Tracking brain palmitoylation change: predominance of glial change in a mouse model of Huntington's disease

Abstract

Protein palmitoylation, a reversible lipid modification of proteins, is widely used in the nervous system, with dysregulated palmitoylation being implicated in a variety of neurological disorders. Described below is ABE/SILAM, a proteomic strategy that couples acyl-biotinyl exchange (ABE) purification of palmitoyl-proteins to whole animal stable isotope labeling (SILAM) to provide an accurate tracking of palmitoylation change within rodent disease models. As a first application, we have used ABE/SILAM to look at Huntington's disease (HD), profiling palmitoylation change in two HD-relevant mouse mutants: the transgenic HD model mouse YAC128 and the hypomorphic Hip14-gt mouse, which has sharply reduced expression for HIP14 (Zdhhc17), a palmitoyl-transferase implicated in the HD disease process. Rather than mapping to the degenerating neurons themselves, the biggest disease changes instead map to astrocytes and oligodendrocytes (i.e., the supporting glial cells).

FIGURE 1

Example data for flottilin-1 (Flot1) from the ABE/SILAM analysis of palmitoylation in Hip14-gt and WT littermate brains. (A) Example chromatograms are shown for two Flot1 peptides from a 14N/15N-WT sample run (at left) and from a 14N/15N-Hip14-gt sample run (at right). Each chromatogram compares the abundance of the 14N-test peptide (red trace) deriving from either the WT littermate (at left) or Hip14-gt animal (at right) to the corresponding 15N-reference peptide (black trace). The ratio of the peak volumes for the test and reference peptides is reported below. (B) Graphical summary of the ABE/SILAM peptide data for Flot1, for the eight MS/MS runs that comprise the Hip14-gt analysis. For the two test genotypes analyzed, i.e. Hip14-gt and WT littermate, four mixed 14N/15N-ABE samples were prepared from three mice (m1, m2, and m3) plus one technical replicate (m1-t2). 14N/15N-peak ratios are reported for each Flot1 peptide, identified from each MS/MS run are reported. At right, the data aggregated from the four component MS/MS runs is shown with the median value and standard deviation indicated (***, P = 4 × 10⁻¹²). See related Figure S1.

FIGURE 2

Flot1 and Flot2 expression levels and DHHC PAT specificities. (A) Quantitative immunoblotting was used to analyze expression-level change in Hip14-gt and WT brains for carbonic anhydrase II (CA II), flotillin 1 (Flot1), flotillin 2 (Flot2), and glutamine synthetase (GS). Whole brain homogenates from Hip14-gt and WT animals were analyzed by immunoblotting with specific antibodies. SNAP-25 was used as the normalization control. Example immunoblots are shown at left and quantified results represented as means +/− SD are shown at right (n = 3, * P = 0.01, ** P = 0.003). (B) Analysis of HIP14- and DHHC5-mediated palmitoylation of Flot1 and Flot2 in yeast. Yeast cells were co-transformed with two plasmids, a plasmid that drives constitutive expression of a DHHC PAT (HIP14, DHHC5, or empty vector control) and a plasmid for GAL1 promoter-inducible expression of the test substrate protein (SNAP-25, Flot1, or Flot2). Palmitoylation was assessed using a click chemistry-based approach in the expressing yeast cells were metabolically labeled with the alkynated palmitate analog ODYA (see Experimental Procedures). Substrate and enzyme proteins, anti-FLAG immune precipitated from protein extracts, were click-reacted with azido-Alexa647 allowing fluorographic detection. Anti-HA immunoblotting was used to monitor the levels of these proteins both within the immune precipitation (at left) and within the initial protein extracts (at right). See related Figure S3.

FIGURE 3

Analysis of carbonic anhydrase II (CA II) and glutamine synthetase (GS) palmitoylation. (A) CA II and GS both show hydroxylamine-dependent ABE purification indicative of palmitoylation. WT mouse brain homogenates were processed through parallel acyl-RAC purifications, either in the presence or absence of hydroxylamine (HAm). The two purified samples were blotted with antibodies specific to the two proteins (“palm.”). (B) Fractional palmitoylation was assessed by thiol-Sepharose-mediated depletion of indicated proteins, following acyl-RAC work-up of WT whole brain homogenates. As for panel A, homogenates were processed through parallel plus- and minus-HAm protocols, with the portion of the samples that failed to bind to the thiol-Sepharose (unbound) being compared to the starting homogenate (total) by immunoblotting with the indicated specific antibodies. See related Figure S4.

FIGURE 4

Carbonic anhydrase II (CA II) and glutamine synthetase (GS) expression level changes (A) Expression-level analysis of CA II and GS in 12 month-old YAC128 and WT littermate whole brain homogenates. An immunoblot analysis identical to that in Fig. 1C was employed (WT, n=2; YAC128, n=4). (B) Brain distribution of CA II and GS expression reductions. Homogenates from striatum, cortex and cerebellum dissected from five 15 month-old YAC128 and WT littermate brains were subjected to quantitative immunoblot analysis. Results are depicted as means +/− SD. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

FIGURE 5

Correlating the Hip14-gt and YAC128 palmitoylation profiles. (A) Hip14-gt: YAC128 comparison. The thirteen proteins identified with the most significant ABE/SILAM change in Hip14-gt relative to WT littermates (from Table 1; red bars) were analyzed for change in YAC128 brain relative to its WT littermates (blue bars are indicative of significant change, while light blue bars indicate non-significant change). Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001. Proteins are denoted by gene symbol, except for CA II (Car2; carbonic anhydrase II) and GS (Glul; glutamine synthetase). (B) YAC128:Hip14-gt comparison. The ten proteins identified with the most significant ABE/SILAM change in YAC128 (Table 2; blue bars) were analyzed for change in Hip14-gt (red bars for significant change, while change denoted by light red bars does not pass the significance threshold). Note that the change reported for each protein is change in mutant ABE palmitoyl-proteome levels relative to that derived from isogenic, age-matched WT littermates.