The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias

Mice and genotyping

The tTA:CHMP2BIntron5 and tTA:CHMP2BWT mice used in this study have been described [7]; both males and females were used. All procedures involving mice were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts Chan Medical School.

Drosophila genetics

Flies were maintained on a 12-h light/12-h dark cycle on standard cornmeal-yeast agar medium at 25 °C. UAS-CHMP2BIntron5 flies used were described previously [1]. GMR-Gal4, UAS-RNAi SPAST (#27,570), and UAS-RNAi_SPAST (#53,331) fly lines were from the Bloomington Drosophila Stock Center. For genetic interaction studies, the recombined fly line (GMR-Gal4:UAS-CHMP2BIntron5) was crossed with UAS-RNAi_SPAST flies. To quantify the retinal degeneration phenotype, we classified the eye phenotype, with or without SPAST downregulation, into three groups: severe (+ + +), medium (+ +), and weak ( +). This classification was based on the relative abundance of black spots on the eye, ranging from a dozen or so scattered spots ( +) to spots covering approximately 50–70% or more of the eye surface (+ + +).

Mammalian cell culture, siRNAs, constructs, transfection and immunoprecipitation

HEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal calf serum (Life Technologies) and maintained in a humidified incubator at 37 °C with 5% CO2. All siRNAs for gene silencing were from Qiagen (Additional File 4: Table S1). pCMV-3/FLAG-CHMP2BIntron5 and pCMV-3/FLAG-CHMP2BWT plasmids were generated as described [10]. Full-length human spastin M87 plasmids were generated by cutting the pCMV-Tag 3A/WT myc-M1 (Addgene, Cat. no. 87719) and pCMV-Tag 3B/WT myc-M87 (Addgene, Cat. no. 87722) and then subcloned into the pEGFP-C1 vector (Addgene). Full length spastin M87 is used throughout this study. siRNAs or constructs were transiently transfected into cells with RNAiMAXor Lipofactmine3000 (Invitrogen), as recommended by the manufacturer, for 48 h.

Three 100-mm tissue culture dishes of HEK293 cells at 70% confluency were transfected with pCMV-3/FLAG-CHMP2BIntron5, pCMV-3/FLAG-CHMP2BWT, or pCMV-3/FLAG empty vector with Lipofectamine 3000. After 48 h, transfected cells were collected and homogenized in immunoprecipitation (IP) lysis buffer (Thermo Fisher, Cat. no. 87787) with protease and phosphatase inhibitors (CST, catalog no. 5872). Homogenates were centrifuged at 4 °C for 10 min at 13,000g, to obtain supernatants. Protein concentrations of supernatants were determined with the Bradford assay (Bio-Rad). For co-IP experiments, supernatants of CMV-3/FLAG-CHMP2BIntron5, pCMV-3/FLAG-CHMP2BWT, or pCMV-3/FLAG with the same amount of total proteins were preabsorbed with anti-FLAG M2 affinity gel (Sigma, catalog no. A2220), incubated overnight at 4 °C, centrifuged and washed three times for 5 min each with washing buffer (50 mM Tris–HCl, pH 7.4, and 150 mM NaCl), and suspended in FLAG elution solution (Sigma catalog no. F4799) for 30 min at 4 °C. The supernatants were used for western blot.

Proteomic analysis of CHMP2BIntron5 interacting proteins

To identify proteins that interact with CHMP2BIntron5, proteins in experimental and control IP samples were electrophoresed a short distance into a polyacrylamide–sodium dodecyl sulfate gel and stained with the Coomassie Brilliant Blue (Bio-Rad). In-gel digestion and liquid chromatography–tandem mass spectrometry analysis were done by the Mass Spectrometry Facility at the University of Massachusetts Chan Medical School. Protein abundance was estimated with IBAQ quantification, in which summed peptide intensities are normalized to the number of theoretically observable peptides of the protein. pCMV-3/FLAG served as a control to exclude nonspecific interacting proteins. Interacting proteins that were not associated with FLAG proteins but bound more to FLAG-CHMP2BIntron5 than FLAG-CHMP2BWT were selected for further analyses. Total proteins were further ranked by iBAA value from most to least abundant. Mass spectrometry (MS) analysis was done by the UMass Chan Medical School Mass Spec Core with a standard protocol as published before [12].

Western blots

The mouse cortex was dissected, quickly frozen at  − 80 °C, homogenized, and sonicated in RIPA buffer with proteinase and phosphatase inhibitors (CST, catalog no. 5872). The cultured cells were lysed in RIPA buffer (Thermo Scientific). The protein extract was centrifuged to remove tissue debris, and boiled for 5 min. Protein (20 μg) from each sample was subjected to SDS-PAGE using 4–20% precast gels (Bio-Rad) and immunobloted with the following primary antibodies: rabbit anti-spastin (Proteintech, catalog no. 22792–1-AP; 1:1000) and mouse anti-β-actin (Sigma-Aldrich, catalog no. A2228; 1:3000), overnight at 4 °C. After incubation, immunoblots were washed and incubated with IRDye fluorescent anti-rabbit and anti-mouse secondary antibodies (LI-COR Biosciences). Images were acquired with a LI-COR CLx Odyssey System.

