CNS myelination requires cytoplasmic dynein function

Background: Cytoplasmic dynein provides the main motor force for minus‐end‐directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration, and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. Results: We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. Conclusions: In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination. Developmental Dynamics 244:134–145, 2015. © 2014 Wiley Periodicals, Inc.     

Dynein intermediate chains DYCI-1 and WDR-60 have specific functions in Caenorhabditis elegans

Dynein is a microtubule-dependent motor protein required for cell division, retrograde intracellular transport, and intraflagellar transport (IFT). Dynein 1 and dynein 2 serve as molecular motors in the cytoplasm and cilia, respectively. Each dynein consists of multiple subunits. Although the components of dynein 1 and dynein 2 are different and specific in most species, a previous study has suggested that dynein intermediate chain subunit DYCI-1 is shared by both dynein 1 and 2 in Caenorhabditis elegans (C. elegans). Here, we show that C. elegans has two dynein intermediate chains—DYCI-1 and WDR-60—and their functions are different. Mutational analysis showed that dyci-1 is essential for the retrograde axonal transport of synaptic vesicles. In the same mutant allele, IFT is not affected at all. Instead, wdr-60 is essential for IFT. Thus, we suggest that dynein 1 and dynein 2 have specific intermediate chains in C. elegans as in other organisms.     

Axonal protein synthesis and transport

Recent evidence has challenged our ideas about the nature of axonal protein synthesis and transport. Previous metabolic labeling evidence supported the idea that all axonal proteins were synthesized in the cell body and then transported as formed cytoplasmic structures into the axon. Recent evidence suggests that neither the synthesis nor the transport of axonal proteins is that simple. Though most axonal proteins do appear to be synthesized in the neuronal cell body, a small amount of protein appears to be synthesized intra-axonally in some axons. Though small in amount, intra-axonal protein synthesis may be important functionally in some axons. Recent experiments have also begun to identify the presence of a rich array of transport motors in axons, including many members of the kinesin, dynein and myosin families. Progress is being made in identifying which cargoes are being transported by which of these motors. Finally, recent experiments have addressed an old question about whether axoplasmic proteins are transported as filamentous polymers or as soluble components in axons. The answer is that both mechanism can be used in axons. For example, neurofilament protein can move in its particulate or polymeric state, while tubulin can move in its soluble or unpolymerized state.     

Transport and egress of herpes simplex virus in neurons

The mechanisms of axonal transport of the alphaherpesviruses, HSV and pseudorabies virus (PrV), in neuronal axons are of fundamental interest, particularly in comparison with other viruses, and offer potential sites for antiviral intervention or development of gene therapy vectors. These herpesviruses are transported rapidly along microtubules (MTs) in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterogradely in the opposite direction. Retrograde transport follows fusion and deenvelopment of the viral capsid at the axonal membrane followed by loss of most of the tegument proteins and then binding of the capsid via one or more viral proteins (VPs) to the retrograde molecular motor dynein. The HSV capsid protein pUL35 has been shown to bind to the dynein light chain Tctex1 but is likely to be accompanied by additional dynein binding of an inner tegument protein. The mechanism of anterograde transport is much more controversial with different processes being claimed for PrV and HSV: separate transport of HSV capsid/tegument and glycoproteins versus PrV transport as an enveloped virion. The controversy has not been resolved despite application, in several laboratories, of confocal microscopy (CFM), real-time fluorescence with viruses dual labelled on capsid and glycoprotein, electron microscopy in situ and immuno-electron microscopy. Different processes for each virus seem counterintuitive although they are the most divergent in the alphaherpesvirus subfamily. Current hypotheses suggest that unenveloped HSV capsids complete assembly in the axonal growth cones and varicosities, whereas with PrV unenveloped capsids are only found travelling in a retrograde direction.     

