Affiliations: [a] Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC, Australia
| [b] Department of Neurology, University of California San Francisco, San Francisco, CA, USA
| [c] KynuRex, 634 Los Palmos Drive, San Francisco, CA, USA
Correspondence to: Danny M. Hatters, Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC 3010, Australia. Tel.: +61 3 8344 2530; E-mail: [email protected].
Abstract: Background: A hallmark of Huntington’s disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. Objective: It remains unclear how the length of the N-terminal fragments relates to polyQ – mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). Methods: N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. Results: N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. Conclusions: PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.