Subcellular fractionation and solubility analysis

HEK293 cells were collected 48 h after transfection and subjected to subcellular fractionation with a ProteoExtract Subcellular Proteome Extraction Kit (Millipore, catalog no. 539790), according to the manufacturer’s protocol for adherent cells. If some cells became nonadherent during the protocol, the cytosolic, membrane, and nuclear fractions were spun at 750 g, 5500 g, and 6800 g, respectively, for 10 min at 4 °C, to remove any contamination from later fractions. Proteins were resolved by SDS–PAGE and immunoblotted with spastin antibody (Proteintech, catalog no. 22792–1-AP; 1:1000).

For SPAST solubility analysis, cells were seeded into six-well dishes at 250,000 cells/well; 48 h after transfections, cells were washed with PBS, released with 0.25% trypsin, and resuspended in DMEM pre-warmed to 37 °C. The cells were then spun down, washed with PBS, and resuspended in 20 μl of PBS. The cells were lysed by two cycles of flash freezing on dry ice and rapidly thawing at 42 °C. The lysate was spun at 1000 g, and the resulting supernatant was transferred to a new tube and re-spun to remove any insoluble material. The pellet was rinsed 3 times with PBS and resuspended in the corresponding volume of supernatant and briefly sonicated with a tip sonicator (Sonopuls, catalog no. 2070). Equivalent fractions of total volume for 100 ng of supernatant and resuspended pellet were boiled with SDS loading buffer (50 mm Tris–Cl, pH 6.8, 2% (2 w/v) SDS, 0.1% (w/v) bromophenol blue) and 10 mm dithiothreitol, separated by SDS-PAGE on 10% polyacrylamide–sodium dodecyl sulfate gels and immunoblotted with spastin antibody (Proteintech, catalog no. 22792-1-AP; 1:1000).

Immunofluorescence analysis of cultured cells

HeLa cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 5 min, blocked with 5% bovine serum albumin for 30 min, and incubated overnight with the following primary antibodies: rabbit anti-spastin (Proteintech, catalog no. 22792–1-AP; 1:200), mouse anti-FLAG (Sigma, catalog no. F1804; 1:1000), rabbit anti-p62 (Proteintech, catalog no. 18420–1-AP; 1:2000). After incubation, the cells were washed three times with PBS, incubated first with donkey anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, catalog no. A-21202; 1:500) and then with goat anti-rabbit Alexa Fluor 568 secondary antibody (Invitrogen, catalog no. A-11011; 1:500) for 1 h at room temperature, and mounted with HardSet Mounting Medium with DAPI (Vectashield, catalog no. H-1500). Confocal images were acquired with a ZEISS LSM 800 laser-scanning confocal microscope and processed with ZEISS ZEN microscope software. Fluorescence images were acquired with a ZEISS inverted microscope (LSP T PMT).

Immunostaining of mouse brain sections

Paraffin-embedded tissue sections were deparaffinized and hydrated in a series of graded alcohols. After antigen retrieval with citrate buffer (Sigma, C9999), the sections were washed once with water, treated with BLOXALL Endogenous Blocking Solution (Vector Lab, SP-6000–100) for 10 min washed with PBST for 10 min, blocked with Dako blocking reagent for 24 h, and incubated overnight with guinea pig anti-p62 (Progen, catalog no. GP62-C) and polyclonal anti-SPAST (Proteintech, catalog no. 22792) and 0.1% Triton-X 100; the antibodies were diluted 1:200 in DAKO antibody diluent (Agilent, S302283-2) overnight. The sections were washed three times with PBST for 10 min each and incubated with Alexa-conjugated secondary antibodies (Invitrogen, catalog nos. A-11075 and A32790) in detergent-supplemented DAKO antibody diluent buffer for 2 h in the dark. The sections were washed three times with PBST for 10 min each and mounted with DAPI Fluoromount-G Mounting Medium (Invitrogen). The total surface of stained brain sections from three mice per genotype group was scanned (Sanderson Center for Optical Experimentation) (SCOPE) (UMass Chan Medical School). Images from each channel were exported with TissueFACSL viewer software and processed in ImageJ. JACop plugin in Image J was used to calculate Mander’s overlap coefficient [3, 15]. P62 was considered as an aggregate marker to reveal the extent to which two signals occupy the same place. Manual thresholding was applied to exclude the background signals from all images. Representative figures were obtained with a confocal microscope (Leica SP8).

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