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Glutamine Proline-Rich PQE-1 Proteins Protect Caenorhabditis Elegans Neurons from Huntingtin Polyglutamine Neurotoxicity

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Glutamine Proline-Rich PQE-1 Proteins Protect Caenorhabditis Elegans Neurons from Huntingtin Polyglutamine Neurotoxicity Glutamine͞proline-rich PQE-1 proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine neurotoxicity Peter W. Faber*†, Cindy Voisine*, Daphne C. King, Emily A. Bates, and Anne C. Hart‡ Massachusetts General Hospital Cancer Center, 149-7202 13th Street, Charlestown, MA 02129; and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115 Edited by H. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA, and approved October 30, 2002 (received for review September 6, 2002) Huntington’s disease is a progressive neurodegenerative disease polyQ expansion diseases. Several Drosophila and C. elegans model caused by a polyglutamine (polyQ) repeat expansion in the hunting- systems have been established in which expression of expanded tin protein [Huntington’s Disease Collaborative Research Group glutamine tracts in huntingtin and other polyQ disease proteins (1993) Cell 72, 971–983]. To understand the mechanism by which (e.g., ataxin 1 and ataxin 3), lead to neurodegeneration (17–25). We polyQ repeats cause neurodegeneration and cell death, we modeled established a C. elegans model system where expression of the N polyQ neurotoxicity in Caenorhabditis elegans. In our model, expres- terminus of human huntingtin was directed to a limited set of sion of N-terminal fragments of human huntingtin causes polyQ- neurons, including the well characterized glutamatergic ASH sen- dependent degeneration of neurons. We conducted a genetic screen sory neurons using the osm-10 promoter (17, 25–28). In our model to identify proteins that protect neurons from the toxic effects of system, expression of the N terminus of human huntingtin carrying expanded polyQ tracts. Loss of polyQ enhancer-1 (pqe-1) gene func- a polyQ tract consisting of 150 glutamines (Htn-Q150) leads to tion strongly and specifically exacerbates neurodegeneration and cell progressive degeneration, but not death, of the ASH neurons in death, whereas overexpression of a pqe-1 cDNA protects C. elegans aged (8-day-old) animals (17). neurons from the toxic effects of expanded huntingtin fragments. A To identify cellular pathways involved in polyQ neurotoxicity, glutamine͞proline-rich domain, along with a charged domain, is we performed a genetic screen for mutations that enhanced critical for PQE-1 protein function. Analysis of pqe-1 suggests that degeneration of ASH neurons expressing expanded huntingtin proteins exist that specifically protect neurons from the toxic effects fragments. We demonstrate that loss of polyQ enhancer-1 (pqe-1) of expanded polyQ disease proteins. gene function dramatically exacerbates polyQ toxicity in C. elegans neurons, whereas its overexpression promotes neuron t least eight hereditary neurodegenerative disorders, including survival. Overt phenotypes have not been observed in pqe-1 AHuntington’s disease (HD), have been identified in which the mutant animals other than enhancement of polyQ neurotoxicity. disease locus encodes a protein containing an expanded glutamine Alternative splicing of pqe-1 yields multiple nuclear localized tract (1). HD patients carry expanded glutamine repeats in the N PQE-1 proteins that contain a glutamine͞proline-rich domain terminus of huntingtin, a widely expressed protein of unknown and͞or an exonuclease domain. Genetic analysis of polyQ neu- function (2, 3). Although huntingtin is expressed in many cell types, rotoxicity in C. elegans reveals that PQE-1 proteins normally the primary cellular pathology of HD is degeneration of neurons of provide dramatic protection against expanded polyQ tracts. the striatum and cortex, leading to dramatic personality changes and motor dysfunction in early stages of the disease (4, 5). Gen- Materials and Methods erally, the onset of disease symptoms is inversely related to the Assessment of ASH Degeneration and Survival. ASH survival was length of the glutamine expansion. However, additional factors assessed using GFP reporters expressed in ASH. osm-10::GFP NEUROSCIENCE distinct from polyglutamine (polyQ) length influence the age of (pHA#29) expresses GFP in ASH and three other sensory onset. For example, genotypic variation linked to a region encoding neurons (28). The presence of pHA#29 in transgenic animals the GluR6 kainate receptor accounts for Ϸ10% of the variance in caused dye-filling defects in 0–5% of ASH neurons, independent the age of onset (6, 7). of age and͞or genotype. Aging of animals to 8 days was Molecular aspects of HD provide clues toward understanding performed as previously described (17). ASH survival in the the mechanism by which mutant huntingtin causes neurotoxicity. L1͞L2 stage of pqe-1(rt13) animals was assessed by collecting Expanded glutamine tracts in the huntingtin protein alter its eggs for 5 h and incubating for 24 h before scoring; Htn-Q150 physical properties (8, 9). PolyQ-containing N-terminal frag- and GFP were expressed from extrachromosomal arrays by using ments of mutant