Neuronal depolarization modifies motor protein mobility

Active neuronal transport along microtubules participates in the targeting of mRNAs, proteins and organelles to their sites of action. Cytoplasmic dynein represents a minus-end-directed microtubule-dependent motor protein. Due to the polarity of microtubules in axonal and distal dendritic compartments, with microtubule minus-ends pointing toward the inside of the cell, dyneins mainly mediate retrograde transport pathways in neurons. Since dyneins transport synaptic proteins, we asked whether changes in neuronal activity would in general influence dynein transport. KCl-induced depolarization, a condition that mimics the effects of neuronal activity, or pharmacological blockade of neuronal action potentials, respectively, was combined with neuronal live cell imaging, using an autofluorescent dynein intermediate chain fusion (monomeric red fluorescent protein [mRFP]–dynein intermediate chain [DIC]) as a model protein. Notably, we found that induced activity significantly reduced dynein particle mobility, as well as both the total distance and velocity of movements in mouse cultured hippocampal neurons. In contrast, blockade of neuronal action potentials through TTX did not alter any of the parameters analyzed. Neuronal depolarization processes therefore represent candidate mechanisms to regulate intracellular transport of neuronal cargoes.     

Lysosomal proliferation and distal degeneration in motor neurons expressing the G59S mutation in the p150Glued subunit of dynactin

An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.     

Dynein motors transport activated Trks to promote survival of target-dependent neurons

Mutations that alter dynein function are associated with neurodegenerative diseases, but it is not known why defects in dynein-dependent transport impair neuronal survival. Here we show that dynein function in axons is selectively required for the survival of neurons that depend on target-derived neurotrophins. Stimulation of axon terminals with neurotrophins causes internalization of neurotrophin receptors (Trks). Using real-time imaging of fluorescently tagged Trks, we show that dynein is required for rapid transport of internalized, activated receptors from axon terminals to remote cell bodies. When dynein-based transport is inhibited, neurotrophin stimulation of axon terminals does not support survival. These studies indicate that defects in dynein-based transport reduce trafficking of activated Trks and thereby obstruct the prosurvival effect of target-derived trophic factors, leading to degeneration of target-dependent neurons.     

Microtubule motors, phosphorylation and axonal transport of neurofilaments

The recent demonstration that the axonal transport motors kinesin and dynein participate in axonal transport of neurofilaments (NFs), and that the association of NFs with these motors is regulated by phosphorylation provides new insight into several aspects of axonal transport and NF biology. This review juxtaposes older and more recent findings on NF dynamics, and speculates on the organization of axonal NFs as suggested by real-time analyses of NF transport.     

Novel Dynein DYNC1H1 Neck and Motor Domain Mutations Link Distal Spinal Muscular Atrophy and Abnormal Cortical Development

DYNC1H1 encodes the heavy chain of cytoplasmic dynein 1, a motor protein complex implicated in retrograde axonal transport, neuronal migration, and other intracellular motility functions. Mutations in DYNC1H1 have been described in autosomal‐dominant Charcot–Marie–Tooth type 2 and in families with distal spinal muscular atrophy (SMA) predominantly affecting the legs (SMA‐LED). Recently, defects of cytoplasmic dynein 1 were also associated with a form of mental retardation and neuronal migration disorders. Here, we describe two unrelated patients presenting a combined phenotype of congenital motor neuron disease associated with focal areas of cortical malformation. In each patient, we identified a novel de novo mutation in DYNC1H1: c.3581A>G (p.Gln1194Arg) in one case and c.9142G>A (p.Glu3048Lys) in the other. The mutations lie in different domains of the dynein heavy chain, and are deleterious to protein function as indicated by assays for Golgi recovery after nocodazole washout in patient fibroblasts. Our results expand the set of pathological mutations in DYNC1H1, reinforce the role of cytoplasmic dynein in disorders of neuronal migration, and provide evidence for a syndrome including spinal nerve degeneration and brain developmental problems.     

The amount of slow axonal transport is proportional to the radial dimensions of the axon

Summary Axons are fundamentally cylindrical and their geometry is defined by two basic parameters, i.e. diameter and length. The average cross-sectional diameter of an axon is determined primarily by the number and density of cytoskeletal structures (i.e. microtubules and neurofilaments) in the axon. The proteins that constitute these structures are synthesized in the nerve cell body and are conveyed through the axon by slow axonal transport. In particular, slow component a (SCa) supplies all of the axonal neurofilament proteins and most of the microtubule proteins to the axon. To study the relationship between slow axonal transport and axonal diameter, the slowly transported proteins were radiolabelled in rat dorsal root ganglion (DRG) cells. The amount of radiolabelled SCa proteins transported in individual unmyelinated and myelinated DRG axons was measured by the electron microscopic autoradiographic method. We found that the amount of SCa transported in the axons is proportional to axonal cross-sectional area. These results indicate that slow axonal transport of microtubules and neurofilaments is a primary determinant of axonal diameter.     

