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Motor neurons rely on motor proteins
Review
TRENDS in Cell Biology
Vol.14 No.5 May 2004
Motor neurons rely on motor proteins Erika L.F. Holzbaur University of Pennsylvania, D400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA
The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot–Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington’s disease and Alzheimer’s disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases. The recent renaissance in the microscopic imaging of living cells has revealed what cell biologists have long suspected – the inside of a cell is a very dynamic environment. Molecular motor proteins are continuously shuttling vesicles and organelles along the microtubules and actin filaments that make up the cellular cytoskeleton. Nowhere is this transport more essential than in the neuron. These are highly polarized cells with axonal extensions that can reach up to a meter in length. Although axons can represent . 99% of the volume of a cell, protein and lipid synthesis occur almost exclusively in the cell body, and active transport is, therefore, required to supply the axon with newly synthesized material. The microtubule motor kinesin, and additional kinesin-related motor proteins, drive this anterograde transport. In return, material targeted for degradation is actively returned to the cell body by the microtubule motor cytoplasmic dynein (Box 1). Kinesin and cytoplasmic dynein were both initially characterized in brain extracts [1,2]. Although they are now known to be essential for the normal function of many cell types, it is clear that microtubule motors have specialized, crucial roles in neurons. Recent progress in the analysis of the effects of mutations in motor proteins on the nervous system has provided a new appreciation of the importance of active transport in the neuron, and has implicated slowing of this transport in a growing array of neurodegenerative diseases. Here, I highlight recent developments linking defects in microtubule motor function to developmental and degenerative disorders of the nervous system.
Although progress has been rapid, it is also clear that a more thorough understanding of neuronal cell biology will be required to develop rational interventions in degenerative diseases, diseases that together affect millions worldwide.
Box 1. Microtubule motor proteins Kinesin Kinesin is a microtubule motor that uses the energy of ATP hydrolysis to move along microtubules. Microtubules are polarized polymers, with a plus (fast-growing) end and a minus (slow-growing) end. Conventional kinesin, also known as kinesin I, moves unidirectionally towards the plus end of the microtubule. Kinesin I is a heterodimer composed of two heavy chains (kif5A, kif5B or kif5C) and two light chains (KLC1 or KLC2). The motor domain that binds to and hydrolyzes ATP, as well as binding in a nucleotide-dependent manner to microtubules, is localized to the N terminus of the kinesin heavy chain. The C terminus of the heavy chain as well as the kinesin light chains mediate binding of the motor to its vesicular cargo [60].
Kinesin superfamily Members of the kinesin superfamily share homology with the , 340 amino acid motor domain of conventional kinesin. 45 distinct members of this superfamily, known as KIFs, have been identified in the human genome [7]. Although many of the KIFs are microtubule motor proteins that move cargo towards microtubule plus ends (anterograde transport), other KIFs have been shown to move towards microtubule minus ends. Furthermore, some kinesin superfamily members have been shown to destabilize microtubules, affecting cytoskeletal dynamics rather than transport.
Dyneins Dynein was first identified as the motor protein responsible for the motility of eukaryotic cilia and flagella, for example tracheal cilia and sperm-tail flagella. The cytoplasmic form of dynein is similar in structure to the flagellar enzyme and also uses ATP hydrolysis to generate force toward microtubule minus ends. Both the cytoplasmic and flagellar enzymes are large multisubunit complexes; cytoplasmic dynein is composed of two heavy chains, each . 500 kDa, with consensus AAA domains, as well as intermediate, light intermediate and light chains. Cytoplasmic dynein is the major motor for retrograde axonal transport in neurons but also is required for a wide range of cellular functions, including endoplasmicreticulum-to-Golgi vesicular transport, neurofilament transport, mRNA localization and mitotic spindle assembly.
Dynactin Dynactin is a dynein-activator complex required for most of the cellular functions of cytoplasmic dynein. Dynactin acts both to increase the efficiency (processivity) of the motor and to couple the motor to vesicular cargos. Dynactin is composed of seven to nine polypeptides, including the p150Glued polypeptide that forms the sidearm of the complex and binds both to microtubules and to dynein; the Arp1 polypeptide that forms an actin-like filament at the base of the complex; and the dynamitin, or p50, subunit that localizes to the shoulder between side-arm and base.
Corresponding author: Erika L.F. Holzbaur ([email protected]). www.sciencedirect.com 0962-8924/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.03.009
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Microtubule motors are key during development Kinesins Initial observations in invertebrate models clearly showed the crucial role of kinesin during the development of the nervous system [3]. Although major defects in the organization of the neuromuscular system are not observed in Drosophila expressing mutations in kinesin heavy chain, at the cellular level axons develop marked swellings packed with vesicles, synaptic membrane proteins and mitochondria. Dystrophic neuromuscular junctions were also observed [4]. Mouse knockouts of two of the three genes encoding isoforms of kinesin I (conventional kinesin) result in embryonic lethal phenotypes [5,6]. Developmental defects have also been observed to result from mutations in other members of the kinesin superfamily, known as KIFs. There are more than 45 proteins in the human genome that include a domain homologous to the kinesin motor domain [7], and many of these proteins are also motors with essential functions in neurons. For example, the protein known as Unc104 in Caenorhabditis elegans and KIF1A in mice is required for the transport of synaptic vesicle precursors [8,9]. In the mouse, loss of KIF1A leads to a dramatic loss of synaptic vesicle density, resulting in both motor and sensory phenotypes, and early death [9]. Although KIF1A transports some synaptic vesicle proteins (such as synaptotagmin, synaptophysin and Rab3A), it does not appear to transport other synaptic vesicle proteins (such as SV2), indicating that there is specificity in anterograde transport – specific cargos are transported by specific motors [10]. Thus, kinesin-like proteins in the nervous system show functional specification, and mutations and knockouts have revealed specific roles. Dyneins By contrast, cytoplasmic dynein has multiple crucial roles in the cell, both during division and during interphase. The dynein-activator complex dynactin is also required for most of these dynein-dependent cellular functions. Therefore, inhibition of either dynein or dynactin function leads to an embryonic lethal phenotype in both invertebrates and mice [11,12]. Detailed studies have been performed on a Drosophila mutant, Gl 1, which expresses a truncated form of the largest polypeptide in the dynactin complex. This subunit, Glued in Drosophila and p150Glued in mammalian cells, binds to both cytoplasmic dynein and microtubules [13,14], and increases the efficiency of dynein-mediated transport [15]. Flies expressing the Gl 1 mutation have a dominant negative phenotype with both anatomical and functional disruption of the nervous system, including defects in axonal pathfinding during development [16,17]. Similarly, defects in axon projections result from mutations in a dynein light chain [18]. In Drosophila, dynein and dynactin are not required for normal axon growth but are required for the processes of terminal branching and arborization, synaptogenesis, and synapse stabilization, as well as axonal transport [19– 21]. Both dynein and the dynein-associated protein Lis1 have also been linked to defects in neuronal development in higher eukaryotes. Mice homozygous for subtle missense mutations in cytoplasmic dynein heavy chain www.sciencedirect.com
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demonstrate defects in neuronal migration, branching and arborization, and die within 1 day after birth [22]. Defects in neuronal migration are also seen in the human developmental disorder lissencephaly (smooth brain), which is caused by mutations in the gene Lis1. Elegant studies in filamentous fungi have shown that the Lis1 homolog is in the same pathway as cytoplasmic dynein [23]. By analogy, both cytoplasmic dynein and Lis1 might be required for the migration of neurons to the cortex during embryogenesis [24]. Interdependency of microtubule motors Axonal transport is bidirectional, and accumulating evidence suggests that anterograde and retrograde motors function in an interdependent manner. Antibodies to either kinesin or dynactin result in a bidirectional inhibition of transport of vesicular motility in axoplasm in vitro [25 –27]. In Drosophila, dominant genetic interactions have been noted among kinesin, cytoplasmic dynein and dynactin [28]. We have observed a direct biochemical interaction between kinesin and cytoplasmic dynein that would allow for coordination of bidirectional transport [29]. A similar direct link has been observed between another member of the kinesin superfamily, kinesin II, and dynactin [30]. This coordination of oppositely directed motor complexes is clearest in the axonal transport of neurofilaments. The net movement of neurofilaments from the neuronal cell body out along the axon occurs significantly more slowly than the movement of membranous vesicles via fast anterograde transport. The mechanism driving slow axonal transport was mysterious until Wang et al. [31] succeeded in directly imaging the movement of fluorescently labeled neurofilaments in cultured neurons. The net slow anterograde movement results from rapid movements in either the anterograde or retrograde directions, interrupted by pauses in which the neurofilament remains stationary. Recent work suggests that the motors kinesin (KIF5A [6]) and dynein [32] both interact with neurofilaments, and the net slow movement might result from a tug-of-war mechanism between two oppositely directed motors. Transport defects linked to motor neuron degeneration Although it is not surprising that microtubule motor proteins are required for the complex cytoskeletal changes occurring as neurons differentiate, migrate, extend growth cones and establish appropriate synaptic connections during development, the ongoing function of mature neurons is also dependent on active transport. This transport might be essential to maintain the health of all neurons but becomes particularly key in cells with extended axons such as those of the peripheral nervous system. These neurons might be uniquely vulnerable to defects or slowing in axonal transport, resulting in progressive neurodegenerative disease. The first evidence to support this hypothesis came from an analysis of the degenerative Charcot– Marie-Tooth disease, a heritable peripheral neuropathy with multiple genetic determinants. Patients develop progressive weakness and atrophy of distal muscles. Type 1 forms of this
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TRENDS in Cell Biology
Kinesin
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Figure 1. Microtubule motors drive axonal transport in neurons. Neurons rely on anterograde transport, powered by kinesin and proteins of the kinesin superfamily (KIF), to supply newly synthesized material to the axon and presynapatic terminal. Conventional kinesin (KIF5) is a heterodimer of two heavy chains and two light chains. Defects in anterograde transport have been shown to cause Charcot– Marie-Tooth disease type 2A, hereditary spastic paraplegia type 10 and congenital fibrosis of the extraocular muscles type 1 [34,35,37]. Retrograde axonal transport, driven by cytoplasmic dynein and dynactin, returns material targeted for degradation back to the cell body. Neurotrophic factors and their receptors are also actively transported from synapse to cell body by dynein and dynactin. Cytoplasmic dynein and dynactin are both large multisubunit complexes that interact to produce effective motility. Mutations in dynein and dynactin result in motor neuron disease in mice and humans, respectively [22,43].
