Integrative Characterization of the R6/2 Mouse Brain Model of Huntington Disease Reveals Dysfunctional Neurotransmitter Metabolism
Huntington disease (HD) is a fatal neurodegenerative disease, where dysfunction and loss of striatal and cortical neurons are central to the pathogenesis of the disease. Here, we integrated quantitative studies to investigate the underlying mechanisms behind HD pathology in a systems-wide manner. To this end, we used state-of-the-art mass spectrometry (MS) to establish a spatial brain proteome from late-stage R6/2 mice and compared this to wild-type (WT) counterparts. From the quantitative analysis, we observed altered expression of proteins in pathways related to energy metabolism, synapse function, and neurotransmitter homeostasis. To support these findings, metabolic 13C labelling studies confirmed a compromised astrocytic regulation of glutamate-GABA-glutamine cycling resulting in impaired release of glutamine and GABA synthesis. In recent years increasing attention has been focused on the role of astrocytes in HD, and our data supports that therapeutic strategies to improve astrocytic glutamine metabolism may help ameliorate symptoms in HD.
Sample Processing Protocol
Mice were euthanized by cervical dislocation and the brain was removed for dissection. Striatum, cortex, hippocampus, and midbrain were dissected and lyzed using RIPA buffer supplemented with protease and phosphatase inhibitors. Peptides were desalted on C18 Sep-Pak columns. Preparation of proteome library for matching was done using 2 mg of peptides from cortex for concatenated high pH fraction. All peptide samples were analyzed with nanoflow Easy-nLC 1000 coupled to Q Exactive HF mass spectrometers. Peptides were separated on in-house packed column with 1.9 μm C18 beads using 265 min gradients. Data was collected data dependent acquisition methods.
Data Processing Protocol
All data files were analyzed using the MaxQuant software suite 220.127.116.11 (ww.maxquant.org) with the Andromeda search engine. MS/ MS spectra were searched against an in silico tryptic digest of Mus musculus proteins from the UniProt sequence database (release version April 2014). All MS/MS spectra were searched with the following MaxQuant parameters for peptide identification: acetyl (protein N-terminus) and methionine oxidation were searched as variable modifications; cysteine carbamidomethylation was set as fixed modification; max 2 missed cleavages; and precursors were initially matched to 4.5 ppm tolerance and 20 ppm. The false discovery rate (FDR) for protein and peptide matches was set to 1% based on Andromeda score, peptide length, and individual peptide mass errors. Peptide length was minimum 7 amino acids, minimum Andromeda score was 40, and maximal peptide mass was 4600Da. The second peptide feature was enabled. The match between runs option was also enabled with a match time window of 0.5 min and an alignment time window of 20 min.
Niels Henning Skotte, University of Copenhagen
Michael L. Nielsen, Faculty of Health Sciences, Department of Proteomics, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark ( lab head )
Skotte NH, Andersen JV, Santos A, Aldana BI, Willert CW, Nørremølle A, Waagepetersen HS, Nielsen ML. Integrative Characterization of the R6/2 Mouse Model of Huntington's Disease Reveals Dysfunctional Astrocyte Metabolism. Cell Rep. 2018 23(7):2211-2224 PubMed: 29768217