3 November 2009. The long axons of neurons act as intracellular highways, with motor proteins shuttling their cargo up and down microtubule tracks. Block that traffic—by any number of ways—and the result is often feeble, dying neurons. The impairment of fast axonal transport (FAT) in a variety of neurodegenerative diseases was the theme at a mini-symposium held Sunday, 18 October 2009, at the Society for Neuroscience annual meeting in Chicago, Illinois (reviewed in Morfini et al., 2009).
“These diseases…share several common characteristics,” said Gerardo Morfini, who co-chaired the session with Gustavo Pigino. Both work at the University of Illinois in Chicago. A frequent pattern, Morfini said, is that defects in axonal transport and synapse function lead to a “dying back” axonal pathology, loss of connectivity between neurons, and, much later on, neuronal cell death.
Researchers recapped the impairment of FAT in models of Parkinson disease (see ARF related news story on Morfini et al., 2007), Alzheimer’s (see ARF related news story on Pigino et al., 2009), and hereditary spastic paraplegia (Edgar et al., 2004). They discussed new studies as well. For example, Daryl Bosco of the University of Massachusetts in Worcester presented data showing that two proteins associated with amyotrophic lateral sclerosis (ALS)—superoxide dismutase 1 (SOD1) and Fused in Sarcoma (FUS)—inhibit FAT. Skip Binder of the Northwestern University Medical School in Chicago shared results on a phosphorylation site that regulates tau’s interference in axonal trafficking. And, in a separate session on Huntington disease held October 20, Sarah Pollema of the University of Illinois at Chicago showed which part of polyglutamine-expanded huntingtin interferes with transport. (Hint: It’s not where you might think.)
For their experiments, the scientists depended on North Atlantic squid (Loligo pealii), netted off the coast of the Marine Biological Laboratory in Woods Hole, Massachusetts, so researchers could harvest their giant axons. “This animal seems to have been created by nature for neuroscientists,” quipped Morfini in a presentation last month at the André-Delambre Foundation Symposium on ALS in Québec City. Their giant axons are half a millimeter in diameter, and researchers can extrude the axoplasm “like a sausage,” Morfini said, onto a microscope slide. They can then watch molecular motors cart material up and down the microtubules, and perfuse proteins and drugs to see if they affect transport.
SOD1 and FUS: Each Blocks Transport in Its Own Way
Axonal transport has long been a topic of interest in ALS. Mutations in dynein cause motor neuron degeneration in mice (see ARF related news story on Hafezparast et al., 2003). And in a recent genomewide association study, researchers found an allele of kinesin-associated protein 3 (KIFAP3) that lengthened survival time among people with the disease (see ARF related news story on Landers et al., 2009).
Bosco, Morfini, and colleagues added SOD1 protein—mutations to the SOD1 gene are the most common cause of inherited ALS—to squid axoplasm. Wild-type protein had no effect, but G93A mutant SOD1 inhibited anterograde transport. Retrograde transport proceeded unimpeded. The same was true for other ALS-linked SOD1 mutants H46R, A4V, and G85R. To explore the mechanism by which SOD1 slowed transport, the researchers infused the squid axoplasm with various kinase inhibitors in addition to the mutant protein. They found that inhibiting p38 MAP kinase restored normal transport in the presence of mutant SOD1. To the authors, the data suggest that mutant SOD1 activates p38, which is known to phosphorylate kinesin, knocking the motor off the microtubules.
Mutant SOD1 is implicated in only 2 percent of ALS cases; other inherited mutations likely account for a further 8 percent, with the remaining instances currently thought to be sporadic. However, some scientists suspect wild-type SOD1 of involvement in motor neuron pathology in sporadic ALS, too, as mutations in the DNA sequence are not the only way to compromise a protein. Bosco suggested that the protein’s structure could be modified in various ways in disease. The protein normally functions as a dimer, with an intramolecular disulfide bond and zinc and copper cofactors—but any of those characteristics could change in disease, she said. Altered wild-type SOD1 might be just as bad for motor neurons as the mutant forms.
Bosco hypothesized that antibodies raised to mutant SOD1 (Urushitani et al., 2007) might also interact with wild-type protein in people with sporadic ALS. Among CNS tissue samples from 10 people who died of sporadic ALS, she found that four stained positive with the mutant SOD1 antibodies. Four did not and a further two had no evident motor neurons to examine. The researchers are currently using mass spectrometry to discover which SOD1 modifications are present in the immunoreactive samples.