disease result from defects in myelination, leading to myelinopathy. Type 2 forms result from molecular defects in the neuron, leading to axonal degeneration [33]. Hirokawa and colleagues have found that patients with Charcot – Marie-Tooth disease type 2A (CMT2A) have a loss-of-function mutation in the motor domain of the neuronal kinesin KIF1B [34] (Figure 1). KIF1B transports synaptic vesicle precursors along the axon. KIF1B knockout mice die shortly after birth, with multiple neurological defects, including alterations in the number and morphology of brain neurons (Figure 2b). More informative was the analysis of heterozygous kif1B þ/2 mice, which showed significant defects in the transport of synaptic vesicle precursor proteins by 2 months after birth, and progressive muscle weakness and motor discoordination by 1 year, indicating that haploinsufficiency of KIF1B leads to peripheral neuropathy (Figure 2a). This observation in the mouse is consistent with the observation that, in humans, the Q98L point mutation in the ATP binding site of the motor causes an autosomal dominant form of CMT2A [34]. A mutation in conventional kinesin has also been linked to human disease (Figure 1). Reid et al. [35] identified a N256S missense mutation in the KIF5A gene, a conventional kinesin gene expressed in neurons (Figure 1). This mutation causes autosomal dominant hereditary spastic paraplegia type 10 (SPG10) in a large family. This mutation occurs at an invariant asparagine residue in the motor domain of the protein, and similar mutations in other kinesins have been shown to inhibit motor activity. Patients with the mutation develop axonal degeneration of both motor and sensory neurons. KIF21A is an another neuronally expressed member of www.sciencedirect.com
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the kinesin superfamily, and a probable plus-end-directed motor involved in anterograde axonal transport [36]. Somewhat surprisingly, whereas KIF21A is expressed throughout the nervous system in mice [36], mutations in human KIF21A appear exclusively to affect the development and/or function of the oculomotor nerve [37]. The autosomal dominant strabismus disorder congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is due to heterozygous missense mutations in KIF21A [37]. Six different mutations have been identified in unrelated patient families. Interestingly, these mutations map to a mutational hot spot that encodes part of the heptad repeat sequence in the coiled-coil stalk of the motor protein, and are hypothesized to disrupt the dimerization of the motor. Although kinesin and other KIFs supply the axon and presynaptic terminal with new material, cytoplasmic dynein drives transport back to the cell body. This retrograde transport returns material to the cell body for degradation and recycling but is also required for the transport of neurotrophic factors necessary to the survival and proper functioning of the neuron [38]. These factors appear to require active transport to appropriately stimulate signal transduction pathways [39]. This transport might be mediated by a direct interaction between cytoplasmic dynein and neurotrophic factor receptors [40]. Therefore, neurons might be particularly susceptible to defects in retrograde transport. Cytoplasmic dynein has multiple essential roles in the cell, so the hypothesis that active dynein-mediated retrograde transport is required for the continued health of motor neurons was initially difficult to test. In both Drosophila and mouse, null mutations in cytoplasmic dynein are lethal early in development [11,12]. To specifically test the role of dynein/dynactin in motor neurons, LaMonte et al. [41] generated transgenic mice that overexpress the dynamitin subunit of dynactin in postnatal motor neurons. Previous cellular studies have shown that overexpression of dynamitin disrupts the integrity of the dynactin complex, and effectively blocks dynein-mediated functions [42]. The resulting mice overexpressing dynamitin developed late-onset progressive motor neuron degeneration and muscle atrophy [41] (Figure 2e,f). Although this study demonstrated that disruption of dynein/dynactin is sufficient to cause motor neuron disease in a mouse model, it did not address the issue of cellular susceptibility, because transgene expression was targeted to motor neurons. However, recent work from Hafezparast et al. [22] has shown that motor neurons are uniquely sensitive to disruptions in dynein function. Mice heterozygous for either of two N-ethyl-N-nitrosourea (ENU)-generated mutations, Loa and Cra1, demonstrate a motor neuron degenerative phenotype similar to that described above [41]. Positional cloning revealed that both mutations are in the cytoplasmic dynein heavy chain gene. Neither mutation results in a significant change in the expression levels or localization of dynein. Instead, each of the mutations appears to inhibit dynein function more subtly, perhaps reducing the integrity of the dynein molecule, because they both map to the dynein heavy chain dimerization domain. Although the mutant heavy
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Figure 2. Mouse models of neurodegenerative disease. (a) KIF1B heterozygote knockout mice display a phenotype of chronic peripheral neuropathy, characterized by progressive muscle weakness, a staggering gait and motor discoordination [34]. (b) Homozygous KIF1B-knockout mice have lethal neuronal defects at birth. Cultured primary hippocampal neurons from the kif1B 2/2 mouse show delays in differentiation and increased neuronal cell death [34]. (c) KIF5A conditional null (Kif5A null/KIF5A flox; Cre synapsin) mice show abnormal hind limb posture and gait (bottom) compared with controls (top) [6]. (d) Progressive degeneration of sensory, but not motor, axons is observed in KIF5A conditional null (Kif5A null/KIF5A flox;Cre synapsin) mice compared with littermate control mice [6]. The figure shows cross-sections of dorsal roots of threeweek- and five-and-a-half-month-old control and mutant mice. (e) Transgenic (Tg) mice overexpressing the dynamitin subunit of dynactin [Tg (dynamitin)] display progressive hind end weakness and abnormal gaits [41]. (f) Progressive degeneration of motor neurons in the ventral root is observed in Tg (dynamitin) mice compared with wild-type littermate control mice [41]. The figure shows cross-sections from the L5 ventral roots of 10– 14-month-old wild-type and transgenic mice. (g) Mice heterozygous for either the Loa or the Cra1 mutation in cytoplasmic dynein heavy chain display progressive locomotor disorders as well as abnormal twisting of the body and clenching of the hindlimbs when suspended by their tails [22]. (h) Both the Loa heterozygous (Loa/þ) and homozygous (Loa/Loa) mice exhibit reduced nerve branching in developing hindlimbs compared with wild-type mice. Nerve branching was visualized in hind limbs of mice at embryonic day 13.5 with an anti-neurofilament antibody. Scale bars: 20 mm, (b,d); 200 mm, (h). Images in (a,b) reprinted, with permission, from Ref. [34]. Images in (c,d) reprinted, by copyright permission of The Rockefeller University Press, from Ref. [6]. Images in (e,f) reprinted, with permission, from Ref. [41]. Image in (g) courtesy of Gabriele Stumm and Elizabeth Fisher. Image in (h) reprinted, with permission, from Ref. [22].
