 combination are essential for RAB-3 transport but not for SNB-1. In the same way, the distance travelled by RAB-3 particles from axonal hillock to distal segments of ALM neuron was affected in both syd-2 and unc-10 mutants but SNB-1 travel was affected only in syd-2 mutant background. In another approach, assuming that an additional linker between RAB-3 and UNC-104 exists, deletion of the motor’s PH domain may affect the colocalization between UNC-104 and RAB-3 to a lesser extent as compared to SNB-1 and UNC-104. Indeed, the colocalization between SNB-1 and UNC-104 was largely affected when deleting the PH domain, while RAB-3 and UNC-104 colocalization remained unaffected. Consistently with our model, in syd-2 and unc-10 mutant animals, UNC-104 (with a deleted PH domain) and RAB-3 colocalization was strongly diminished. RAB-3 and SNB-1 also exhibited interactions in BiFC assays and this complex further colocalizes with UNC-104 in somas. From these results, we may indeed conclude that UNC-10/RAB-3 complex is required to additionally strengthen the association of SYD-2 with UNC-104, further enhancing the connectivity of motor to its cargo. Abstract II A plethora of human diseases are based on cilia dysfunctions including polycystic kidney disease, Bardet-Biedl and Meckel-Gruber syndrome. Fortunately, basic mechanisms underlying cilia development and intraflagellar transport (IFT) became more understandable in recent years. Though at the same time a complex ciliary machinery was unraveled leading to new questions, specifically, how IFT cargo assembles at the cilia base, how it localizes to cilia, and how “IFT trains” are regulated. One intriguing recent finding describes the ciliary regulating function of two C. elegans kinases DYF-5 and DYF-18, and we hypothesized that even more kinases and phosphatases may be uncovered. We therefore employed data mining tools to identify kinases and phosphatases specifically expressing in C. elegans ciliated sensory neurons. We then used a broad range of methods such as dye-filling, chemotaxis, IFT component expression pattern etc. to investigate the effects of selected kinases and phosphatases from a candidate screen on ciliogenesis and IFT, and have identified PKG-1 as well as GCK-2 as potential candidates significantly affecting cilia development as well as intraflagellar cargo transport. In pkg-1 mutants, severe accumulation of homodimeric kinesin-2 OSM-3 at the cilia tip was observed in conjunction with an overall reduction in retrograde speeds of ciliary dynein XBX-1, leading to abnormal cilia morphology, likely as a function of reduced tubulin acetylation. While in gck-2 mutants OSM-3 and IFT-particle A (CHE-11) motility was significantly elevated in conjunction with increased tubulin acetylation, KAP-1 motility was decreased, confirming a recent model in which the slow KAP-1 motor restricts the motility of the fast OSM-3 motor. Crucially, all observed effects in mutant animals can be rescued by overexpressing the respective protein PKG-1 or GCK-2 (under the cilia specific Posm-5 promoter). Both, PKG-1 and GCK-2 follow similar expression pattern in cilia and also display 75 movements owing to their colocalization with other investigated IFT machinery components. Because PKG-1 is related to cGMP pathways, we also studied the effect of upstream effectors DAF-11 and ODR-1, respectively, leading to similar observations compared with the pkg-1 mutants. Vice versa, since GCK-2 is related to the mTOR pathway, we used rapamycin to inhibit this pathway leading to similar effects as seen in gck-2 mutants. Importantly, we also manipulated the histone deacetylases HDA-4 and SIR-2.1 to understand their role in regulating cGMP and mTOR pathways. In summary, we identified and characterized the distinct functions of two novel kinases affecting ciliogenesis and IFT in C. elegans. Abstract III Various neurological diseases bearing defects in neurofilaments (NF’s) are known including Parkinson’s disease, Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth disease and Tauopathies. Though a critical role of NF’s has been ascertained in neuropathological diseases, little is known about how these diseases develop on the molecular level, critical to facilitate future drug design. Model organisms, such as Zebrafish, Drosophila and C.elegans, are employed to study and understand how these diseases develop. However, whether a neurofilament (NF) homolog exist in the nematode still remains unclear. Therefore, the major goal of this study was to identify and characterize a putative NF-like protein in C. elegans. Using bioinformatics tools, we identified TAG-63 with numerous sequence homologies to NEFH (BLAST e-value of 5E-09), three coiled coils as well as various phosphorylation sites. We then employed a broad range of techniques such as Western blotting, transmission electron microscopy (TEM), worm imaging, motility analysis etc. to delineate the role of tag-63 in C.elegans. To identify NF homologs, using KEGG database, we received hits for NEFH orthologs from various animals. Using this bioinformatics tool, we also note a cluster of NEFH orthologs in a rooted phylogenetic tree emerging from a common TAG-63 ancestor. Though we cannot detect KSP repeats in TAG-63, we identified nine potential phosphorylation sites using Scansite tool. Furthermore, the coiled coil prediction tool identified three potential coiled coils in TAG-63. To understand if tag-63 is expressed in neuronal tissues, we generated a transcriptional tag-63 reporter that expresses in a broad range of body, head and tail neurons. Most importantly, TAG-63 also exhibits features of NF-L such as molecular weight of around 70 kDa, lack of KSP 134 repeats and the ability to form filamentous structures when viewed under transmission electron microscopy. Moreover, as it has been reported that NF’s affect axonal transport, we investigated the effect of tag-63 knockout on synaptic vesicle transport. We found that anterograde transport of UNC-104 is significantly reduced in tag-63 mutant animals pointing to a motor-activating role of TAG-63. Though we do not reveal similar effects on UNC-104’s cargo SNB-1, we did measure increased retrograde movements of this cargo. Further, velocity and flux of UNC-104(KIF1A) are largely diminished in tag-63 knockout worms. In addition, fluorescence intensity of SNB-1 increases in the cell body while near the synapse of HSN neuron it decreases in tag-63 mutant background. In summary, we identified and characterized a NF-like protein in C.elegans, and also demonstrate that lack of this protein limits axonal transport efficiencies, suggesting that this model organism may be used for studying neurofilament-based neurological diseases.

