Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

Abstract

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca²⁺ or Mg²⁺ but not pulses of cAMP. Although hd⁻ cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd⁻ cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein. Genetic evidence in humans and mouse models of Huntington's disease suggests that the disease mutation confers a deleterious gain-of-function on huntingtin that acts through the deregulation of some aspect of the protein's normal function(s). While huntingtin's function is poorly understood, its evolutionary conservation makes investigation of its physiological role in lower organisms an attractive route that has yet to be fully exploited. Therefore, we have used Dictyostelium discoideum to study the consequences of huntingtin (hd) deficiency. Developing Dictyostelium cells chemotax to form a multicellular slug that forms a fruiting body, comprising dormant spores encased above dead stalk cells. We found that hd⁻ cells were hypersensitive to hypoosmotic stress. When starved, hd⁻ cells aggregate by accretion, showed disorganized F-actin, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic signaling was rescued with Ca²⁺ or Mg²⁺ but not pulses of cAMP. Development of hd⁻ mutants produced small fruiting bodies with round, defective spores, and when mixed with wild-type cells they didn't differentiate into spores. Our results are consistent with mammalian studies that show huntingtin is a multifunctional protein involved in many biochemical processes; and, importantly, they establish Dictyostelium as a valuable experimental organism for exploring in biochemical detail huntingtin's normal function(s).