chains are ubiquitously expressed, the observed phenotype appears to be specific to motor neurons (Figure 2g,h). These observations support the hypothesis that motor neurons are uniquely sensitive to retrograde transport defects. A link between dynein/dynactin and human disease has now been observed. A North American human pedigree with an autosomal dominant form of lower motor neuron disease was found to have a mutation in the p150Glued subunit of dynactin (Figure 1). A G59S mutation in the www.sciencedirect.com
highly conserved CAP-Gly domain of the protein was sufficient to induce late-onset progressive motor neuron degeneration and muscle atrophy [43]. The phenotype of affected patients is milder than motor neuron diseases such as amyotrophic lateral sclerosis (ALS), consistent with the decrease but not complete loss of function for the mutant protein observed in in vitro binding assays. As with the murine Loa and Cra1 mutations, it is likely that a loss of function would be lethal earlier in development. The clinical effects of the dynactin mutation are limited to
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motor neurons, again suggesting that motor neurons are uniquely sensitive to defects in dynein/dynactin function. However, their relative susceptibility might be more complex than a strict dependence on axon length, because muscle weakness was most pronounced in the hands rather than the lower limbs in this family. Multiple factors such as axon length, level of trophic factor support and degree of innervation might all contribute to the severity of motor neuron degeneration observed. So how do these observations relate to more common motor neuron degenerative diseases such as ALS? The cause or causes of most cases of ALS, a progressive and fatal motor neuron disease with a prevalence of 6 in 100 000 worldwide, are not known. About 90% of ALS cases are sporadic, whereas ,10% are familial. 15 –20% of familial ALS cases are due to missense mutations in the gene encoding superoxide dismutase 1 (SOD1). Although more than 100 different mutations in SOD1 have been shown to cause ALS, these mutations seem to cause a toxic gain of function rather than simply a loss of function. An elegant study by Subramaniam et al. [44] has suggested that the toxic gain of function does not involve alterations in SOD1 activity but instead is more likely to be due to the propensity of mutant SOD1 to form toxic aggregates [45], which might be particularly harmful to motor neurons. Transport studies have shown defects in slow axonal transport in mouse models of ALS. The cytoskeletal polymer neurofilaments are moved outward via slow axonal transport and the rate of this outward movement has been shown to slow before the onset of neurodegenerative symptoms in mouse models of ALS [46,47]. The observed accumulation of neurofilaments along the axons of motor neurons observed in these mouse models mirrors the pathology observed in human patients, and has led to the suggestion that neurofilament aggregation might further damage motor neurons by ‘axonal strangulation’ [46] or the nonspecific slowing of transport. However, the observation that the net outward slow transport of neurofilaments is due to the combined action of both fast anterograde (kinesin) and retrograde (dynein) motors discussed above suggests that the observed aggregation of neurofilaments might be a result of, rather than a cause of, defects in the axonal transport machinery. In contrast to ALS, which has an average age of onset of 55, the other major motor neuron disease, spinal muscular atrophy (SMA), is an early-onset disease. SMA is caused by mutations in the SMN gene [48]. In mice, there is a single copy of SMN, which is essential. In humans, a duplication resulted in two tandem copies of the gene, SMN1 and SMN2. SMN1 is fully functional but SMN2 expresses a truncated isoform of the protein that does not function well during development. The SMN protein is required for RNA processing in all
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cell types, so the particular susceptibility of motor neurons to defects in this gene was surprising. However, recent studies have shed new light on the problem and might reveal mechanistic similarities to other motor neuron diseases. SMN is actively transported in neurons [49], where it is associated with b-actin-encoding RNA. Studies on motor neurons isolated from an SMA mouse model demonstrate reductions in axon growth and smaller growth cones. These deficiencies correlate with reduced levels of b-actin-encoding mRNA localized to distal axons and growth cones, suggesting that SMN is required for the proper transport of b-actin message down the axon, leading Rossoll et al. [50] to suggest that cytoskeletal defects might be the primary cellular defect that leads to the loss of motor neurons in SMA. Although this disease is clearly distinct from later onset degenerative diseases, it is intriguing that the pathogenesis might involve defects in axonal transport. Is transport the Achilles’ heal of the neuron? If neurons are particularly dependent on axonal transport then both direct and indirect inhibition of this transport might be either causative or contributory factors to neurodegenerative disease. Although direct inhibition might be due to the types of motor mutations described above and summarized in Table 1, indirect effects might include the slowing of transport by protein aggregates or cytoskeletal disorder. For example, as described above, the accumulation of neurofilaments in the axon of motor neurons is a key pathological marker for ALS and these neurofilament aggregates might clog the axon, leading to neuronal cell death via axonal strangulation. Although motor neurons might be particularly susceptible to inhibition of axonal transport, slowing of transport might also be a contributory factor in other degenerative diseases. Accumulations of an abnormally phosphorylated form of the microtubule associated protein tau into paired helical filaments are a key marker of Alzheimer’s disease. The resulting disruption of the microtubule cytoskeleton might affect transport and, therefore, contribute in part to the neuronal degeneration observed [51]. There might also be a more direct link between microtubule motor proteins and Alzheimer’s disease. A direct interaction between kinesin light chains and the amyloid precursor protein (APP) has been observed [52]. APP is normally processed in a complex pathway in the cell. Abnormal processing leads to the accumulation of an amyloidogenic peptide that is toxic to cells, and the aggregation of this peptide forms the plaques that are a characteristic pathological feature of brains from Alzheimer’s patients. It has been proposed that APP is the kinesin receptor on transport vesicles moving outward from the cell body that also carry b-secretase (BACE) and
Table 1. Mutations in motors or associated proteins linked to human neurodegenerative disease Gene/locus
Polypeptide
Disease
Refs
KIF1B SPG10 FEOM1 DCTN1
KIF1B, anterograde motor KIF5A, anterograde motor KIF21A, presumed anterograde motor p150Glued subunit of the dynein –dynactin retrograde motor complex
Charcot –Marie-Tooth disease type 2A Hereditary spastic paraplegia Congenital fibrosis of the extraocular muscles type 1 Motor neuron disease
[34] [35] [37] [43]
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presenilin-1, and that cleavage of APP releases kinesin from the membrane. The possible role of this interaction in the pathogenesis of Alzheimer’s disease is not yet clear. The progressive degeneration observed in Huntington’s disease (HD) might also be linked to disruptions in motor protein function. HD is caused by the expansion of trinucleotide repeats, resulting in the incorporation of extended glutamine tracts in the protein huntingtin (htt). Like other polyglutamine diseases, expansion of these glutamine tracks has a pathological effect that might be mediated by the developmental of intracellular aggregates. HD is autosomal dominant, suggesting that the extended glutamine sequences cause a toxic gain of function. Somewhat surprisingly, however, the normal cellular role of the htt protein is not well understood. Based on cellular localization studies, htt is proposed to be a vesicular protein and has been shown to be transported in both the anterograde and retrograde directions in axons [53]. Previously, an interaction has been shown between htt-associated protein 1 (HAP1) and dynactin [54,55]. Now, further progress has been made linking htt and axonal transport. In an in vitro assay for axonal transport, a fragment of the htt protein with expanded repeats was found to inhibit vesicular motility, whereas a similar protein fragment lacking the expanded triplet repeats associated with HD did not [56]. Inhibition of both anterograde and retrograde transport was observed. Using Drosophila as a model system, Goldstein and colleagues have shown that expression of a cytoplasmic form of htt with expanded polyglutamine repeats results in retinal degeneration in adults and axonal blockages in larval neurons [57]. Although these data provide a promising new lead on the pathogenesis of HD, they do not