Dynamitin in Motor Neurons: Dynamite for ALS Research?
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In yesterday’s Neuron, researchers show that disruption of axonal transport systems in transgenic mice leads to a late-onset, slowly progressive motor neuron disease reminiscent of those human motor neuron diseases. A major impediment to developing therapeutics for amyotrophic lateral sclerosis (ALS) and related motoneuron diseases is that the molecular basis for the neuronal damage remains poorly understood. Some familiar cases of ALS are related to mutations in the copper/zinc superoxide dismutase (SOD) gene, but exactly how this affects neurons is still uncertain. One theory holds that mutant SOD restricts transport along neuronal axons. Indeed, scientists have been looking more and more to these essential transport systems-which carry proteins as far as several feet from the cell body to the tip of the axon-to find clues to the etiology of ALS. (See ARF news item on axonal transport in AD.)
Erika Holzbaur and colleagues at the University of Pennsylvania in Philadelphia and Wyeth Research in Princeton, New Jersey, overexpressed the protein dynamitin in transgenic mice. Dynamitin throws a spanner in the transport works by disrupting the dynactin protein complex. Dynactin acts like an alternator. Without it, the motor protein dynein, which drives the transport of neurofilaments along axons, cannot function. The researchers engineered the mice to begin expressing dynamitin about two weeks after birth, as functional dynein is essential for development.
The transgenic mice appeared normal until they were about 5 to 9 months old, whereupon they began to show symptoms of motor neuron loss, including awkward gait, poor locomotion, weakness, and atrophy of the skeletal muscles. These symptoms correlated with dynactin disruption and with accumulation of neurofilaments in the axons. Furthermore, mice with late symptomatic signs of neuron disease (>16 months old) had lost about 25 percent of their axons. Those with intermediate-stage symptoms exhibited dramatically poorer retrograde transport from the nerve terminal to the cell body, exactly the type of defect that would be expected if dynein function was compromised.
Meanwhile, in the same issue of Neuron, researchers in Graeme Davis’ lab at University of California, San Francisco, report that in Drosophila, dynactin is also essential for stabilizing synapses at the neuromuscular junction. The making and breaking of these synapses, or synapse remodeling, is known to be essential during development and also later in life, as it facilitates the rewiring that is essential for activity-based learning.
Davis et al. devised an assay to measure retreating synapses based on a protein footprint they left in the muscle. The Discs-large protein, for example, is recruited to the muscle side of the neuromuscular junction; its presence in the absence of neural markers indicates a retracted synapse.
Using this assay, the authors found that several proteins that contribute to the dynactin complex, including Arp-1 and Glued-1, are essential for maintening these synapses. Inhibiting the expression of Arp-1 by RNA interference, or expressing dominant-negative mutants of Glued-1, increased the frequency and extent of synaptic retraction. Not all synapses retract, however, and the cells otherwise seemed healthy. It would be interesting to apply this retraction assay to mammalian models of dynactin dysfunction.—Tom Fagan
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Primary Papers
- LaMonte BH, Wallace KE, Holloway BA, Shelly SS, Ascaño J, Tokito M, Van Winkle T, Howland DS, Holzbaur EL. Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron. 2002 May 30;34(5):715-27. PubMed.
- Eaton BA, Fetter RD, Davis GW. Dynactin is necessary for synapse stabilization. Neuron. 2002 May 30;34(5):729-41. PubMed.
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Response by Erika Holzbaur
The multifunctional nature of the dynein/dynactin pathway in vertebrates, including key roles in mitotic spindle assembly, axonal transport, and organelle positioning, makes it challenging to dissect the specific functional mechanisms involved in each process. The role of dynein and dynactin in transport to the aggresome discussed by Ramesh is yet another function for the motor complex, and clearly may be disrupted in the M21 line. It will certainly be of interest to examine the contribution of the aggresome pathway to the development of neurodegeneration. However, the established role for dynein and dynactin in axonal transport, both fast retrograde transport and the net slow anterograde transport of neurofilaments, suggest that disruption of these processes are key to the observed phenotype. For now, the demonstration of a significant inhibition of retrograde transport, as well as the neurofilament accumulations observed in the transgenic mice, clearly support this interpretation. In future it will be of particular interest to carefully assess the role of aggresome disruption, and synaptic stability, as suggested by the work of Eaton Fetter, and Davis, in the onset of neurodegeneration observed in the dynamitin mice.
The paper by Eaton et al. elegantly demonstrates a role for dynactin in synapse stablization. Disruption of dynactin resulted in significantly increased synapse retraction at neuromuscular junctions during synaptic growth. While this work focused on development, it will clearly be of interest to examine the role of dynactin in maintaining the stability of neuromuscular junctions in adults. Our demonstration that the post-developmental disruption of dynactin in mice leads to motor neuron degeneration and muscle atrophy supports a key role for dynactin in maintaining neuronal health. It will be of particular interest to examine synaptic stability in dynamitin-overexpressing transgenic mice in order to assess the relevance of Eaton et al.'s finding to the pathogenesis of late-onset neurodegenerative diseases such as ALS.
Ohio State University
The paper by LaMonte et al. adds a new twist to the complex history of axonal transport in ALS. The demonstration that disruption of axonal transport can cause ALS-like disease is very interesting.
