The mutant protein that causes Huntington disease blocks axonal traffic by putting the molecular equivalent of the wheel-locking “boot” on motor proteins, according to a paper published online June 14 in Nature Neuroscience. Principal investigators Gerardo Morfini and Scott Brady, both of the University of Illinois in Chicago, and colleagues teased out the specifics of huntingtin’s attack on kinesin-1, showing that mutant huntingtin activates cJun N-terminal kinase 3 (JNK3) to phosphorylate the motor protein and prevent it from binding to the microtubule tracks along the axon. The work suggests that JNK3, or the upstream kinases that likely activate it, could be drug targets for Huntington disease.
“This mutant protein looks like it tweaks one particular signaling pathway, and then all hell breaks loose,” said Peter Hollenbeck of Purdue University in West Lafayette, Indiana, who was not involved in the current research. The pathway is currently incomplete with the cascade between huntingtin and JNK3 remaining a bit of a black box.
Neurons depend on reliable fast axonal transport to supply the synapses at the end of long axons, making transport a vulnerable point for nerve cells. Scientists already knew that the pathogenic proteins in several neurodegenerative diseases activate kinases to gum up axonal transport. Huntingtin has been repeatedly implicated in vesicular trafficking (for review, see Caviston and Holzbaur, 2009), and Brady’s group and others have shown that in Alzheimer disease, Aβ inhibits transport via casein kinase 2 (see ARF related news story; Pigino et al., 2009; Moreno et al., 2009).
The huntingtin gene causes disease when it contains too many CAG trinucleotide repeats, causing an extra long succession of glutamine (polyQ expansion) in the protein. Morfini and Brady suspected JNK might interact with huntingtin because they had already shown that another polyglutamine-expanded protein, the androgen receptor, causes spinal and bulbar muscular atrophy via JNK’s action on fast axonal transport (see ARF related news story; Morfini et al., 2006). JNK phosphorylates the heavy chain of kinesin, inhibiting microtubule binding.
The researchers found that polyQ-expanded huntingtin (polyQ-Htt) inhibited both retrograde and anterograde transport in isolated squid axons, whereas the wild-type protein did not. When JNK inhibitors were included, the effect of polyQ-expanded huntingtin was blocked, suggesting JNK carries out huntingtin’s dirty work. In mouse neuroblastoma cells, polyQ-Htt transfection increased the levels of phosphorylated, active JNK.
There are three isoforms of JNK, and using inhibitors that affect them differentially, the scientists determined that JNK3 was the most important for huntingtin’s effect on transport. Many kinases come in more than one isoform, and researchers should consider that fact, Brady said: “They’re not interchangeable.” The authors used mass spectrometry to map the residue that JNK3 phosphorylates, serine-176 in the kinesin heavy chain.
As powerful as the squid assay is, the important question is what happens in mammalian neurons, said Erika Holzbaur of the University of Pennsylvania in Philadelphia, who was not part of the current research. Morfini and colleagues transfected cultured hippocampal cells with GFP-tagged kinesin heavy chain constructs that included wild-type serine-176, a glutamate at position 176 (to mimic constant phosphorylation), or an alanine at 176 (an construct that cannot be phosphorylated). To evaluate transport, they observed how much GFP-tagged kinesin reached the axon tips. The S176E mutant allowed approximately 55 percent of kinesin to travel to the tips, while the other constructs permitted approximately 75 percent or more to reach the final destination. However, the error bars were large. “It’s not day and night,” Holzbaur said, and she suggested that JNK3 serine-176 may contribute to kinesin transport, but is an insufficient explanation on its own.
The paper offers a tantalizing explanation as to why Huntington’s affects only the nervous system. Although huntingtin expression is ubiquitous, JNK3 expression is limited to the brain and testes. Brady suspects that huntingtin, and JNK3, are involved in normal regulation of kinesin-based transport, but that the poly-Q expanded huntingtin throws the system off balance. “It’s when you get activation at too high a level, or in the wrong subcellular compartment, that you get into trouble,” Brady said. The researchers are currently working on defining the pathway between huntingtin and JNK3 activation; any member of that cascade could be a potential new target for HD therapies.—Amber Dance
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