address the question of specificity. Although htt protein is widely expressed in brain, HD pathology is confined to striatal neurons. Why are the expanded repeats so toxic to these cells and not to other cell types sensitive to transport defects, such as motor neurons? It is clear that there is much we do not yet understand. Concluding remarks and outstanding questions Progress in understanding the mechanisms that drive axonal transport has been steady since the discovery of the intracellular microtubule motors kinesin and cytoplasmic dynein. These motors clearly play a role in the normal development of the nervous system, affecting neuronal migration, axonal pathfinding and synapse stabilization. However, studies of neurodegenerative diseases have highlighted the role of active axonal transport in maintaining the continued health of neuronal cells with extended projections such as motor neurons. Although the observation that motor neurons degenerate in response to inhibition of axonal transport is consistent with the remarkable length of axons in these cells, the accumulating data on transport defects in other types of neurons suggest that slowing of transport might be a general phenomenon in neurodegenerative disease. In some cases, such as CMT2A, slowing of transport might be causative and, in other cases, it might be contributory. In ALS for instance, there might be a range of different factors that can insult a neuron but these might cause a www.sciencedirect.com
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common degenerative pathway once the bottleneck of transport becomes affected. Because disruption of transport is sufficient to cause motor neuron degeneration, disease progression might then follow a common path. Accumulating data suggest that both anterograde and retrograde transport are crucial. Inhibition of anterograde transport leads to depletion of synaptic proteins at the presynaptic terminal and, thus, to degeneration and muscle atrophy. However, inhibition of retrograde transport has similar effects on motor neuron survival and muscle atrophy, presumably owing to inhibition of neurotrophic factor transport and signaling. Further work is required to reveal whether bulk inhibition is involved or whether it is the disruption in the transport of specific cargo that is key. Many questions remain. In a few cases, the effects on transport are clearly direct, owing to mutations in motor proteins such as kinesin or interacting proteins such as dynactin. In other cases, inhibition of transport might be indirect. For example, the overall regulation of axonal transport might be affected by an upstream disruption of signal transduction pathways that regulate these motors in vivo. Little is known about how microtubule motors are regulated within the neuron. Finally, in some cases there might be a nonspecific physical block in transport, such as impedance of transport owing to the accumulation of neurofilaments or other aggregated proteins. An example of this might be the SOD1 mutations observed in familial ALS, which inhibit axonal transport, but whether the mutant SOD1 inhibits motors directly, acts to perturb motor regulation or inhibits nonspecifically owing to the accumulation of aggregated protein remains to be determined. Another key question is the different cellular sensitivities that are observed. Motor neurons appear to be uniquely sensitive to mutations in cytoplasmic dynein or dynactin, for example. However, mutations in other cytoskeletal proteins appear specifically to inhibit axonal transport in sensory neurons, which might have axons as long as those of motor neurons. For example, loss of the cytoskeletal cross-linking protein BPAG1n4 leads to defects in retrograde transport in sensory but not motor neurons [58]. Although, in general, the mechanisms of axonal transport might be similar in motor and sensory neurons, there appear to be cell-type-specific mechanisms as well. We must also continue to consider alternate hypotheses. For example, a mutation in dynactin is sufficient to cause motor neuron disease. Although the most likely hypothesis is that this disease results from a defect in transport, the observation of synapse destabilization resulting from RNAi inhibition of dynactin in Drosophila [20] suggests that there might be more than one mechanism contributing to the degenerative phenotype. Finally, there clearly might be environmental contributions to transport defects. Several viruses preferentially infect the nervous system and actually highjack microtubule motors to facilitate the course of infection. Does this highjacking in turn affect the efficiency of transport, leading in future to degenerative disease? Although this is a disquieting idea, there might also be some hope in this
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approach. The retrograde viral delivery of insulin-like growth factor 1 was recently shown to prolong survival in a SOD1 mouse model for ALS [59]. IGF was specifically administered to motor neurons using a viral vector that preferentially affects neurons and uses retrograde transport to move rapidly to the cell body. Motor neuron diseases such as ALS are currently incurable but transport might provide some hope on the horizon.
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Acknowledgements I thank Lee Ligon, Kevin K. Pfister, Karen Wallace, Mariko Tokito and David Howland for their intellectual input. I also thank Lee Ligon and the laboratories of Nobutaka Hirokawa, Larry Goldstein, Elizabeth Fisher and Gabriele Strumm for providing figures. My work was supported by a National Institutes of Health grant (GM48661) and the ALS Association.
References 1 Vale, R.D. et al. (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39 – 50 2 Paschal, B.M. and Vallee, R.B. (1987) Retrograde transport by the microtubule-associated protein MAP 1C. Nature 330, 181– 183 3 Saxton, W.M. et al. (1991) Kinesin heavy chain is essential for viability and neuromuscular functions in Drosophila, but mutants show no defects in mitosis. Cell 64, 1093– 1102 4 Hurd, D.D. and Saxton, W.M. (1996) Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila. Genetics 144, 1075 – 1085 5 Tanaka, Y. et al. (1998) Targeted disruption of mouse conventional kinesin heavy chain, Kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93, 1147– 1158 6 Xia, C-H. et al. (2003) Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A. J. Cell Biol. 161, 55– 66 7 Miki, H. et al. (2001) All kinesin superfamily protein, KIF, genes in mouse and human. Proc. Natl. Acad. Sci. U. S. A. 98, 7004– 7011 8 Hall, D.H. and Hedgecock, E.M. (1991) Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell 65, 837 – 847 9 Yonekawa, Y. et al. (1998) Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice. J. Cell Biol. 141, 431– 441 10 Okada, Y. et al. (1995) The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. Cell 81, 769 – 780 11 Gepner, J. et al. (1996) Cytoplasmic dynein function is essential in Drosophila melanogaster. Genetics 142, 865– 878 12 Harada, A. et al. (1998) Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein. J. Cell Biol. 141, 51 – 59 13 Karki, S. and Holzbaur, E.L. (1995) Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J. Biol. Chem. 270, 28806 – 28811 14 Waterman-Storer, C.M. et al. (1995) The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp1). Proc. Natl. Acad. Sci. U. S. A. 92, 1634– 1638 15 King, S.J. and Schroer, T.A. (2000) Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20 – 24 16 Garen, S.H. and Kankel, D.R. (1983) Golgi and genetic mosaic analyses of visual system mutants in Drosophila melanogaster. Dev. Biol. 96, 445 – 466 17 Reddy, S. et al. (1997) Mutant molecular motors disrupt neural circuits in Drosophila. J. Neurobiol. 33, 711 – 723 18 Phillis, R. et al. (1996) Mutations in the 8 kDa dynein light chain gene disrupt sensory axon projections in the Drosophila imaginal CNS. Development 122, 2955– 2963 19 Murphey, R.K. et al. (1999) Dynein – dynactin function and sensory axon growth during Drosophila metamorphosis: a role for retrograde motors. Dev. Biol. 209, 86 – 97 20 Eaton, B.A. et al. (2002) Dynactin is necessary for synapse stabilization. Neuron 34, 729 – 741 21 Bowman, A.B. et al. (1999) Drosophila Roadblock and Chlamydomonas www.sciencedirect.com
