Article type: Research Article
Authors: Valencia, Antonio | Sapp, Ellen | Kimm, Jeffrey S. | McClory, Hollis | Ansong, Kwadwo A. | Yohrling, George | Kwak, Seung | Kegel, Kimberly B. | Green, Karin M. | Shaffer, Scott A. | Aronin, Neil | DiFiglia, Marian
Affiliations: Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA | Huntington's Disease Society of America, New York, NY, USA | CHDI Management/CHDI Foundation, Princeton, NJ, USA | Department of Biochemistry and Molecular Pharmacology and The Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA, USA | Departments of Medicine and Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA
Note: [] Equal contribution.
Note: [] Equal contribution.
Note: [] Correspondence to: Marian DiFiglia, Department of Neurology, Massachusetts General Hospital East, 114 16th Street, Room 2002, Charlestown, MA 02129, USA. Tel.: +1 617 726 8446; Fax: +1 617 726 1264; E-mail: difiglia@helix.mgh.harvard.edu
Abstract: Background: Synaptic connections are disrupted in patients with Huntington's disease (HD). Synaptosomes from postmortem brain are ideal for synaptic function studies because they are enriched in pre- and post-synaptic proteins important in vesicle fusion, vesicle release, and neurotransmitter receptor activation. Objective: To examine striatal synaptosomes from 3, 6 and 12 month old WT and Hdh140Q/140Q knock-in mice for levels of synaptic proteins, methionine oxidation, and glutamate release. Methods: We used Western blot analysis, glutamate release assays, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Striatal synaptosomes of 6 month old Hdh140Q/140Q mice had less DARPP32, syntaxin 1 and calmodulin compared to WT. Striatal synaptosomes of 12 month old Hdh140Q/140Q mice had lower levels of DARPP32, alpha actinin, HAP40, Na+/K+-ATPase, PSD95, SNAP-25, TrkA and VAMP1, VGlut1 and VGlut2, increased levels of VAMP2, and modifications in actin and calmodulin compared to WT. More glutamate released from vesicles of depolarized striatal synaptosomes of 6 month old Hdh140Q/140Q than from age matched WT mice but there was no difference in glutamate release in synaptosomes of 3 and 12 month old WT and Hdh140Q/140Q mice. LC-MS/MS of 6 month old Hdh140Q/140Q mice striatal synaptosomes revealed that about 4% of total proteins detected (>600 detected) had novel sites of methionine oxidation including proteins involved with vesicle fusion, trafficking, and neurotransmitter function (synaptophysin, synapsin 2, syntaxin 1, calmodulin, cytoplasmic actin 2, neurofilament, and tubulin). Altered protein levels and novel methionine oxidations were also seen in cortical synaptosomes of 12 month old Hdh140Q/140Q mice. Conclusions: Findings provide support for early synaptic dysfunction in Hdh140Q/140Q knock-in mice arising from altered protein levels, oxidative damage, and impaired glutamate neurotransmission and suggest that study of synaptosomes could be of value for evaluating HD therapies.
Keywords: Mutant huntingtin, HD, synaptosomes, oxidative damage, methionine oxidation, glutamate release, synaptic proteins, mass spectrometry
DOI: 10.3233/JHD-130080
Journal: Journal of Huntington's Disease, vol. 2, no. 4, pp. 459-475, 2013
