Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

Tamara Ratovitski,Marjan Gucek,Haibing Jiang,Ekaterine Chighladze,Elaine Waldron,James D'Ambola,Zhipeng Hou,Yideng Liang,Michelle A Poirier,Ricky R Hirschhorn,Rona Graham,Michael R Hayden,Robert N Cole,Christopher A Ross

doi:10.1074/jbc.m804813200

Abstract

Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.

Highlights

Way and the molecules involved in it may represent potential therapeutic targets for intervention in Huntington disease (HD)
Cleavage of Htt at position 586 by caspase 6 is of particular importance for HD pathogenesis, as alterations of this site in a YAC128 HD mouse model strikingly ameliorate the phenotype [25]
Htt cleavage may play an important role in HD, it is possible that not all Htt proteolytic fragments contribute to toxicity

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Mutagenesis—All Htt constructs used represent N-terminal fragments and are referred to as N followed by a number of amino acids present (e.g. N511). Htt-FLAG fusion proteins were immunoprecipitated using anti-FLAG M2 affinity gel (Sigma) according to the manufacturer’s protocol, followed by fractionation on NuPAGE 4 –12% BisTris polyacrylamide gels (Invitrogen), and detection with an antibody to exon 1 of Htt. For cell fractionation in HEK293 cells, M-PER lysates were centrifuged at 15,000 ϫ g. Following elution with a wells were lysed in 200 ␮l of M-PER buffer (Pierce), and lucif- FLAG peptide, the proteins were further purified using size erase activity was measured in 1/5 of the lysate using the Lucif- exclusion chromatography This step was performed under erase Assay System (Promega) according to the manufacturer’s denaturing conditions to disrupt Htt protein complexes and protocol. The two fragmentation spectra patterns and masses cp-2 fragments, even though the N511 band contains ϳ10 matched closely, confirming that the peptide detected in the times more Htt protein than the band corresponding to the cp-2 fragment digest is MDSNLPR. HT22 (Fig. 4B) cells, confirming cp-2 cp-2 the mass spectrometry results.

C N511-52Q N511-82Q

Findings

DISCUSSION