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LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis. J. Cell Biol. 146, 165– 179 Hafezparast, M. et al. (2003) Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 300, 808 – 812 Xiang, X. et al. (1995) NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol. Biol. Cell 6, 297– 310 Kato, M. and Dobyns, W.B. (2003) Lissencephaly and the molecular basis of neuronal migration. Hum. Mol. Genet. 12, R89– R96 Brady, S.T. et al. (1990) A monoclonal antibody against kinesin inhibits both anterograde and retrograde fast axonal transport in squid axoplasm. Proc. Natl. Acad. Sci. U. S. A. 87, 1061– 1065 Stenoien, D.L. and Brady, S.T. (1997) Immunochemical analysis of kinesin light chain function. Mol. Biol. Cell 8, 675– 689 Waterman-Storer, C.M. et al. (1997) The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport. Proc. Natl. Acad. Sci. U. S. A. 94, 12180 – 12185 Martin, A.A. et al. (1999) Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport. Mol. Biol. Cell 10, 3717 – 3728 Ligon et al. (2004) A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity. J. Biol. Chem. 10.1074/ jbc.M313472200 (www.jbc.org) Deacon, S.W. et al. (2003) Dynactin is required for bidirectional organelle transport. J. Cell Biol. 160, 297 – 301 Wang, L. et al. (2000) Rapid movement of axonal neurofilaments interrupted by prolonged pauses. Nat. Cell Biol. 2, 137 – 141 Shah, J.V. et al. (2000) Bidirectional translocation of neurofilaments along microtubules mediated in part by dynein/dynactin. Mol. Biol. Cell 11, 3495– 3508 Tanaka, Y. and Hirokawa, N. (2002) Mouse models of Charcot – MarieTooth disease. Trends Genet. 18, S39– S44 Zhao, C. et al. (2001) Charcot – Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bb. Cell 105, 587– 597 Reid, E. et al. (2002) A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10). Am. J. Hum. Genet. 71, 1189– 1194 Marszalek, J.R. et al. (1999) Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B. J. Cell Biol. 145, 469 – 479 Yamada, K. et al. (2003) Heterozygous mutations of the kinesin KIF21A in congenital fibrosis of the extraocular muscles type 1 (CFEOM1). Nat. Genet. 35, 318 – 321 Chao, M.V. (2003) Retrograde transport redux. Neuron 39, 1 – 2 Ye, H. et al. (2003) Evidence in support of signaling endosomes-based retrograde survival of sympathetic neurons. Neuron 39, 57– 68 Yano, H. et al. (2001) Association of Trk neurotrophin receptors with components of the cytoplasmic dynein motor. J. Neurosci. 21 RC125 (1 – 7) LaMonte, B.H. et al. (2002) Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron 34, 715– 727 Echeverri, C.J. et al. (1996) Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132, 617– 633 Puls, I. et al. (2003) Dynactin mutation in motor neuron disease. Nat. Genet. 33, 455– 456 Subramaniam, J.R. et al. (2002) Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading. Nat. Neurosci. 5, 301– 307 Elam, J.S. et al. (2003) Amyloid-like filaments and water-filled nanotubes formed by SOD1 mutant proteins linked to familial ALS. Nat. Struct. Biol. 10, 461 – 467 Williamson, T.L. and Cleveland, D.W. (1999) Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons. Nat. Neurosci. 2, 50 – 56 Zhang, B. et al. (1997) Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation. J. Cell Biol. 139, 1307– 1315 Frugier, T. et al. (2002) The molecular basis of spinal muscular atrophy. Curr. Opin. Genet. Dev. 12, 294– 298
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49 Zhang, H.L. et al. (2003) Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization. J. Neurosci. 23, 6627 – 6637 50 Rossoll, W. et al. (2003) SMN, the spinal muscular atrophydetermining gene product, modulates axon growth and localization of b-actin mRNA in growth cones of motoneurons. J. Cell Biol. 163, 801 – 812 51 Mandelkow, E. et al. (2003) Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses. Neurobiol. Aging 24, 1079 – 1085 52 Kamal, A. et al. (2000) Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I. Neuron 28, 449 – 459 53 Block-Galarza, J. et al. (1997) Fast transport and retrograde movement of huntingtin and HAP 1 in axons. Neuroreport 8, 2247 – 2251 54 Engelender, S. et al. (1997) Huntingtin-associated protein 1 (HAP1)
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interacts with the p150Glued subunit of dynactin. Hum. Mol. Genet. 6, 2205– 2212 Li, S-H. et al. (1998) Interaction of huntingtin-associated protein with dynactin p150Glued. J. Neurosci. 18, 1261 – 1269 Szebenyi, G. et al. (2003) Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport. Neuron 40, 41 – 52 Gunawardena, S. et al. (2003) Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40, 25 – 40 Liu, J-J. et al. (2003) BPAG1n4 is essential for retrograde axonal transport in sensory neurons. J. Cell Biol. 163, 223 – 229 Kaspar, B.K. et al. (2003) Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301, 839 – 842 Vale, R.D. (2003) The molecular motor toolbox for intracellular transport. Cell 112, 467 – 480
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information. www.sciencedirect.com
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Motor neurons rely on motor proteins Erika L.F. Holzbaur University of Pennsylvania, D400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA
The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot–Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington’s disease and Alzheimer’s disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases. The recent renaissance in the microscopic imaging of living cells has revealed what cell biologists have long suspected – the inside of a cell is a very dynamic environment. Molecular motor proteins are continuously shuttling vesicles and organelles along the microtubules and actin filaments that make up the cellular cytoskeleton. Nowhere is this transport more essential than in the neuron. These are highly polarized cells with axonal extensions that can reach up to a meter in length. Although axons can represent . 99% of the volume of a cell, protein and lipid synthesis occur almost exclusively in the cell body, and active transport is, therefore, required to supply the axon with newly synthesized material. The microtubule motor kinesin, and additional kinesin-related motor proteins, drive this anterograde transport. In return, material targeted for degradation is actively returned to the cell body by the microtubule motor cytoplasmic dynein (Box 1). Kinesin and cytoplasmic dynein were both initially characterized in brain extracts [1,2]. Although they are now known to be essential for the normal function of many cell types, it is clear that microtubule motors have specialized, crucial roles in neurons. Recent progress in the analysis of the effects of mutations in motor proteins on the nervous system has provided a new appreciation of the importance of active transport in the neuron, and has implicated slowing of this transport in a growing array of neurodegenerative diseases. Here, I highlight recent developments linking defects in microtubule motor function to developmental and degenerative disorders of the nervous system.
Although progress has been rapid, it is also clear that a more thorough understanding of neuronal cell biology will be required to develop rational interventions in degenerative diseases, diseases that together affect millions worldwide.
Box 1. Microtubule motor proteins Kinesin Kinesin is a microtubule motor that uses the energy of ATP hydrolysis to move along microtubules. Microtubules are polarized polymers, with a plus (fast-growing) end and a minus (slow-growing) end. Conventional kinesin, also known as kinesin I, moves unidirectionally towards the plus end of the microtubule. Kinesin I is a heterodimer composed of two heavy chains (kif5A, kif5B or kif5C) and two light chains (KLC1 or KLC2). The motor domain that binds to and hydrolyzes ATP, as well as binding in a nucleotide-dependent manner to microtubules, is localized to the N terminus of the kinesin heavy chain. The C terminus of the heavy chain as well as the kinesin light chains mediate binding of the motor to its vesicular cargo [60].
Kinesin superfamily Members of the kinesin superfamily share homology with the , 340 amino acid motor domain of conventional kinesin. 45 distinct members of this superfamily, known as KIFs, have been identified in the human genome [7]. Although many of the KIFs are microtubule motor proteins that move cargo towards microtubule plus ends (anterograde transport), other KIFs have been shown to move towards microtubule minus ends. Furthermore, some kinesin superfamily members have been shown to destabilize microtubules, affecting cytoskeletal dynamics rather than transport.
Dyneins Dynein was first identified as the motor protein responsible for the motility of eukaryotic cilia and flagella, for example tracheal cilia and sperm-tail flagella. The cytoplasmic form of dynein is similar in structure to the flagellar enzyme and also uses ATP hydrolysis to generate force toward microtubule minus ends. Both the cytoplasmic and flagellar enzymes are large multisubunit complexes; cytoplasmic dynein is composed of two heavy chains, each . 500 kDa, with consensus AAA domains, as well as intermediate, light intermediate and light chains. Cytoplasmic dynein is the major motor for retrograde axonal transport in neurons but also is required for a wide range of cellular functions, including endoplasmicreticulum-to-Golgi vesicular transport, neurofilament transport, mRNA localization and mitotic spindle assembly.
Dynactin Dynactin is a dynein-activator complex required for most of the cellular functions of cytoplasmic dynein. Dynactin acts both to increase the efficiency (processivity) of the motor and to couple the motor to vesicular cargos. Dynactin is composed of seven to nine polypeptides, including the p150Glued polypeptide that forms the sidearm of the complex and binds both to microtubules and to dynein; the Arp1 polypeptide that forms an actin-like filament at the base of the complex; and the dynamitin, or p50, subunit that localizes to the shoulder between side-arm and base.
