Participants and samples
Patients, family members and unrelated controls were recruited under informed written consent as approved by the institutional review boards of Macquarie University, King’s College London, Imperial College London, University of Massachusetts at Worcester, Stanford University, Emory University, McGill University Health Center, University of New South Wales, Mayo Clinic, Queen Elizabeth Hospital (South Birmingham Research Ethics Committee), Beaumont Hospital, Hospital Universitario 12 de Octubre, Fundación CIEN, IRCCS Istituto Auxologico Italiano and Fondazione IRCCS Istituto Neurologico ‘C. Besta’, and the University of Tokyo. Patients were diagnosed with definite or probable ALS according to El Escorial criteria27. Patients had previously been screened for mutations/expansions in known ALS genes including TARDBP, SOD1, FUS, UBQLN2, OPTN, VCP, PFN1 and C9ORF72. Formalin-fixed, paraffin-embedded cervical spinal cord sections from Australian patients and neurologically normal controls were provided by New South Wales Tissue Resource Centre (Sydney, Australia).
Genetic linkage analysis
Genomic DNA was extracted from peripheral blood using standard protocols. For SNP-based genetic linkage analysis, genotyping was undertaken using the Affymetrix GeneChip Mapping 10Kv2.0 XbaI Array containing 10,204 SNP markers following the manufacturer’s instructions and apparatus (Affymetrix, Santa Clara, CA, USA). The raw microarray feature intensities were processed using the Affymetrix Genotyping Tools software package (GCOS/GTYPE) to derive SNP genotypes, marker order and linear chromosomal location. Parametric multipoint linkage analysis of SNP data was performed using Merlin v1.1 with intermarker distances obtained from the Marshfield (http://research.marshfieldclinic.org/) and deCODE (http://www.decode.com/) sex-averaged SNP linkage maps.
For microsatellite-based genetic linkage analysis, genotyping was performed by deCODE genetics using 539 microsatellite markers spaced at an average of 8 cM. Two-point and multipoint linkage analysis was performed using the FASTLINK v4.1 program of the easyLINKAGE v5.08 package28,29 and graphed using R package lodplot v1.2. All available family members were used for analysis. Parameters for linkage analysis included autosomal dominant inheritance, age-dependent penetrance (0–30 years, 1%; 31–40 years, 30%; 41–50 years, 50%; 51–60 years, 70%; 61–70 years, 85%, >70 years, 90%), a disease allele frequency of 0.0001, equal male and female recombination, and equal marker allele frequencies. For fine mapping across the chr16p13.3 haplotype, control allele frequencies and, where available, CEPH allele frequencies were used to calculate two-point and multipoint LOD scores. Multipoint linkage analysis was performed under three penetrance models including the age-dependent penetrance classes described above, as well as 100 and 70% disease penetrance.
Sequencing and mutation analysis
Exomes were captured using TruSeq Exome Enrichment kit or Agilent SureSelectXT Human All Exon V4. Paired-end sequencing was performed using the Illumina HiSeq2000 instrument.
CCNF exons were sequenced using Fluidigm Access Array target enrichment and the MiSeq sequencing platform (Illumina). Amplicon primers were designed using the Fluidigim D3 Design Studio to produce 150 bp amplicons targeting the exons of CCNF.
Validation and analysis of the CCNF mutations was performed by direct DNA sequencing following PCR amplification of coding exons (NM_001761). PCR products were Sanger sequenced using Big-Dye terminator sequencing and an ABI 3730XL DNA analyser (Applied Biosystems).
SNP genotyping in control individuals was performed using a custom TaqMan SNP genotyping assay according to the manufacturer’s instructions (Life Technologies) and analysed using a Viia 7 real-time PCR system (Life Technologies).
Control exome data from 967 neurologically healthy individuals of predominantly Western European descent obtained from the Diamantina Institute, University of Queensland (Diamantina Australian Control Collection).
