|Brett Lauring and Katia Evans from the Department of Pathology.
Ms. Evans will be finishing her Ph.D shortly. Dr. Lauring, M.D./Ph.D., is assistant professor of pathology and a researcher at the Taub Institute for Research in Alzheimer's Disease and the Aging Brain.|
BY RICHARD KESSIN, PH.D.,
ASSOCIATE DEAN FOR GRADUATE AFFAIRS
It is a long way from the body of a neuron in the spinal cord to a synapse in a limb. The nucleus and the cell body have to maintain a distant colonial outpost in the nerve terminal and so constant traffic flows down the neuron and back.
The route is paved (well, not really, but this is a metaphor), and machines lumber along it carrying mitochondria, secretory vesicle precursors, and other supplies needed at the synapse.
The paving is the domain of tubulin, which, as its name implies, forms a largish cable called a microtubule, with one end near the center of the cell (called the minus end) and another (the plus end) extending into the periphery.
This is true in most cells, but in the case of a neuron, the periphery is far away.
The truck, to continue our road metaphor, is a complex of proteins containing dynein, which can literally walk along the tubulin cable, but only in one direction, from the center out.
It is called a molecular motor and it is literally a motor.
Let's leave transporting things from the periphery to the center for another time; that is the responsibility of another motor.
Bound to the dynein is a group of proteins, the dynactin complex, which connects dynein to its cargo — a mitochondrion or a vesicle — which is sped along to the nerve terminal.
Over time, as we all know, roads need repair or rerouting.
So with neurons, which remodel as demands change.
Neurons expend a lot of energy maintaining their axons, most famously regenerating them when they are severed.
Axonopathies of various kinds are known, but the mechanism behind one movement disorder — autosomal dominant hereditary spastic paraplegia, whose major symptom is a defective gait — was not known until recently.
In 1999, Hazen and colleagues discovered that the affected gene coded for an ATPase of a particular kind, but this brought us no closer to knowing how a mutation in this ATPase, which is called Spastin, caused disease.
Enter graduate student Katia Evans and her mentor, Brett P. Lauring.
They knew that members of the large family of ATPases had certain sections in common, but other parts of the protein differed from one family member to the next.
Katia and Brett noticed that Spastin had a sequence of amino acids in this nonconserved domain that had a few close cousins, one of which was called Katanin.
This protein has been known for a decade to play a role in microtubule reorganization during mitosis and to do so by severing microtubules.
To determine whether Spastin had a similar role, they began by purifying the protein and doing basic biochemistry, including measurements of ATPase activity.
Katia asked what mutations are known in the human victims of hereditary spastic paraplegia.
They turned out to be changes of particular amino acids so Katia engineered these mutations into normal Spastin and expressed the constructs in bacteria.
None of them had any ATPase activity in a biochemical assay.
Certain of these amino acid substations result in a protein to which ATP binds but is not hydrolyzed.
These mutant AAA ATPases bind their associated proteins tenaciously.
Using this trick and elegant fluorescence microscopy Katia, Brett, and their colleagues, Edgar Gomes, Steven Reisenweber, and Gregg G. Gundersen, showed that Spastin bound tightly to microtubules.
This result showed that Spastin interacted with the tubulin in microtubules but gave no hint as to what the normal protein was doing to the microtubules.
|ABOVE: The work was considered so significant that the editors of the Journal of Cell Biology put an image contributed by Katia Evans, Brett Lauring, and their colleagues on the cover. Details and references can be found in Evans et al. Journal of Cell Biology, Volume 168, pages 599-606, Feb. 14, 2005.
Next the group asked whether wild-type Spastin would sever microtubules in tissue-culture cells.
To do this they permeabilized cells by adding small amounts of detergent and the inhibitor taxol, which stabilizes the microtubules.
If Spastin is then added it cleaves the tubulin, but only if ATP is also added.
None of the Spastins that were modified to be identical to the disease variants caused severing of the microtubules.
To be absolutely sure, Katia, Brett, and their colleagues purified each of the components — Spastin or Spastin mutants, microtubules, and ATP.
Here again, the normal Spastin plus ATP caused a breakage of the microtubules, but the mutant Spastins did not.
Spastin alone (plus ATP) is sufficient for severing microtubules and requires no other proteins.
The disease phenotype in which axons of some long neurons degenerate after normal central nervous system development suggests that the severing activity is important for normal maintenance.
There is a second severing activity, namely Katanin, but this is apparently not sufficient and it does not compensate for Spastin deficiency.
An increasing number of neurodegenerative disorders are associated with microtubule function.
Mutations in dynein or other motors cause motor neuron degeneration or another form of hereditary spastic paraplegia.
A microtubule-associated protein called Tau is mutated in chromosome 17-linked fronto-temporal dementia and accumulates in the neurofibrillary tangles of Alzheimer's disease.
As a result of the current work, microtubule destabilizing enzymes also are implicated in neurodegeneration.