The authors demonstrate that overexpression of dynamitin, a modulator of cytoplasmic dynein, can induce motor neuron disease in mice. This shares some similarity with the SOD1 mouse model and also many sporadic forms of ALS. The swelling of neuronal processes and presence of spheroids are observed in both mouse and human ALS. However, the disease is also different. The onset and progression of the disease is slower as compared to the more rapid degeneration observed in the SOD1 mouse model. The variable progression coupled with periods of regenerative changes also makes this distinct from the SOD1 model. Additionally, pathological differences between the SOD1 mouse and this model suggest that different mechanisms may be involved.
Dynamitin and cytoplasmic dynein are involved in a variety of cellular process that include ER to Golgi trafficking, axonal transport and also mitotic spindle assembly. Although the involvement of dynein in the motor neuron disease process is demonstrated, it still remains unclear how that leads to neurodegeneration. Defective axonal transport may be one mechanism but other pathways may also be involved.
Intracellular structures called aggregosomes are observed in cells carrying CFTR or PS1. Aggregosome is an intracellular structure associated with the microtubule organization center (MTOC), which is thought to enlarge in response to proteasomal inhibition or the presence of misfolded proteins. Proteasomal subunits, HSPs and other chaperones are recruited to the aggregosome, and it is thought that this cellular structure may play a role in the digestion of misfolded proteins.
In an in-vitro model that mimics protein conformational disease, the transport of aggregated GFP-labeled proteins to aggregosomes was observed. Overexpression of dynamitin prevented aggregosome formation, and small GFP-reactive aggregates were scattered within the cytosol. The effect of dynamitin on protein aggregate sequestration and proteasomal breakdown suggest that dynamitin may be involved in aggregate disposal pathways that are common to protein conformation diseases. Further exploration of this pathway using this model would shed more light on this process.
The interaction between neurofilament and dynamitin also needs to be understood, especially with the surprising life-extending effect of NF-H overexpression in the SOD1 mice. It would be interesting to test if crossing this animal to the SOD1 mice would also extend the life of the SOD1 mice. The history of neurofilament research in ALS suggests that the modulation of axonal transport is complex."
Alzheon
On the Davis paper: The combination of technologies used to identify proteins that play a role in synapse retraction is innovative and powerful. This group has taken advantage of the wealth of information provided by yeast genetics about the identity of genes important in cytoskeletal regulation and budding. This information led to the identification of 74 homologous genes in Drosophila. The protein products of these genes are now being examined for their ability to cause synaptic retraction.
To date this work has led to the identification of Arp-1, a protein component of the dynactin complex. This complex is part of the dynein molecular motor system involved in microtubule-based axonal transport. The Davis group used RNAi to decrease the level of functional Arp-1 expression, and hence to disrupt the dynactin complex. This caused synaptic retraction at presynaptic boutons in the Drosophila NMJ. A mutation in another dynactin protein, Glued1, was found to have similar effects.
This study should serve as a catalyst to highlight the relationship between axonal transport and synapse stability. Decreases in stability are likely to have profound effects on the functional properties of these specialized structures. The work by Davis' group has already provided indications that neurotransmitter release may be compromised at such 'destabilized' synapses. A further possibility is that retrograde transport involving the dynein/dynactin molecular motor may also be compromised. This could, in fact, lead to a failure of neurotrophins to be transported back to the cell body. Although these workers do not directly address this question, assays examining retrograde movement of specific proteins could be developed.
On the Holzbaur paper: A major challenge facing neuroscientists today is to understand the process of neurodegeneration, particularly as it relates to major incurable diseases, such as Alzheimer's, Parkinson's, ALS, and a number of trinucleotide repeat disorders including Huntington's disease, and spinocerebellar ataxias. Very little is known about the mechanistic basis underlying neurodegeneration, so addressing the hypothesis that a disruption of axonal transport can cause neurodegeneration is both timely and significant.
A clue these and other workers followed is that motor neurons in ALS patients have an accumulation of neurofilaments in the cell body and axons. These filaments are synthesized in the cell body and transported along axons by microtubule-based slow axonal transport. Hence, it is possible that defects in axonal transport lead to motor neuron degeneration. To test whether or not disruption of axonal transport can cause motor neuron degeneration as observed in ALS, these workers generated a mouse model to disrupt the dynein/dynactin complex, a particular type of microtubule 'molecular motor'. It is known that overexpression of dynamitin, a protein within this complex, can disrupt the dynactin complex, and inhibit dynein-mediated processes in cells. Hence, the authors generated a transgenic mouse model in which dynamitin is overexpressed.
These mice exhibited evidence of motor neuron degeneration, skeletal muscle atrophy, as well as neurofilament accumulation. The authors conclude that inhibition of transport results in cumulative damage to motor neurons, observed as a swelling of processes, and eventually leads to cell death.
These findings may represent a general model relevant for a number of human diseases in which defects of protein movement between cell body and synaptic terminals is defective. A particular property of neurons is their long axon separating the cell body from synaptic terminal, which requires the processing and transport of proteins from cell body to terminals and back. It may be expected that defects in transport mechanisms will be detrimental to neurons. Even a slight change in efficiency or rate of transport may lead to cumulative effects that could change neurotransmitter release or the rate of retrograde transport of essential molecules such as neurotrophins. Eventually such defects could lead to changes in synaptic efficacy, synapse retraction, and even cell death.
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