Fast axonal transport of membrane protein and intra-axonal diffusion of free leucine in a neuron of Aplysia

We injected radioactive leucine into a neuron soma of Aplysia, and found that the leucine and synthesized membrane proteins moved in the axon, but soluble proteins did not. The movement of membrane proteins showed typical characteristics of fast axonal transport, whereas that of leucine could be explained totally due to intra-axonal diffusion by the following observations: the migration profiles of the leucine closely coincided with the theoretical diffusion; the migration continued even when the fast axonal transport of membrane proteins was stopped by local cooling of the axon; and a damming phenomenon, an indication of axonal transport, seen in membrane proteins was not observed when the axon was occluded or when the axonal transport was blocked by the local cooling.     

Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and β-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases.     

Anatomical correlations of intrinsic axon repair after partial optic nerve crush in rats.

About 15% of retinal ganglion cells survive diffuse axonal injury of the optic nerve in adult rats. Following initial blindness, discrimination of visual stimuli in behavioral tests recovers within three weeks. To investigate the mechanisms promoting this functional recovery the axonal transport and the neurofilaments were studied. Intraocularly applied MiniRuby is transported until the place of crush and accumulated in enlarged axon terminals. Three weeks after lesion the anterograde transport of MiniRuby recovers distal to the place of crush. At the same point in time the retrograde transport of surviving retinal ganglion cells is restored which was visualized by horseradish peroxidase injected into the superior colliculus. The heavy neurofilament was stained immunohistochemically and analyzed statistically up to three weeks after optic nerve crush. The stained filaments in the axon fibers of retinal ganglion cells appear wavelike and/or fragmented up to day 8, but first signs of heavy neurofilament restitution in the fibers of the optic nerve are seen at day 12 after axonal injury. Because these results cannot be explained by longlasting axon regeneration, the present results provide convincing evidence for intrinsic axon repair soon after diffuse axonal injury that correlates in time with recovery of vision.     

Selective axonal transport in a single cholinergic axon of Aplysia--role of colchicine-resistant microtubules

Substance-specific selective axonal transport was examined in a single axon by injecting [3H]leucine and [14C]acetylcholine simultaneously into the cell body of a giant cholinergic neuron (R2) in the abdominal ganglion of Aplysia kurodai. The ganglion and attached nerves were cultured for several hours after the injection and the migration of radioactive substances along the axons of the injected neuron was examined. The substances examined were 3H labeled membrane proteins and soluble proteins synthesized in the cell body, 14C labeled bound acetylcholine formed in the cell, injected [3H]leucine and soluble [14C]acetylcholine. Membrane proteins and bound acetylcholine (plus a part of soluble acetylcholine) moved along the axon somatofugally at maximum velocities of 2.4 and 1.7 mm/h, respectively, at 25 degrees C. Soluble proteins, free leucine and most of the soluble acetylcholine did not move by fast axonal transport but diffused inside the axon of the neuron R2 at rates predicted from their expected diffusion constants in the axoplasm [Koike H. and Nagata Y. (1979) J. Physiol. 295, 397-417]. The diffusion kinetics of these substances were analysed and used for determination of true axon length, and to separate axonal transport components from diffusing components. An antimitotic drug, colchicine, selectively suppressed the axonal transport of membrane proteins but not of acetylcholine at 1-5 mM concentration, though it finally blocked the axonal transport of acetylcholine at 20 mM. When 1-5 mM colchicine was separately perfused only to the distal axon of the neuron R2, the migration of membrane proteins was stopped just proximal to the colchicine perfusion zone but acetylcholine migration was not disturbed by the drug. The moving component of acetylcholine was recovered by sucrose density centrifugation from a compartment previously reported as that of vesicular acetylcholine. As a possible mechanism of this selective axonal transport, it is proposed that there are two groups of microtubules: a colchicine-sensitive group of microtubules which may transport membrane proteins, and a colchicine-resistant group which may preferentially transport the transmitter substance acetylcholine at a slower rate.     