Corresponding author: Erika L.F. Holzbaur ([email protected]). www.sciencedirect.com 0962-8924/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.03.009
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Microtubule motors are key during development Kinesins Initial observations in invertebrate models clearly showed the crucial role of kinesin during the development of the nervous system [3]. Although major defects in the organization of the neuromuscular system are not observed in Drosophila expressing mutations in kinesin heavy chain, at the cellular level axons develop marked swellings packed with vesicles, synaptic membrane proteins and mitochondria. Dystrophic neuromuscular junctions were also observed [4]. Mouse knockouts of two of the three genes encoding isoforms of kinesin I (conventional kinesin) result in embryonic lethal phenotypes [5,6]. Developmental defects have also been observed to result from mutations in other members of the kinesin superfamily, known as KIFs. There are more than 45 proteins in the human genome that include a domain homologous to the kinesin motor domain [7], and many of these proteins are also motors with essential functions in neurons. For example, the protein known as Unc104 in Caenorhabditis elegans and KIF1A in mice is required for the transport of synaptic vesicle precursors [8,9]. In the mouse, loss of KIF1A leads to a dramatic loss of synaptic vesicle density, resulting in both motor and sensory phenotypes, and early death [9]. Although KIF1A transports some synaptic vesicle proteins (such as synaptotagmin, synaptophysin and Rab3A), it does not appear to transport other synaptic vesicle proteins (such as SV2), indicating that there is specificity in anterograde transport – specific cargos are transported by specific motors [10]. Thus, kinesin-like proteins in the nervous system show functional specification, and mutations and knockouts have revealed specific roles. Dyneins By contrast, cytoplasmic dynein has multiple crucial roles in the cell, both during division and during interphase. The dynein-activator complex dynactin is also required for most of these dynein-dependent cellular functions. Therefore, inhibition of either dynein or dynactin function leads to an embryonic lethal phenotype in both invertebrates and mice [11,12]. Detailed studies have been performed on a Drosophila mutant, Gl 1, which expresses a truncated form of the largest polypeptide in the dynactin complex. This subunit, Glued in Drosophila and p150Glued in mammalian cells, binds to both cytoplasmic dynein and microtubules [13,14], and increases the efficiency of dynein-mediated transport [15]. Flies expressing the Gl 1 mutation have a dominant negative phenotype with both anatomical and functional disruption of the nervous system, including defects in axonal pathfinding during development [16,17]. Similarly, defects in axon projections result from mutations in a dynein light chain [18]. In Drosophila, dynein and dynactin are not required for normal axon growth but are required for the processes of terminal branching and arborization, synaptogenesis, and synapse stabilization, as well as axonal transport [19– 21]. Both dynein and the dynein-associated protein Lis1 have also been linked to defects in neuronal development in higher eukaryotes. Mice homozygous for subtle missense mutations in cytoplasmic dynein heavy chain www.sciencedirect.com
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demonstrate defects in neuronal migration, branching and arborization, and die within 1 day after birth [22]. Defects in neuronal migration are also seen in the human developmental disorder lissencephaly (smooth brain), which is caused by mutations in the gene Lis1. Elegant studies in filamentous fungi have shown that the Lis1 homolog is in the same pathway as cytoplasmic dynein [23]. By analogy, both cytoplasmic dynein and Lis1 might be required for the migration of neurons to the cortex during embryogenesis [24]. Interdependency of microtubule motors Axonal transport is bidirectional, and accumulating evidence suggests that anterograde and retrograde motors function in an interdependent manner. Antibodies to either kinesin or dynactin result in a bidirectional inhibition of transport of vesicular motility in axoplasm in vitro [25 –27]. In Drosophila, dominant genetic interactions have been noted among kinesin, cytoplasmic dynein and dynactin [28]. We have observed a direct biochemical interaction between kinesin and cytoplasmic dynein that would allow for coordination of bidirectional transport [29]. A similar direct link has been observed between another member of the kinesin superfamily, kinesin II, and dynactin [30]. This coordination of oppositely directed motor complexes is clearest in the axonal transport of neurofilaments. The net movement of neurofilaments from the neuronal cell body out along the axon occurs significantly more slowly than the movement of membranous vesicles via fast anterograde transport. The mechanism driving slow axonal transport was mysterious until Wang et al. [31] succeeded in directly imaging the movement of fluorescently labeled neurofilaments in cultured neurons. The net slow anterograde movement results from rapid movements in either the anterograde or retrograde directions, interrupted by pauses in which the neurofilament remains stationary. Recent work suggests that the motors kinesin (KIF5A [6]) and dynein [32] both interact with neurofilaments, and the net slow movement might result from a tug-of-war mechanism between two oppositely directed motors. Transport defects linked to motor neuron degeneration Although it is not surprising that microtubule motor proteins are required for the complex cytoskeletal changes occurring as neurons differentiate, migrate, extend growth cones and establish appropriate synaptic connections during development, the ongoing function of mature neurons is also dependent on active transport. This transport might be essential to maintain the health of all neurons but becomes particularly key in cells with extended axons such as those of the peripheral nervous system. These neurons might be uniquely vulnerable to defects or slowing in axonal transport, resulting in progressive neurodegenerative disease. The first evidence to support this hypothesis came from an analysis of the degenerative Charcot– Marie-Tooth disease, a heritable peripheral neuropathy with multiple genetic determinants. Patients develop progressive weakness and atrophy of distal muscles. Type 1 forms of this
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Figure 1. Microtubule motors drive axonal transport in neurons. Neurons rely on anterograde transport, powered by kinesin and proteins of the kinesin superfamily (KIF), to supply newly synthesized material to the axon and presynapatic terminal. Conventional kinesin (KIF5) is a heterodimer of two heavy chains and two light chains. Defects in anterograde transport have been shown to cause Charcot– Marie-Tooth disease type 2A, hereditary spastic paraplegia type 10 and congenital fibrosis of the extraocular muscles type 1 [34,35,37]. Retrograde axonal transport, driven by cytoplasmic dynein and dynactin, returns material targeted for degradation back to the cell body. Neurotrophic factors and their receptors are also actively transported from synapse to cell body by dynein and dynactin. Cytoplasmic dynein and dynactin are both large multisubunit complexes that interact to produce effective motility. Mutations in dynein and dynactin result in motor neuron disease in mice and humans, respectively [22,43].
disease result from defects in myelination, leading to myelinopathy. Type 2 forms result from molecular defects in the neuron, leading to axonal degeneration [33]. Hirokawa and colleagues have found that patients with Charcot – Marie-Tooth disease type 2A (CMT2A) have a loss-of-function mutation in the motor domain of the neuronal kinesin KIF1B [34] (Figure 1). KIF1B transports synaptic vesicle precursors along the axon. KIF1B knockout mice die shortly after birth, with multiple neurological defects, including alterations in the number and morphology of brain neurons (Figure 2b). More informative was the analysis of heterozygous kif1B þ/2 mice, which showed significant defects in the transport of synaptic vesicle precursor proteins by 2 months after birth, and progressive muscle weakness and motor discoordination by 1 year, indicating that haploinsufficiency of KIF1B leads to peripheral neuropathy (Figure 2a). This observation in the mouse is consistent with the observation that, in humans, the Q98L point mutation in the ATP binding site of the motor causes an autosomal dominant form of CMT2A [34]. A mutation in conventional kinesin has also been linked to human disease (Figure 1). Reid et al. [35] identified a N256S missense mutation in the KIF5A gene, a conventional kinesin gene expressed in neurons (Figure 1). This mutation causes autosomal dominant hereditary spastic paraplegia type 10 (SPG10) in a large family. This mutation occurs at an invariant asparagine residue in the motor domain of the protein, and similar mutations in other kinesins have been shown to inhibit motor activity. Patients with the mutation develop axonal degeneration of both motor and sensory neurons. KIF21A is an another neuronally expressed member of www.sciencedirect.com
