20 July 2003. In this week’s Neuron, researchers report that polyglutamine-expanded huntingtin (Htt)-the direct cause of Huntington's disease-exacerbates inositol triphosphate-mediated calcium signaling. The paper's principal. author, Ilya Bezprozvanny, University of Texas Southwestern Medical. Center, Dallas, together with collaborators at the University of British Columbia, Vancouver, discovered the connection when they used yeast two-hybrid screens to identify proteins that bind to rat inositol triphosphate receptor 1 (InsP3R1), the major neuronal. receptor for this intracellular signaling molecule.
First author Tie-Shan Tang found two InsP3R1-binding proteins in rat cDNA libraries, neuronal. cytoskeleton 4.1N, and huntingtin-associated protein 1 (HAP1), which is one of several. proteins shown to bind huntingtin (see ARF related news story). Focusing on the latter, Tang and colleagues used immunoprecipitation experiments to show that InsP3R1 and HAP1 interact in cultured cells and also in lysates from rat cerebellum and cortex. To determine if HAP1 could bind simultaneously to the inositol receptor and huntingtin, the authors used a similar strategy, showing that antibodies to the receptor could precipitate huntingtin in the presence of HAP1. This suggests that the three proteins can interact to form a complex. Perhaps more significantly, Tang and colleagues found that huntingtin containing 82 glutamine repeats was precipitated more readily than one with only 23 repeats, suggesting that glutamine expansion of the protein strengthens the ternary complex.
Taking this observation one step further, Tang and colleagues asked if polyglutamine-expanded huntingtin has any effect on inositol triphosphate signaling. To answer this, the authors tested the activity of the InsP3 receptors embedded in a lipid bilayer. When Tang measured receptor-induced electric currents across the bilayer, he found that InsP3 activation of the receptor was amplified by about 10-fold when a huntingtin variant containing 138 glutamines (Htt-138Q) was present. In contrast, huntingtin containing 15 glutamines had little or no effect on receptor activity. This experiment suggests that expanded Htt and the receptor interact directly, a conclusion that was confirmed by immunoprecipitation experiments. But the ternary complex may also be physiologically relevant, because when Tang added both the Htt-138Q and HAP1 to the bilayer, he measured an even greater activation (13-fold) of the receptor.
But what about medium spiny striatal. neurons (MSNs), the ones most affected by Huntington's disease? Do these protein interactions have any relevance there? Tang and colleagues transfected MSNs with Htt-138Q, then measured InsP3 signaling. InsP3 is usually released by the action of phospholipase C, whereupon the inositol phosphate diffuses to the endoplasmic reticulum and mobilizes intracellular stores of calcium. When Tang and colleagues challenged the Htt-138Q transfected cells with small amounts of an agonist (3,5-dihydroxyphenylglycine or DHPG) to the phospholipase C-linked metabotropic glutamate receptor, they found a significant increase in free intracellular calcium (about 50 percent), whereas the same amount of DHPG elicited a very minor release of calcium in normal. MSN cells.
All told, these experiments seem to link expanded huntingtin with the endoplasmic reticulum and calcium homeostasis. The increased basal levels of calcium measured by Tang et al. in the Htt-138Q-transfected MSN cells may also explain why mitochondria membranes from HD patients are more susceptible to calcium-induced depolarization (see ARF related news story), while in an accompanying preview, Anurag Varshney and Barbara Ehrlich from Yale University propose that expanded Htt is "poised to make the cells more responsive to stimulation by glutamate receptor agonists, eventually leading to neuronal. degeneration and Huntington's disease." Varshney and Ehrlich also conclude that the metabotropic glutamate receptor mGluR5, which is expressed in the spiny neurons, "should be considered a potential drug target for Huntington's disease."-Tom Fagan.
Tang T-S, Tu H, Chan EYW, Maximov A, Wang Z, Wellington CL, Hayden MR, Bezprozvanny I. Huntingtin and huntingtin-associated protein 1 influence neuronal. calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron 2003 July 17;39:227-239. Abstract
Varshney A, Ehrlich BE. Intracellular Ca2+ signalling and human disease: the hunt begins with Huntington's. Neuron 2003 July 17;39:195-199. Abstract