Computational protocol: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

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[…] Three independent coronal brain sections were used for each mouse, containing the dorsal striatum (bregma 0.5–1.1 mm) and were stained with presynaptic VGlut1 or VGlut2 (Chemicon, anti-guinea pig, 1:500) and postsynaptic PSD95 (Zymed, Rabbit, 1:500) markers as described previously. Secondary antibodies used were goat anti-guinea pig Alexa 488 (VGlut1/2) dilution 1:200 and goat anti-rabbit Alex 594 (PSD95) dilution 1:200 (Invitrogen). Three mice for each genotype; WT, CK2α′(+/−), KIQ175 and KIQ175/CK2α′(+/−) were evaluated in a double-blinded fashion. The 5 μm-thick confocal scans (optical section depth 0.33 μm, 15 sections per scan, imaged area per scan=20.945 μm2) of the synaptic zone in dorsal striatum were performed at 63 × magnification on a Leica SP5 confocal laser-scanning microscope. Maximum projections of three consecutive optical sections (corresponding to 1 μm depth) were generated. The Puncta Analyzer Plugin for ImageJ (available upon request; [email protected]) was used to enumerate co-localized synaptic puncta. This assay takes the advantage of the fact that presynaptic and postsynaptic proteins reside in separate cell compartments (axons and dendrites, respectively), and they would appear to co-localize at synapses because of their close proximity. The number of animals used in our study was 12 mice for each genotype group and sex. At least 5 optical sections per brain section and at least 3 brains sections per animal were analysed, making a total of 45–60 image data sets per brain region in each genotype/age. [...] Golgi Cox staining was performed on WT, CK2α′(+/−), KIQ175 and KIQ175/CK2α′(+/−) mice (three mice per genotype) using FD Rapid GolgiStain Kit (FD NeuroTechnologies). Dye-impregnated brains were embedded in Tissue Freezing Medium (TFM, TBS), rapidly frozen on ethanol pretreated with dry ice, cryo-sectioned coronally at 200 μm thickness and mounted on gelatin-coated microscope slides (Southern Biotech). Sections were stained according to directions provided by the manufacturer. Sections that contain the dorsal striatum were imaged and MSNs in the striatum were identified by their morphology. Secondary and tertiary apical dendrites were imaged for spine analysis as follows: z-stacks (30 μm total on z-axis, single section thickness=0.5 μm) of Golgi-stained dendrites were taken at 63 × magnification on a Zeiss AxioImager M1 microscope. A series of TIFF files corresponding to each image stack were loaded into the Reconstruct programme (http://synapses.clm.utexas.edu) and spine analyses performed as previously described. The clasification of spines is based on width, length, and length:width ratio measurements taken using the Reconstruct software (http:// synapses.clm.utexas.edu; RRID:nif-0000-23420) designed and validated. Spines were identified and classified by choosing 10 μm segments of dendrites and identified on selected dendritic stretches. The z-length (spine length) and spine head width were measured for each spine. Measurements were exported to a custom Microsoft Excel macro that was used to classify spines based on the width, length, and length:width ratio measurements taken in Reconstruct. Spines were categorized based on the following hierarchical criteria: (1) more than one spine head=‘branched spine,' (2) head width>0.7 μm=‘mushroom spine,' (3) length>2 μm=‘filopodia,' (4) length:width >1=‘thin spine,' and (5) length:width ≤1=‘stubby spine.' Branched and mushroom spines were identified as mature spines, thin and stubby spines were categorized as intermediate spines, and filopodia were classified as immature spines. Statistical analyses of changes in spine density, length, width and spine type were conducted in the Statistica programme (StatSoft): A total of 3 animals per genotype, 15 dendrites per animal, 45 dendrites per genotype were analysed in MSNs in the dorsal striatum in a blinded fashion. The number of spines analysed per neuron type per age per genotype exceeded 1,000. [...] Quantification of the specific activity (U per ml) of GST-CK2α (Addgene pDB1-27083) and GST-CK2α′ (Addgene pDB6-27084) purified recombinant enzymes was performed using casein kinase assay kit (CycLex) and commercial CK2 holoenzyme (New England Biolabs) as positive control for phosphorylation. Quantification was carried out following per manufacture's instructions. Purified recombinant human HSF1 trimer (1 μg) was incubated with recombinant CK2 (Holoenzyme or GST purified subunits CK2α or CK2α′) with 5,000 U ml−1. As negative controls for the assay we used samples without ATP or samples without enzyme. The reaction was carried out at 37 °C during 30 min (control conditions) or at 37 °C during 20 min followed by 10 min at 42 °C (Heat shock conditions). 6 × SDS was added and boiled for 2 min to terminate the reaction. Samples were analysed by phosphoproteomics. [...] Phosphoproteomic analysis was performed by the Proteomics and Metabolomics Shared Resource at Duke University. Protein samples were mixed with loading buffer and reduced with 10 mM DTT at 70 °C for 10 min before SDS–PAGE separation on a 4–12% bis-tris acrylamide gel (NuPAGE, Invitrogen) with colloidal coomassie staining. Bands corresponding to HSF1 were excised and subjected to standardized in-gel trypsin digestion (http://www.genome.duke.edu/cores/proteomics/sample-preparation/documents/In-gelDigestionProtocolrevised.pdf). Extracted peptides were lyophilized to dryness and resuspended in 12 μl of 0.2% formic acid/2% acetonitrile. Phosphopeptides were enriched using GL Biosciences p10 TiO2-derivatized tips according to the manufacturer's protocol. Each sample was subjected to chromatographic separation on a Waters NanoAquity UPLC equipped with a 1.7 μm BEH130 C18 75 μm I.D. X 250 mm reversed-phase column. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Following a 4 μl injection, peptides were trapped for 3 min on a 5 μm Symmetry C18 180 μm I.D. X 20 mm column at 5 μl per min in 99.9% A. The analytical column was then switched in-line and a linear elution gradient of 5% B to 40% B was performed over 30 min at 400 nl per min. The analytical column was connected to a fused silica PicoTip emitter (New Objective, Cambridge, MA) with a 10 μm tip orifice and coupled to a QExactive Plus mass spectrometer through an electrospray interface operating in a data-dependent mode of acquisition. The instrument was set to acquire a precursor MS scan from m/z 375–1675 with MS/MS spectra acquired for the ten most abundant precursor ions. For all experiments higher-energy collisional dissociation (HCD) energy settings were 27v and a 120 s dynamic exclusion was employed for previously fragmented precursor ions.Raw LC-MS/MS data files were processed in Proteome Discoverer (Thermo Scientific) and then submitted to independent Mascot searches (Matrix Science) against a SwissProt database (Human taxonomy) containing both forward and reverse entries of each protein (20,322 forward entries). Search tolerances were 5 p.p.m. for precursor ions and 0.02 Da for product ions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M), deamidation (+0.98 Da on NQ), and phosphorylation (+79.99 Da on STY) were considered dynamic mass modifications. All searched spectra were imported into Scaffold (v4.3, Proteome Software) and scoring thresholds were set to achieve a peptide false discovery rate of 1% using the PeptideProphet algorithm. Raw data can be found in . […]

Pipeline specifications

Software tools Statistica, cycleX
Databases Addgene
Applications Miscellaneous, scRNA-seq analysis
Organisms Mus musculus, Homo sapiens