Intracellular protein aggregation is a common theme in neurodegenerative disease. The formation of aggregates is blamed on protein overproduction, a failure of clearance, or both. Score one for the clearance side with new data showing that mutations in the cell motor protein dynein can drive the formation of intracellular protein aggregates by slowing protein degradation. Inhibiting dynein enhances the aggregation and toxicity of poly-Q expanded huntingtin protein in mice and fly models of Huntington disease, according to new work from David C. Rubinsztein at the Cambridge Institute for Medical Research in England and his colleagues at the MRC and Cambridge University. The results, which appeared online on June 26 in Nature Genetics, show that dynein plays an important role in autophagy, the process by which cells collect defective proteins into lysosomes and destroy them. The mutations also slowed clearance of the Parkinson protein α-synuclein, suggesting that dynein-powered autophagy could regulate protein inclusion formation in a number of neurodegenerative diseases, including possibly Alzheimer disease.
Mutations in dynein cause a late-onset motor neuron disease resembling amyotrophic lateral sclerosis (ALS) in mice, a pathology that was attributed to defective dynein-driven retrograde transport of vesicles along microtubules in neurons. But the mice also show intracellular protein aggregates, and Rubinsztein and colleagues hypothesized that dynein mutations might interfere with protein clearance via autophagy, another process thought to involve microtubule-dependent vesicle transport. Rubinsztein’s previous work showed that both pathogenic poly-Q expanded huntingtin (Ravikumar et al., 2002) and mutant α-synuclein (Webb et al., 2003) were cleared by this pathway.
To ask if dynein was involved in the clearance of toxic proteins, co-first authors Brinda Ravikumar and Abraham Acevedo-Arozena first looked at the turnover of huntingtin and α-synuclein in cells. Using cells with inducible expression of a GFP-huntingtin fusion containing 74 Q repeats, or an A53T synuclein mutant, allowed the researchers to switch on protein production, then turn it off and watch the decay as protein was cleared. When they did this, they found that dynein inhibitors or dominant-negative mutants of dynein components, but not kinesin inhibitors, slowed the breakdown of either α-synuclein or huntingtin. The persistence of huntingtin led to increased intracellular aggregation. The effects were specific for mutant, disease-causing proteins, and were not seen with either wild-type huntingtin or synuclein. Elevation of huntingtin aggregates was also associated with nuclear fragmentation and cell death.
Turning to autophagy, the authors found that the effect of dynein inhibition on a specific autophagosome marker, LC3, showed similar effects to the pharmacological blocking of autophagosome-lysosome fusion. Autophagosomes became larger and the number of autophagosome-lysosome fusion vesicles was cut in half. These results suggested autophagosomes were not migrating and fusing normally with lysosomes in the dynein-deficient cells, which would explain the slower clearance of synuclein and huntingtin.
But is any of this physiologically relevant? Ravikumar et al. showed that dynein mutations enhance the toxicity of huntingtin in two different animal models of HD. In fruit flies, crossing dynein mutants with huntingtin flies expressing a Q120 expansion in exon 1 resulted in enhanced loss of photoreceptor neurons in the eye. In mice, the Ross/Borchelt mouse HD phenotype was also exacerbated by a dynein heavy chain mutation. When combined with the huntingtin mutation, the dynein mutant mice showed a worsening of the neurological symptoms and a shorter lifespan compared to the dynein-intact HD mice. Huntingtin aggregates were already visible in mice by the time they were 10 weeks old, suggesting that the dynein mutation had in fact impaired clearance of the mutant protein. Finally, the mice had increased levels of the LC3 autophagosome marker, just as the researchers had seen in their cell experiments.
The authors conclude that dynein mutations enhance the neurodegenerative phenotype in HD mice primarily via effects on autophagy. Loss of dynein function has other effects, most notably on retrograde transport, and while the investigators cannot rule out a role for these effects, they write that “reduced autophagy is sufficient to explain the enhanced phenotype of the Huntington disease mutation in combination with dynein mutations.”
Autophagy has been independently linked to the clearance of other neurodegenerative disease-linked proteins, including amyloid-β. Forty years ago, Bob Terry showed that lysosomes accumulate in AD (Suzuki and Terry, 1967), and early work from Charlie Glabe’s lab has also linked lysosomes to Aβ clearance. Research on autophagy for clearance of Aβ and other neurotoxic proteins is currently booming (see ARF meeting report on the San Diego protein misfolding conference).
Could autophagy present a new therapeutic possibility for protein aggregation diseases? Rubinsztein has presented intriguing results for rapamycin, an immunosuppressive drug which stimulates autophagy, enhances clearance of mutant huntingtin protein, and improves neurological function in HD mice (see ARF related news story).—Pat McCaffrey
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