huntingtin form cytosolic and nuclear aggre- the osm-10 promoter. All animals were cultured at 25°C unless gates in affected tissue (10). Therefore, the expanded glutamine otherwise stated. tract in mutant huntingtin may elicit neurotoxicity by altering interactions of huntingtin with critical cellular constituents. For Genetic Screening and Mapping. dpy-20(e1282); rtEx102 example, in the yeast two-hybrid system, normal huntingtin [osm-10::Htn-Q150, dpy-20(ϩ)] were generated by injection of associates with Hip1, a human homolog of the yeast cytoskeletal pHA#16 and pDPY20 at 65 and 140 ng͞␮l, respectively (17). protein, Sla2 (11–13). Loss of a normal interaction between Hip1 Mutant alleles were isolated as animals with ASH-specific and mutant huntingtin may disrupt cytoskeletal integrity, lead- dye-filling defects among the F2 progeny of rtEx102 animals ing to degeneration. Furthermore, transcription factors are sequestered by mutant huntingtin, interfering with normal gene transcription (14–16). Thus, the mechanisms of pathogenesis of This paper was submitted directly (Track II) to the PNAS office. HD may be triggered by changes or alterations of interactions Abbreviations: HD, Huntington’s disease; polyQ, polyglutamine; DiD, 1,1Ј-dioctadecyl- with normal or abnormal protein partners. 3,3,3Ј,3Ј-tetramethylindodicarbocynanine perchlorate. The molecular and functional similarities between Caenorhab- *P.W.F. and C.V. contributed equally to this work. ditis elegans and Homo sapiens nervous systems suggest that inver- †Present address: Exploratory Biology, Athersys Incorporated, Cleveland, OH 44115-2634. tebrate genetics can be used to address basic questions regarding ‡To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.262544899 PNAS ͉ December 24, 2002 ͉ vol. 99 ͉ no. 26 ͉ 17131–17136 Downloaded by guest on September 29, 2021 Table 1. Progressive enhancement of expanded polyQ toxicity in pqe-1 analyses were done with homozygous pqe-1(rt13) animals pqe-1 mutant animals unless otherwise stated. % defective, % defective, Genetic linkage of pqe-1 to chromosome III was determined Genotype Lines 3 days 8 days using RW7000 (32). dpy-1(e1) pqe-1(rt13) unc-32(e189) and the strain CB4856 were used for recombination mapping by using Htn-Q150 13 0 13ϩ͞Ϫ4 single-nucleotide polymorphisms identified by Wicks et al. (33) pqe-1(rt13) — 00and the C. elegans genome-sequencing project. pqe-1(rt13); Htn-Q2 5 1ϩ͞Ϫ15ϩ͞Ϫ1 pqe-1(rt13); Htn-Q23 5 0 2ϩ͞Ϫ1 Transgenic Strains. In all transgenic strains described, the Htn pqe-1(rt13); Htn-Q95 5 7ϩ͞Ϫ236ϩ͞Ϫ7 fragments were expressed by using the ASH neuron-specific pqe-1(rt13); Htn-Q150 4 73ϩ͞Ϫ588ϩ͞Ϫ4 osm-10 promoter. Htn-Q150 was expressed from rtIs11 in cDNA The genotype and huntingtin transgene are listed in column 1; the number rescue experiments (Table 3). rtIs11 is an integration into of transgenic lines scored is listed in column 2. The huntingtin fragments were chromosome V of the extrachromosomal array rtEx1, which expressed from extrachromosomal arrays by using the osm-10 promoter. The contains pHA#16, pDPY20, and pKP#58 (17). In the over- average percentage of ASH neurons affected (dye-filling defective) in adult expression experiment, Htn-Q150 was expressed from rtIs18 animals is reported (in columns 3 and 4) at 3 and 8 days. More than 150 ASH (Table 4). rtIs18 is an integration into chromosome I of the neurons were scored for each data point. The SEM is listed. extrachromosomal array rtEx362, which contains pHA#16 and elt-2::GFP (30). Cosmids, pqe-1 promoter constructs, or osm-10 promoter constructs (5 ng͞␮l) were coinjected with elt-2::GFP mutagenized with 25 mM ethylmethane sulfonate. Three-day- (30) (Tables 1–3) or rol-6 (34) (Table 4) at 75 ng͞␮l as a marker. old F2 rtEx102 animals were incubated with the vital fluorescent dye 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindodicarbocynanine Immunohistochemistry and Microscopy. Polyclonal antisera were perchlorate (DiD) and examined by fluorescence microscopy for generated in rabbits (Research Genetics, Huntsville, AL) against an ASH-specific dye-filling defects (29). Approximately 29,000 F2 N-terminal peptide VITNNKKKRIDVVTLDEDAPRRVQV and animals were screened. aQ͞P-rich domain (amino acids 96–321) GST fusion protein and pqe-1 alleles were backcrossed at least twice to wild type a C-terminal (amino acids 1546–1670) GST fusion protein. Affin- (N2); rt13 was backcrossed four times. Unmutagenized Htn ity-purified sera recognized the predicted overexpressed transgenes with or without the osm-10::GFP transgene PQE-1B::GFP and PQE-1C::GFP proteins but not PQE-1A::GFP (pHA#29) were reintroduced into pqe-1 animals by using 75 protein in normal animals treated with Bowin’s fixative (35). ng͞␮lofelt-2::GFP (pJM#67) (30) as a marker for backcross- Htn-Q150 was detected by using HP1 (36). PQE-1::GFP fusion ing (Tables 1 and 2). The six alleles of pqe-1 identified are rt13 proteins and Htn-Q150 were expressed in ASH neurons by using (corresponding to Q5613STOP), rt58 (Q6323STOP), rt61 the osm-10 promoter. Images were captured by using OPENLABS (Q5263STOP), rt62 (Q6643STOP), rt64 (M1I), and rt67 software (Improvisions, Coventry, U.K.) and a Zeiss Axioplan.
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