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the kinesin superfamily, and a probable plus-end-directed motor involved in anterograde axonal transport [36]. Somewhat surprisingly, whereas KIF21A is expressed throughout the nervous system in mice [36], mutations in human KIF21A appear exclusively to affect the development and/or function of the oculomotor nerve [37]. The autosomal dominant strabismus disorder congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is due to heterozygous missense mutations in KIF21A [37]. Six different mutations have been identified in unrelated patient families. Interestingly, these mutations map to a mutational hot spot that encodes part of the heptad repeat sequence in the coiled-coil stalk of the motor protein, and are hypothesized to disrupt the dimerization of the motor. Although kinesin and other KIFs supply the axon and presynaptic terminal with new material, cytoplasmic dynein drives transport back to the cell body. This retrograde transport returns material to the cell body for degradation and recycling but is also required for the transport of neurotrophic factors necessary to the survival and proper functioning of the neuron [38]. These factors appear to require active transport to appropriately stimulate signal transduction pathways [39]. This transport might be mediated by a direct interaction between cytoplasmic dynein and neurotrophic factor receptors [40]. Therefore, neurons might be particularly susceptible to defects in retrograde transport. Cytoplasmic dynein has multiple essential roles in the cell, so the hypothesis that active dynein-mediated retrograde transport is required for the continued health of motor neurons was initially difficult to test. In both Drosophila and mouse, null mutations in cytoplasmic dynein are lethal early in development [11,12]. To specifically test the role of dynein/dynactin in motor neurons, LaMonte et al. [41] generated transgenic mice that overexpress the dynamitin subunit of dynactin in postnatal motor neurons. Previous cellular studies have shown that overexpression of dynamitin disrupts the integrity of the dynactin complex, and effectively blocks dynein-mediated functions [42]. The resulting mice overexpressing dynamitin developed late-onset progressive motor neuron degeneration and muscle atrophy [41] (Figure 2e,f). Although this study demonstrated that disruption of dynein/dynactin is sufficient to cause motor neuron disease in a mouse model, it did not address the issue of cellular susceptibility, because transgene expression was targeted to motor neurons. However, recent work from Hafezparast et al. [22] has shown that motor neurons are uniquely sensitive to disruptions in dynein function. Mice heterozygous for either of two N-ethyl-N-nitrosourea (ENU)-generated mutations, Loa and Cra1, demonstrate a motor neuron degenerative phenotype similar to that described above [41]. Positional cloning revealed that both mutations are in the cytoplasmic dynein heavy chain gene. Neither mutation results in a significant change in the expression levels or localization of dynein. Instead, each of the mutations appears to inhibit dynein function more subtly, perhaps reducing the integrity of the dynein molecule, because they both map to the dynein heavy chain dimerization domain. Although the mutant heavy
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Figure 2. Mouse models of neurodegenerative disease. (a) KIF1B heterozygote knockout mice display a phenotype of chronic peripheral neuropathy, characterized by progressive muscle weakness, a staggering gait and motor discoordination [34]. (b) Homozygous KIF1B-knockout mice have lethal neuronal defects at birth. Cultured primary hippocampal neurons from the kif1B 2/2 mouse show delays in differentiation and increased neuronal cell death [34]. (c) KIF5A conditional null (Kif5A null/KIF5A flox; Cre synapsin) mice show abnormal hind limb posture and gait (bottom) compared with controls (top) [6]. (d) Progressive degeneration of sensory, but not motor, axons is observed in KIF5A conditional null (Kif5A null/KIF5A flox;Cre synapsin) mice compared with littermate control mice [6]. The figure shows cross-sections of dorsal roots of threeweek- and five-and-a-half-month-old control and mutant mice. (e) Transgenic (Tg) mice overexpressing the dynamitin subunit of dynactin [Tg (dynamitin)] display progressive hind end weakness and abnormal gaits [41]. (f) Progressive degeneration of motor neurons in the ventral root is observed in Tg (dynamitin) mice compared with wild-type littermate control mice [41]. The figure shows cross-sections from the L5 ventral roots of 10– 14-month-old wild-type and transgenic mice. (g) Mice heterozygous for either the Loa or the Cra1 mutation in cytoplasmic dynein heavy chain display progressive locomotor disorders as well as abnormal twisting of the body and clenching of the hindlimbs when suspended by their tails [22]. (h) Both the Loa heterozygous (Loa/þ) and homozygous (Loa/Loa) mice exhibit reduced nerve branching in developing hindlimbs compared with wild-type mice. Nerve branching was visualized in hind limbs of mice at embryonic day 13.5 with an anti-neurofilament antibody. Scale bars: 20 mm, (b,d); 200 mm, (h). Images in (a,b) reprinted, with permission, from Ref. [34]. Images in (c,d) reprinted, by copyright permission of The Rockefeller University Press, from Ref. [6]. Images in (e,f) reprinted, with permission, from Ref. [41]. Image in (g) courtesy of Gabriele Stumm and Elizabeth Fisher. Image in (h) reprinted, with permission, from Ref. [22].
chains are ubiquitously expressed, the observed phenotype appears to be specific to motor neurons (Figure 2g,h). These observations support the hypothesis that motor neurons are uniquely sensitive to retrograde transport defects. A link between dynein/dynactin and human disease has now been observed. A North American human pedigree with an autosomal dominant form of lower motor neuron disease was found to have a mutation in the p150Glued subunit of dynactin (Figure 1). A G59S mutation in the www.sciencedirect.com
highly conserved CAP-Gly domain of the protein was sufficient to induce late-onset progressive motor neuron degeneration and muscle atrophy [43]. The phenotype of affected patients is milder than motor neuron diseases such as amyotrophic lateral sclerosis (ALS), consistent with the decrease but not complete loss of function for the mutant protein observed in in vitro binding assays. As with the murine Loa and Cra1 mutations, it is likely that a loss of function would be lethal earlier in development. The clinical effects of the dynactin mutation are limited to
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motor neurons, again suggesting that motor neurons are uniquely sensitive to defects in dynein/dynactin function. However, their relative susceptibility might be more complex than a strict dependence on axon length, because muscle weakness was most pronounced in the hands rather than the lower limbs in this family. Multiple factors such as axon length, level of trophic factor support and degree of innervation might all contribute to the severity of motor neuron degeneration observed. So how do these observations relate to more common motor neuron degenerative diseases such as ALS? The cause or causes of most cases of ALS, a progressive and fatal motor neuron disease with a prevalence of 6 in 100 000 worldwide, are not known. About 90% of ALS cases are sporadic, whereas ,10% are familial. 15 –20% of familial ALS cases are due to missense mutations in the gene encoding superoxide dismutase 1 (SOD1). Although more than 100 different mutations in SOD1 have been shown to cause ALS, these mutations seem to cause a toxic gain of function rather than simply a loss of function. An elegant study by Subramaniam et al. [44] has suggested that the toxic gain of function does not involve alterations in SOD1 activity but instead is more likely to be due to the propensity of mutant SOD1 to form toxic aggregates [45], which might be particularly harmful to motor neurons. Transport studies have shown defects in slow axonal transport in mouse models of ALS. The cytoskeletal polymer neurofilaments are moved outward via slow axonal transport and the rate of this outward movement has been shown to slow before the onset of neurodegenerative symptoms in mouse models of ALS [46,47]. The observed accumulation of neurofilaments along the axons of motor neurons observed in these mouse models mirrors the pathology observed in human patients, and has led to the suggestion that neurofilament aggregation might further damage motor neurons by ‘axonal strangulation’ [46] or the nonspecific slowing of transport. However, the observation that the net outward slow transport of neurofilaments is due to the combined action of both fast anterograde (kinesin) and retrograde (dynein) motors discussed above suggests that the observed aggregation of neurofilaments might be a result of, rather than a cause of, defects in the axonal transport machinery. In contrast to ALS, which has an average age of onset of 55, the other major motor neuron disease, spinal muscular atrophy (SMA), is an early-onset disease. SMA is caused by mutations in the SMN gene [48]. In mice, there is a single copy of SMN, which is essential. In humans, a duplication resulted in two tandem copies of the gene, SMN1 and SMN2. SMN1 is fully functional but SMN2 expresses a truncated isoform of the protein that does not function well during development. The SMN protein is required for RNA processing in all
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cell types, so the particular susceptibility of motor neurons to defects in this gene was surprising. However, recent studies have shed new light on the problem and might reveal mechanistic similarities to other motor neuron diseases. SMN is actively transported in neurons [49], where it is associated with b-actin-encoding RNA. Studies on motor neurons isolated from an SMA mouse model demonstrate reductions in axon growth and smaller growth cones. These deficiencies correlate with reduced levels of b-actin-encoding mRNA localized to distal axons and growth cones, suggesting that SMN is required for the proper transport of b-actin message down the axon, leading Rossoll et al. [50] to suggest that cytoskeletal defects might be the primary cellular defect that leads to the loss of motor neurons in SMA. Although this disease is clearly distinct from later onset degenerative diseases, it is intriguing that the pathogenesis might involve defects in axonal transport. Is transport the Achilles’ heal of the neuron? If neurons are particularly dependent on axonal transport then both direct and indirect inhibition of this transport might be either causative or contributory factors to neurodegenerative disease. Although direct inhibition might be due to the types of motor mutations described above and summarized in Table 1, indirect effects might include the slowing of transport by protein aggregates or cytoskeletal disorder. For example, as described above, the accumulation of neurofilaments in the axon of motor neurons is a key pathological marker for ALS and these neurofilament aggregates might clog the axon, leading to neuronal cell death via axonal strangulation. Although motor neurons might be particularly susceptible to inhibition of axonal transport, slowing of transport might also be a contributory factor in other degenerative diseases. Accumulations of an abnormally phosphorylated form of the microtubule associated protein tau into paired helical filaments are a key marker of Alzheimer’s disease. The resulting disruption of the microtubule cytoskeleton might affect transport and, therefore, contribute in part to the neuronal degeneration observed [51]. There might also be a more direct link between microtubule motor proteins and Alzheimer’s disease. A direct interaction between kinesin light chains and the amyloid precursor protein (APP) has been observed [52]. APP is normally processed in a complex pathway in the cell. Abnormal processing leads to the accumulation of an amyloidogenic peptide that is toxic to cells, and the aggregation of this peptide forms the plaques that are a characteristic pathological feature of brains from Alzheimer’s patients. It has been proposed that APP is the kinesin receptor on transport vesicles moving outward from the cell body that also carry b-secretase (BACE) and