A comparison of the density of microtubules in the central and peripheral axonal branches of the pseudounipolar neurons of lizard spinal ganglia

The number and density of microtubules were determined in cross sections of the two branches (central and peripheral) of the bifurcating axon of the pseudounipolar neurons of the lizard thoracic spinal ganglia. In both the central and peripheral branches the average number of microtubules rose, while the microtubular density decreased with an increase in the cross-sectional area of the axonal branch: More precisely, a linear relationship was observed between the logarithm of the microtubular density and the cross-sectional area of the axonal branch. Both the average number of microtubules per cross section of the axonal branch and the microtubular density were found to be significantly lower in the central than in the peripheral branch. Since the amount of material carried by fast transport was found by other authors to be greater in the peripheral than in the central branch, a positive correlation seems to exist between microtubular density and the quantity of material carried by fast transport along the two branches of the axon in pseudounipolar neurons. Such a correlation suggests that microtubules may be somehow involved in the fast transport of material along the axon. The average densities of microtubules were found to be the same comparing two sets of unmyelinated and myelinated central (or peripheral) branches of corresponding size. Therefore, different microtubular densities usually observed in unmyelinated and myelinated axons appear to be correlated with the different size ranges of the two types of axon rather than with the absence or presence of the myelin sheath.     

Kinesin motors and microtubule-based organelle transport in Dictyostelium discoideum

Movement of membrane cargoes and chromosomes is driven by kinesin and dynein motors in most eukaryotic cells. In this review, we describe the known kinesin and dynein genes in Dictyostelium. Dictyostelium primarily utilizes two conventional kinesins, an Unc104/KIF1 kinesin, and cytoplasmic dynein to transport membrane organelles within its cytoplasm. We describe how the biological functions of these motors has been dissected through a combination of biochemical to genetic approaches.     

Fine structure of the Herbst corpuscles in the lingual mucosa of the finch, Lonchura striata.

The ultrastructure of Herbst corpuscles in the lingual mucosa of the finch, Lonchura striata var. domestica, was examined by light and electron microscopy. Numerous Herbst corpuscles were found at the top of connective tissue papillae just beneath the dorsal epithelium. The Herbst corpuscle was composed of an outer capsule, inner core and central axon. The central axon was discoid in shape and immunoreactive for NSE-antiserum. The central axon was surrounded by compactly stacked layers of thin lamellae of lamellar cell processes. Since these lamellae did not completely encircle the axon as seen in cross sections, they displayed a symmetrical longitudinal cleft dividing the inner core into bilateral halves. Numerous axonal spines were seen to extend from the Y-axis of the axolemma into the cleft and occasionally into the cytoplasmic invagination of the lamellar cell body in the inner core. A number of clear and dense-cored vesicles were seen in the axoplasm near the base of axonal spines. Further, the omega-shaped coated invaginations were occasionally found on the axolemma near those places. These findings suggest that the area nearby the axonal spine in the central axon of the Herbst corpuscle is a site active both metabolically and functionally.     

The relation of axonal transport of mitochondria with microtubules and other axoplasmic organelles.

Axonal transport of mitochondria was studied in frog sciatic nerves incubated in agents selected for their known or alleged effect on microtubules or axonal flow. Quantitative data on mitochondria, microtubules, neurofilaments, endoplasmic reticulum, and cross-sectional area of the axon indicate that axonal transport of mitochondria is dependent on microtubules. When more than half of the microtubules are destroyed, the axonal transport of mitochondria is diminished in proportion to the destruction of microtubules. Axonal transport of mitochondria is not related to neurofilaments and endoplasmic reticulum. Changes in the cross-sectional area of axons, even upon reduction to half the normal size, do not noticeably affect mitochondrial transport. Cyanide which blocks oxidative metabolism also blocks axonal transport of mitochondria, but analysis of fine structure indicates that cyanide is destructive to microtubules as well.     

Peripheral axon crush elevates transport of p75NTR in the central projection of sensory neurones of rats

The effect of peripheral axon crush on the axonal transport of the neurotrophin receptors, p75(NTR) and trkA, was studied in dorsal roots of adult rats. Lumbar dorsal roots were crushed for 3-6 h to cause accumulation of p75(NTR) and trkA. Immunohistochemistry showed the presence of the NGF receptors in axons, indicating retrograde and anterograde axonal transport in the dorsal root. Western blots confirmed that the time course of accumulation of p75(NTR) was consistent with fast axonal transport. However, trkA accumulation was too low to indicate significant levels of axonal transport. Sciatic nerve crush induced a 2-fold increase (P<0.05) in the bidirectional axonal transport of p75(NTR) in the dorsal root while trkA transport remained below detectable levels.