That evidence led Bosco to wonder if modified, wild-type SOD1 would also impede axonal trafficking as the mutants did. Sure enough, purified protein from the immunoreactive patient samples did slow FAT in the squid axoplasm.
Earlier this year, researchers linked a new gene to familial ALS. FUS is involved in RNA transcription, splicing, and transport (see ARF related news story on Kwiatkowski et al., 2009 and Vance et al., 2009). When Bosco and colleagues added mutant FUS protein to squid axoplasm, they saw that both anterograde and retrograde transport slowed down. This contrasted with the effects of SOD1, which were solely on anterograde trafficking. The data suggest that FUS’s effects on axonal transport may be mediated by a different mechanism than SOD1’s.
Tau: Presenting PAD
It has been known for some time that tau filaments inhibit anterograde FAT. In previous work, Binder and colleagues discovered that deleting the amino terminus of tau—amino acids 2 through 18—prevented its interference with axonal transport (Lapointe et al., 2009). At the symposium, Binder reported on further research, led by former graduate student Nichole LaPointe, who is now at the University of California-Santa Barbara; Nick Kanaan, currently a post-doc in Binder’s lab; and Morfini. Kanaan wondered if the 2-18 region of tau required the rest of the protein, as well, to inhibit transport. Accordingly, he synthesized a peptide with only those amino acids—and found that this amino-terminal region alone impeded FAT.
Like SOD1, tau exerts its effects on FAT via phosphorylation of the motors. Previously, the researchers found that inhibitors of glycogen synthase kinase-3 (GSK3) and protein phosphatase 1 (PP1) prevent tau from slowing transport. PP1 dephosphorylates GSK3, activating it to dephosphorylate kinesin, detaching the motor from its cargo. The amino terminus of tau corresponds to a consensus sequence for PP1 binding, and the researchers christened amino acids 2 through 18 the Phosphatase Activation Domain (PAD). They do not yet know if this domain directly interacts with PP1 or activates it indirectly, perhaps through an enzymatic cascade.
The PAD contains a phosphorylation site at tyrosine 18, and Kanaan suspected the presence or absence of this phosphate would affect axonal transport. He engineered a mutant with glutamate at position 18 to mimic phosphorylation, and found that the pseudophosphorylated protein did not inhibit FAT. Nor did purified, phosphorylated wild-type tau filaments. Therefore, Kanaan concluded, the PAD’s effect on transport is mediated by phosphorylation at tyrosine 18, and the unphosphorylated form is the one that blocks FAT, presumably through some interaction with PP1.
Binder suspects that in a healthy brain, the PAD is tucked away inside the tau protein, unable to interfere with transport. But when tau is altered in disease, the PAD may stick out. “Anything that presents the PAD region to the cell should inhibit anterograde transport,” Binder said.
Huntingtin: It’s the Ps, Not the Qs
Morfini and colleagues previously showed that poly-glutamine expanded huntingtin, as well, interferes with anterograde transport: It activates cJun N-terminal kinase 3 (JNK3) to phosphorylate kinesin, uncoupling the motor from its tracks (see ARF related news story on Morfini et al., 2009). Pollema, a graduate student in Morfini’s and Brady’s labs, shared her work on which part of huntingtin mediates this effect.
Disease-causing huntingtin harbors an excess of glutamine repeats. Pollema showed that the first exon of the polyQ-expanded protein, containing those repeats, was sufficient to inhibit transport. Yet right next to those glutamines, and also in exon 1, lies a string of prolines. Further along the sequence is a second proline-rich domain, or PRD. To determine which part of the exon slowed axonal traffic, Pollema infused squid axoplasm with exon 1, along with antibodies to block either the glutamate or proline sequences. She found that only the proline antibody prevented the inhibition, indicating that the PRDs, not the polyglutamine repeats themselves, were the culprits. Further confirming the results, she showed that short polyproline peptides were sufficient to inhibit transport.
In conclusion, Morfini wrote in an e-mail to ARF that it might someday be possible to correct axonal transport defects with drugs that modify kinase activity. Several such pharmaceuticals are making their way through clinical trials for a variety of cancers. “Correcting fast axonal transport deficits in neurodegenerative disease by modulating kinase activities appears a promising avenue of research,” Morfini wrote.—Amber Dance.