Bioinformatics
Sequencing reads generated by the Illumina platform were aligned to the hg19 human genome assembly using BWA v0.6.1 (ref. 30), variants were called using SAMtools v0.1.16 (ref. 31) and annotated using ANNOVAR32. Annotated variants were compared among affected family members and controls using R v3.1.1 (http://www.R-project.org/)33. Filtering of variants was performed using dbSNP (releases 131, 132, 134 and 137; https://www.ncbi.nlm.nih.gov/SNP/), 1000 Genomes Project (Nov 2010 release; http://www.1000genomes.org/) and the ESP exome variant server (ESP6500 data release; http://evs.gs.washington.edu/EVS/).
Sequence reads generated by MiSeq were mapped to the hg19 human genome assembly using BWA30. GATK34,35,36 was applied to mapped reads for realigning and recalibration of base quality scores and variant calling. ANNOVAR32 was used for annotation of variants.
Quality filters for rare variant enrichment analysis include read depth ⩾10 in >75% of all samples and genotype quality >20.
Conservation of cyclin F orthologues was examined by aligning sequences from a variety of species (Entrez protein database; http://ncbi.nlm.nih.gov) using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).
Plasmids and cloning
Expression constructs comprising wild type and mutant CCNF cDNA fused with an N-terminal mCherry were developed using pmCherry-C1-CCNF (Addgene, https://www.addgene.org/32975/) and Q5 Site-Directed Mutagenesis kit (NEB) according to the manufacturer’s protocol. All constructs were verified by DNA sequencing.
Antibodies
The following primary antibodies were used in this study: rabbit polyclonal anti-cyclin F (cat # sc-952, Santa Cruz Biotechnology), mouse monoclonal anti-TDP-43 antibody (cat # H00023435-M01, Abnova), rabbit polyclonal anti-ubiquitin (Dako), mouse monoclonal anti-RRM2 (cat # ab57653, Abcam), anti-β actin (cat # A5441 [AC-15], Sigma-Aldrich). All antibodies were commercially sourced and validated according to the antibody data sheets.
Cell lines
Mouse NSC-34 (neuroblastoma/motor neuron-enriched primary spinal cord hybrid) cells were provided by Prof Neil Cashman, University of Toronto. Mouse neuroblastoma Neuro-2a cells, and human neuroblastoma SH-SY5Y cells were from the ATCC repository (ATCC Product Nos. CCL-131 and CRL-2266).
Confocal microscopy
Confocal fluorescence imaging was performed using a Leica DM6000 upright laser-scanning confocal microscope with Leica application suite advanced fluorescence software. Images were acquired with a × 63 (1.4 numerical aperture) oil-immersion objective. Images were acquired using sequential mode to avoid crosstalk between two dyes. Immunohistochemistry imaging was performed with a Zeiss Axio Imager 2 with ZEN pro program, using a × 40 objective.
Cell culture and transfection for UPS assays
NSC-34 cells were maintained in DMEM (Sigma Aldrich) containing 100 U ml−1 penicillin, 100 mg ml−1 streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (Sigma Aldrich). Cells were maintained in a humidified 37 °C incubator with 5% CO2. For the UPS assay and Enzo proteasome assay, NSC-34 cells were plated at a density of 50,000 cells per well in six-well plate. For the Abcam proteasome assay, NSC-34 cells were plated at a density of 2,000 cells per well in 96-plate well plate. Transfections were carried out using Lipofectamine LTX (Life Technologies) according to manufacturer’s protocol. Transfections included 2.5 μg DNA for the UPS assay, 5 μg DNA for the Enzo proteasome assay or 0.1 μg DNA for the Abcam proteasome assay.
