Abstract

BackgroundThe mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown.ResultsDelivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin.ConclusionWe show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.

Highlights

  • The mutation in Huntington’s disease is a polyglutamine expansion near the N-terminus of huntingtin

  • In associated virus (AAV)-infected mouse brains expressing htt1-400-100Q, proteolytic products migrated to cleavage product A (cpA) and cleavage product B (cpB) in HeLa cells. These data show that cpA and cpB accumulate in neurons in a mouse model of Huntington disease (HD)

  • Gammasecretase inhibitors increased survival of HD neurons. These findings suggest that the source of aspartyl protease activity required for cpA production may be cell specific and that gamma secretase activity may have a pathogenic role in HD through formation of cpA

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Summary

Introduction

The mutation in Huntington’s disease is a polyglutamine expansion near the N-terminus of huntingtin. The CAG repeat is translated into a polyglutamine (Q) tract near the N-terminus of huntingtin, which is 3144 amino acids (aa) in length Patients that bear this mutation suffer neurodegeneration resulting in cognitive and personality changes early in the Compelling evidence points to a role for N-terminal huntingtin fragments with an expanded polyQ tract (mutant huntingtin) in HD pathogenesis. N-terminal huntingtin fragments shorter than 342 aa were identified by epitope mapping in degenerating neurons in the brain of HD knock-in mice (HdhCAG150) which express endogenous mutant huntingtin [3]. These stable fragments of mutant huntingtin appear pre-symptomatically as early as 2 weeks postnatal, suggesting their formation precedes the onset of disease in mice [4]. Proteolysis of full-length huntingtin at particular sites may be necessary to produce toxicity

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