Modulation of Mutant Huntingtin N-Terminal Cleavage and Its Effect on Aggregation and Cell Death

Katrin Juenemann 0 1 Christina Weisse 0 1 Denise Reichmann 0 1 Christoph Kaether 0 1 Cornelis F. Calkhoven 0 1 Gabriele Schilling 0 1 0 K. Juenemann (&) Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam , Meibergdreef 15, M3-105, 1105 AZ Amsterdam, The Netherlands 1 K. Juenemann C. Weisse D. Reichmann C. Kaether C. F. Calkhoven G. Schilling Leibniz Institute for Age Research, Fritz Lipmann Institute , 07745 Jena, Germany Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem brain, animal models, and cell culture models. It has been found that N-terminal fragments of the mutant huntingtin protein are more toxic than the full-length protein. Therefore, proteolytic processing of mutant huntingtin may play a key event in the pathogenesis of HD. Here, we present evidence that the region in huntingtin covering amino acids 116 to 125 is critical for N-terminal proteolytic processing. Within this region, we have identified mutations that either strongly reduce or enhance N-terminal cleavage. We took advantage of this effect and demonstrate that the mutation D121-122 within the putative cleavage region enhances N-terminal cleavage of huntingtin and the aggregation of N-terminal fragments. Furthermore, this particular deletion increased the activation of apoptotic processes and decreased neuronal cell viability. Our data indicate that the N-terminal proteolytic processing of mutant huntingtin can be modulated with an effect on aggregation and cell death rate. - Huntingtons disease (HD) is an autosomal-dominant inherited, progressive and fatal neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin gene, coding for polyglutamine (polyQ) (The Huntingtons Disease Collaborative Research Group 1993; Ross et al. 1997). The expansion of the polyQ stretch in the huntingtin protein (htt) to a length greater than 36 consecutive glutamines is causing HD. The onset of disease usually occurs in mid-adult life and lasts 1525 years. Symptoms of HD include involuntary movements, cognitive impairment, and psychiatric disturbances (Vonsattel and DiFiglia 1998). Despite widespread expression of the huntingtin gene (Sharp et al. 1995), there is a selective vulnerability and loss of the medium spiny projection neurons in the striatum (Vonsattel et al. 1985). Transgenic or knock-in mice that express full-length or truncated mutant htt (mhtt) display a neurological phenotype resembling HD with intracellular inclusions (Davies et al. 1997; Hodgson et al. 1999; Reddy et al. 1998; Schilling et al. 1999; Tanaka et al. 2006; Wheeler et al. 2000). Postmortem analysis of brains from HD patients and studies using mouse or cell culture HD models revealed that nuclear and cytoplasmic inclusions contain N-terminal fragments of mhtt (DiFiglia et al. 1997; Gutekunst et al. 1999; Lunkes et al. 2002; Schilling et al. 2007). Moreover, pathological changes are accelerated in HD mouse models overexpressing mhtt N-terminal fragments compared to those with full-length mhtt (Hodgson et al. 1999; Mangiarini et al. 1996; Schilling et al. 1999; Wheeler et al. 2000). One exception is observed with the HD mouse model expressing the first 117 amino acids (aa) of mhtt showing neuronal inclusions but no behavioral abnormalities or neurodegeneration (Slow et al. 2005). Since the correlation between formation of inclusions and neuronal cell death is weak (Gutekunst et al. 1999; Kuemmerle et al. 1999; Saudou et al. 1998), the role of inclusions in HD pathogenesis is not clear. Accumulated evidence suggest that the oligomeric intermediates of mhtt fragments cause cytotoxicity (Arrasate et al. 2004; Ross and Poirier 2005; Sanchez et al. 2003). Htt can be cleaved between residues 400 and 600 at several sites by caspases, calpains, and MMP-10 (Gafni and Ellerby 2002; Goldberg et al. 1996; Kim et al. 2001; Miller et al. 2010; Wellington et al. 2002), which may contribute to toxicity (Gafni et al. 2004; Graham et al. 2006; Wellington et al. 2000). In addition, smaller N-terminal fragments of htt than those generated by caspase or calpain cleavage are found in human HD postmortem brain (DiFiglia et al. 1997; Lunkes et al. 2002) or in mouse models of HD (Li et al. 2000; Schilling et al. 2007). At least two smaller N-terminal fragments, called cp-A and cp-B, have been described (Kim et al. 2006; Lunkes et al. 2002). Deletion of aa 104114 prevented release of cp-A and the production of the longer cp-B cleavage product was reduced after deletion of aa 205214. The release of cp-A and cp-B can be inhibited by pepstatin, suggesting that the protease(s) responsible for the generation of the htt N-terminal cp-A and cp-B belong to the family of aspartyl endopeptidases. In addition, short N-terminal fragments (Cp-1 and Cp-2) can be released by caspase independent proteolytic cleavage of htt in a PC12 cell model expressing full-length mhtt (Ratovitski et al. 2007). Recent studies identified the length of two short htt N-terminal cleavage products. In the HdhQ150 knock-in mouse model htt exon 1 was identified as the smallest cleavage product with a length of 90 aa (Landles et al. 2010) and in vitro studies suggest htt cleavage at position 167 (generating the fragment cp-2) (Ratovitski et al. 2009). The proteolytic pathways that generate short htt N-terminal cleaved fragments are still poorly understood. A better understanding of htt proteolytic processing provides important insights in the early mechanisms of HD pathogenesis and will lead to the development of new HD therapeutics. Our previous work showed that in the HD N171-18Q, -44Q, and -82Q mouse model, cleavage of htt appears to be a normal process (Schilling et al. 1999). We have recently detected an N-terminal fragment of mhtt in the HD N171-82Q mouse model and in human HD postmortem brain lacking the epitope 115129 (Schilling et al. 2007) and being similar to cp-A detected in NG108-15 cells expressing mhtt (Lunkes et al. 2002). Using an in vitro cell culture model, we have now identified a region between aa 116125 in htt where the efficiency of N-terminal cleavage is strongly affected by mutations with the cleavage either being suppressed or enhanced. The mutation D121122 that enhances cleavage, and hence mhtt N-terminal fragment release results in increased aggregation, apoptotic processes and in a decrease of neuronal cell viability. Our data show that the N-terminal cleavage of mhtt can be modulated with an effect on aggregation and cell death rate. Materials and Methods Expression constructs containing htt cDNA encoding the first 171 (N171) and 233 (N233) amino acids with 18 glutamines (wild-type htt) or 82 glutamines (mhtt) were cloned into pcDNA3.1 or pRC/CMV, whereas the numb (...truncated)