In the previous chapters, we studied the spread of the membrane potential in passive or active neuronal structures and the interaction among two or more synaptic inputs. We have yet to give a full account of ionic channels, the elementary units underlying all of this dizzying variety of electrical signaling both within and between neurons. Ionic channels are individual proteins anchored within the bilipid membrane of neurons, glia, or other cells, and can be thought of as water-filled macromolecular pores that are permeable to particular ions. They can be exquisitely voltage sensitive, as the fast sodium channel responsible for the sodium spike in the squid giant axon, or they can be relatively independent of voltage but dependent on the binding of some neurotransmitter, as is the case for most synaptic receptors, such as the acetylcholine receptor at the vertebrate neuromuscular junction or the AMPA and GABA synaptic receptors mediating excitation and inhibition in the central nervous system. Ionic channels are ubiquitous and provide the substratum for all biophysical phenomena underlying information processing, including mediating synaptic transmission, determining the membrane voltage, supporting action potential initiation and propagation, and, ultimately, linking changes in the membrane potential to effective output, such as the secretion of a neurotransmitter or hormone or the contraction of a muscle fiber. Individual ionic channels are amazingly specific. A typical potassium channel can distinguish a K+ ion with a 1.33 Å radius from a Na+ ion of 0.95 Å radius, selecting the former over the latter by a factor of 10,000. This single protein can do this selection at a rate of up to 100 million ions each second (Doyle et al, 1998). At the time of Hodgkin and Huxley’s seminal study in the early 1950s, two broad classes of transport mechanisms were competing as plausible ways for carrying ionic fluxes across the membrane: carrier molecules and pores. At the time, no direct evidence for either one existed. It was not until the early 1970s that the fast ACh synaptic receptor and the Na channel were chemically isolated and purified and identified as proteins.

Many neurodegenerative diseases, such as Alzheimer’s disease, are linked to swellings occurring in long arms of neurons. Many scientists believe that these swellings result from traffic jams caused by the failure of intracellular machinery responsible for fast axonal transport; such traffic jam can plug an axon and prevent the sufficient amount of organelles to be delivered toward the synapse of the axon. Mechanistic explanation of the formation of traffic jams in axons induced by overexpression of tau protein is based on the hypothesis that the traffic jam is caused not by the failure of molecular motors to transport organelles along individual microtubules but rather by the disruption of the microtubule system in an axon, by the formation of a swirl of disoriented microtubules at a certain location in the axon. This paper investigates whether a microtubule swirl itself, without introducing into the model microtubule discontinuities in the traffic jam region, is capable of capturing the traffic jam formation. The answer to this question can provide important insight into the mechanics of the formation of traffic jams in axons.

This paper simulates effects of structural changes in the microtubule (MT) system on mass transfer in an axon. Understanding this process is important for understanding the underlying reasons for many neurodegenerative diseases, such as Alzheimer’s disease. In particular, it is investigated how the degree of polar mismatching in an MT swirl affects organelle trap regions in the axon and inhibiting transport of organelles down the axon. The model is based on modified Smith-Simmons equations governing molecular-motor-assisted transport in neurons. It is established that the structure that develops as a result of a region with disoriented MTs (the MT swirl) consists of two organelle traps, the trap to the left of the swirl region accumulates plus-end oriented organelles and the trap to the right of this region accumulates minus-end oriented organelles. The presence of such a structure is shown to decrease the transport of organelles toward the synapse of the axon. Four cases with a different degree of polar mismatching in the swirl region are investigated; the results are compared with simulations for a healthy axon, in which case organelle traps are absent.