Striatal synaptosomes from Hdh140Q/140Q knock-in mice have altered protein levels, novel sites of methionine oxidation, and excess glutamate release after stimulation.
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.
Antonio
A
Valencia
Ellen
E
Sapp
Jeffrey S
JS
Kimm
Hollis
H
McClory
Kwadwo A
KA
Ansong
George
G
Yohrling
Seung
S
Kwak
Kimberly B
KB
Kegel
Karin M
KM
Green
Scott A
SA
Shaffer
Neil
N
Aronin
Marian
M
DiFiglia
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003342:Corpus Striatum / Q000378:metabolism; D004195:Disease Models, Animal; D055879:Gene Knock-In Techniques; D018698:Glutamic Acid / Q000378:metabolism*; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D010084:Oxidation-Reduction; D013574:Synaptosomes / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D018698:Glutamic Acid; D008715:Methionine