Table 1. Mutations in motors or associated proteins linked to human neurodegenerative disease Gene/locus
Polypeptide
Disease
Refs
KIF1B SPG10 FEOM1 DCTN1
KIF1B, anterograde motor KIF5A, anterograde motor KIF21A, presumed anterograde motor p150Glued subunit of the dynein –dynactin retrograde motor complex
Charcot –Marie-Tooth disease type 2A Hereditary spastic paraplegia Congenital fibrosis of the extraocular muscles type 1 Motor neuron disease
[34] [35] [37] [43]
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presenilin-1, and that cleavage of APP releases kinesin from the membrane. The possible role of this interaction in the pathogenesis of Alzheimer’s disease is not yet clear. The progressive degeneration observed in Huntington’s disease (HD) might also be linked to disruptions in motor protein function. HD is caused by the expansion of trinucleotide repeats, resulting in the incorporation of extended glutamine tracts in the protein huntingtin (htt). Like other polyglutamine diseases, expansion of these glutamine tracks has a pathological effect that might be mediated by the developmental of intracellular aggregates. HD is autosomal dominant, suggesting that the extended glutamine sequences cause a toxic gain of function. Somewhat surprisingly, however, the normal cellular role of the htt protein is not well understood. Based on cellular localization studies, htt is proposed to be a vesicular protein and has been shown to be transported in both the anterograde and retrograde directions in axons [53]. Previously, an interaction has been shown between htt-associated protein 1 (HAP1) and dynactin [54,55]. Now, further progress has been made linking htt and axonal transport. In an in vitro assay for axonal transport, a fragment of the htt protein with expanded repeats was found to inhibit vesicular motility, whereas a similar protein fragment lacking the expanded triplet repeats associated with HD did not [56]. Inhibition of both anterograde and retrograde transport was observed. Using Drosophila as a model system, Goldstein and colleagues have shown that expression of a cytoplasmic form of htt with expanded polyglutamine repeats results in retinal degeneration in adults and axonal blockages in larval neurons [57]. Although these data provide a promising new lead on the pathogenesis of HD, they do not address the question of specificity. Although htt protein is widely expressed in brain, HD pathology is confined to striatal neurons. Why are the expanded repeats so toxic to these cells and not to other cell types sensitive to transport defects, such as motor neurons? It is clear that there is much we do not yet understand. Concluding remarks and outstanding questions Progress in understanding the mechanisms that drive axonal transport has been steady since the discovery of the intracellular microtubule motors kinesin and cytoplasmic dynein. These motors clearly play a role in the normal development of the nervous system, affecting neuronal migration, axonal pathfinding and synapse stabilization. However, studies of neurodegenerative diseases have highlighted the role of active axonal transport in maintaining the continued health of neuronal cells with extended projections such as motor neurons. Although the observation that motor neurons degenerate in response to inhibition of axonal transport is consistent with the remarkable length of axons in these cells, the accumulating data on transport defects in other types of neurons suggest that slowing of transport might be a general phenomenon in neurodegenerative disease. In some cases, such as CMT2A, slowing of transport might be causative and, in other cases, it might be contributory. In ALS for instance, there might be a range of different factors that can insult a neuron but these might cause a www.sciencedirect.com
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common degenerative pathway once the bottleneck of transport becomes affected. Because disruption of transport is sufficient to cause motor neuron degeneration, disease progression might then follow a common path. Accumulating data suggest that both anterograde and retrograde transport are crucial. Inhibition of anterograde transport leads to depletion of synaptic proteins at the presynaptic terminal and, thus, to degeneration and muscle atrophy. However, inhibition of retrograde transport has similar effects on motor neuron survival and muscle atrophy, presumably owing to inhibition of neurotrophic factor transport and signaling. Further work is required to reveal whether bulk inhibition is involved or whether it is the disruption in the transport of specific cargo that is key. Many questions remain. In a few cases, the effects on transport are clearly direct, owing to mutations in motor proteins such as kinesin or interacting proteins such as dynactin. In other cases, inhibition of transport might be indirect. For example, the overall regulation of axonal transport might be affected by an upstream disruption of signal transduction pathways that regulate these motors in vivo. Little is known about how microtubule motors are regulated within the neuron. Finally, in some cases there might be a nonspecific physical block in transport, such as impedance of transport owing to the accumulation of neurofilaments or other aggregated proteins. An example of this might be the SOD1 mutations observed in familial ALS, which inhibit axonal transport, but whether the mutant SOD1 inhibits motors directly, acts to perturb motor regulation or inhibits nonspecifically owing to the accumulation of aggregated protein remains to be determined. Another key question is the different cellular sensitivities that are observed. Motor neurons appear to be uniquely sensitive to mutations in cytoplasmic dynein or dynactin, for example. However, mutations in other cytoskeletal proteins appear specifically to inhibit axonal transport in sensory neurons, which might have axons as long as those of motor neurons. For example, loss of the cytoskeletal cross-linking protein BPAG1n4 leads to defects in retrograde transport in sensory but not motor neurons [58]. Although, in general, the mechanisms of axonal transport might be similar in motor and sensory neurons, there appear to be cell-type-specific mechanisms as well. We must also continue to consider alternate hypotheses. For example, a mutation in dynactin is sufficient to cause motor neuron disease. Although the most likely hypothesis is that this disease results from a defect in transport, the observation of synapse destabilization resulting from RNAi inhibition of dynactin in Drosophila [20] suggests that there might be more than one mechanism contributing to the degenerative phenotype. Finally, there clearly might be environmental contributions to transport defects. Several viruses preferentially infect the nervous system and actually highjack microtubule motors to facilitate the course of infection. Does this highjacking in turn affect the efficiency of transport, leading in future to degenerative disease? Although this is a disquieting idea, there might also be some hope in this
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approach. The retrograde viral delivery of insulin-like growth factor 1 was recently shown to prolong survival in a SOD1 mouse model for ALS [59]. IGF was specifically administered to motor neurons using a viral vector that preferentially affects neurons and uses retrograde transport to move rapidly to the cell body. Motor neuron diseases such as ALS are currently incurable but transport might provide some hope on the horizon.
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Acknowledgements I thank Lee Ligon, Kevin K. Pfister, Karen Wallace, Mariko Tokito and David Howland for their intellectual input. I also thank Lee Ligon and the laboratories of Nobutaka Hirokawa, Larry Goldstein, Elizabeth Fisher and Gabriele Strumm for providing figures. My work was supported by a National Institutes of Health grant (GM48661) and the ALS Association.
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interacts with the p150Glued subunit of dynactin. Hum. Mol. Genet. 6, 2205– 2212 Li, S-H. et al. (1998) Interaction of huntingtin-associated protein with dynactin p150Glued. J. Neurosci. 18, 1261 – 1269 Szebenyi, G. et al. (2003) Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport. Neuron 40, 41 – 52 Gunawardena, S. et al. (2003) Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40, 25 – 40 Liu, J-J. et al. (2003) BPAG1n4 is essential for retrograde axonal transport in sensory neurons. J. Cell Biol. 163, 223 – 229 Kaspar, B.K. et al. (2003) Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301, 839 – 842 Vale, R.D. (2003) The molecular motor toolbox for intracellular transport. Cell 112, 467 – 480
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