Ubiquitinated protein immunoprecipitation
Co-transfected Neuro-2a cells were lysed and total protein was extracted with sonication (10 s, Setting 3, Branson Sonifier 450) in extraction buffer (1% (v/v) Nonidet P-40 in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with protease inhibitor cocktail). Cellular debris was pelleted at 18,000g (30 min at 4 °C). Protein concentration was estimated using the BCA Protein Assay Reagent (Pierce Biotechnology). Typically, 3 μg of anti-ubiquitin (Dako) per 500 μg of protein extract was used for immunoprecipitations. Protein A/G magnetic beads (Pierce Biotechnology) were used to capture the antibody:protein complex. Immunoprecipitates were washed with TBS+1% (v/v) NP-40 (3 × ) to remove non-specifically bound proteins, and then resuspended in 1 × LDS buffer with 50 mM DTT, and heated at 95 °C for 10 min.
UPS reporter assay and 20S proteasome activity assay
For the UPS reporter assay, NSC-34 cells were cultured in six-well plates for 24 h, followed by co-transfection with a UPS-specific degron GFPu and cyclin F constructs. The GFPu reporter contains a CL1 sequence which signals ubiquitination and degradation by the proteasome18,37. The resulting GFP signal is a reporter for rate of protein degradation in the cell. Cells were collected 48 h post transfection by trypsin and resuspended in PBS (Sigma-Aldrich). In order to show the sensitivity of GFPu construct, cells were either transfected with GFPu for 24 h followed by MG132 (Calbiochem) treatment at various doses for a further 24 h or co-transfected with GFPu and mCherry with or without MG132, or lastly co-transfected with GFPu and either Httex125Q or Httex146Q and incubated for 48 h. The fluorescent intensity GFP in collected cells was analysed using flow cytometry Becton Dickinson LSR II. At least 50,000 cells per treatment were collected. Data were gated on mCherry-positive cells with an excitation at 541 nm and an emission at 575 nm. The geometric mean of the GFP signal from this population was collected with an excitation at 488 nm and an emission at 520 nm. Data were from three independent experiments.
The proteasome enzymatic activity of cyclin F was measured using either a 20S proteasome assay kit from Enzo Life Sciences or Abcam following the manufacturer’s instructions. For the Enzo Life Sciences proteasome activity kit, NSC-34 cells were transfected wild-type pmCherry-C1-CCNF or mutant pmCherry-C1-CCNF in six-well plates and protein extract was generated by freeze–thaw lysis 48 h post-transfection. Protein concentration was measured by BCA protein assay (Thermo Scientific) and equal amount of protein was used in the assay. The proteasome activity was measured by hydrolysis of a fluorogenic peptide substrate Suc–Leu–Leu–Val–Tyr–AMC (AMC: 7-amino-4-methylcoumarin). The substrate is cleaved by the 20S proteasome and the release of free AMC fluorophore is used as an indication of proteolytic activity. The fluorescent signal was measured by FLUOstar OPTIMA fluorescence plate reader (BMG Labtech) with an excitation at 360 nm and an emission at 460 nm. The data were collected at 2-min interval for 50 min. For the Abcam proteasome activity kit, NSC-34 cells were transfected with wild-type pmCherry-C1-CCNF or mutant pmCherry-C1-CCNF in 96-well plates for 48 h. A proteasome substrate Leu–Leu–Val–Tyr–R110 was added directly to the cells and incubated at 37 °C for 1 h. The substrate is cleaved by the 20S proteasome and the fluorescent signal generated from the cleavage is used as an indication of proteolytic activity. The fluorescent signal was measured by FLUOstar OPTIMA fluorescence plate reader (BMG Labtech) with an excitation at 490 nm and an emission at 525 nm.
Statistical analysis of in vitro assays
All statistical analyses were performed using GraphPad Prism Software. Two-tail unpaired Student’s t-tests (P<0.05) were used for grouped GFPu data (wild-type, cyclin F mutation (familial and sporadic)). One-way analysis of variance with Dunnett’s multiple comparison tests were used for comparison of GFPu levels in the presence of cyclin F variants, with wild type. Statistical analyses of other in vitro assays were performed using two-tailed unpaired Student’s t-tests (P<0.05). All values were mean±s.e.m. The data met the assumptions of each specific statistical test. Variance was similar between groups. Sample size was chosen based on results from pilot studies.