N-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion.
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.



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).



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.



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.



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.
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC, Australia.
Moore Z
MZ
Chen
Department of Neurology, University of California San Francisco, San Francisco, CA, USA.
Sue-Ann
SA
Mok
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC, Australia.
Angelique R
AR
Ormsby
KynuRex, 634 Los Palmos Drive, San Francisco, CA, USA.
Paul J
PJ
Muchowski
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC, Australia.
Danny M
DM
Hatters
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D042622:DNA Repeat Expansion; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014162:Transfection; D000097573:Red Fluorescent Protein
D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D010455:Peptides; D000097573:Red Fluorescent Protein; C086055:HTT protein, human; C097188:polyglutamine
Aggregation; Huntington’s Disease; inclusion; live cell imaging; polyglutamine; proteolytic fragments