Clinical phenotype of dHMN patients with the HSPB1-P182L mutation
The first reported patient in 2004 carrying an HSPB1-P182L mutation (c.545C > T), patient CMT-391:II.I, belonged to an Austrian family where also a younger sibling was affected. A disease phenotype was reported at the age of 5 and until the last examination at the age of 16, the patient showed gait difficulties, weakness of the distal upper and lower limbs associated with muscular atrophy. In addition, pes cavus and brisk knee reflexes were reported. None of the asymptomatic parents showed the heterozygous HSPB1-P182L mutation but a parental mosaicism for the mutation was identified [11]. This patient was clinically diagnosed by M.A.G.
Creation of constructs and stable cell lines
Constructs used for transient transfection experiments and to generate stable SH-SY5Y cell lines were designed using the Gateway recombination system (Life Technologies). The open reading frames (ORF) of HSPB1 (NM_001540), PCBP1 (NM_006196) and EGFP were amplified by PCR using specific primers flanked by attB recombination sites to allow the insertion of the product in the pDONR221 vector. The described HSPB1 mutations (HSPB1-R127W and HSPB1-P182L) were generated by site-directed mutagenesis. Sequence validated pDONRs were transferred by recombination to the pLenti6/V5 destination vector (Life Technologies) allowing us to generate constructs where the ORF is fused to a V5-tag. To generate the PCBP1-VSV construct, the pDONR was transferred by recombination to a pCR3/VSV destination vector. For the luciferase assays we cloned the PCBP1 and EGFP cDNA’s upstream of a MS2 binding protein ORF containing plasmid [50]. All the final plasmids were verified by Sanger sequencing.
Stable cell lines were generated by lentiviral transduction of the neuronal cell line SH-SY5Y, according to the method described previously [43]. Stable cell lines presented similar expression levels and equal growth rate (data not shown).
Cell culture material and conditions
All cell culture media and supplements were purchased from Life Technologies. The human neuroblastoma cell line SH-SY5Y was purchased from ATCC and cultivated at 37 °C and 5% CO2 in EMEM supplemented with 10% fetal calf serum (FCS), non-essential amino acids, Glutamine and Penicillin-Streptomycin. The HeLa cell line was purchased from ATCC and cultivated at 37 °C and 5% CO2 in DMEM supplemented with 10% FCS, Glutamine and Penicillin-Streptomycin. The HEK293 cell line was purchased from ATCC and cultivated at 37 °C and 5% CO2 in DMEM supplemented with 10% FCS, HEPES, Glutamine and Penicillin-Streptomycin. The NSC-34 cell line was purchased from ATCC and cultivated at 37 °C and 5% CO2 in DMEM supplemented with 10% FCS, HEPES, Glutamine and Penicillin-Streptomycin. From the patients we obtained lymphocytes through venipuncture and lymphoblastoid cell lines were EBV-transformed and cultivated at 37 °C and 6% CO2 in Gibco® RPMI 1640 supplemented with 15% FCS, Sodium Pyruvate and Penicillin-Streptomycin. Besides the described HSPB1-P182L patient we also obtained lymphocytes from an HSPB1-R127W patient (cmt751.01) and a healthy control individual (ceph1454.14). Fibroblasts were obtained from patients by taking skin biopsies and cultivated at 37 °C and 6% CO2 in DMEM/F12 supplemented with 10% FCS, Sodium Pyruvate and Penicillin-Streptomycin. We obtained approval from our local medical ethical committees to perform this study.
Transient transfection
HeLa cell lines were transfected by using Lipofectamine LTX transfection reagent (Life Technologies) according to the manufacturer’s protocol. Briefly, cells were seeded out in 10 cm dishes at 1.5 × 106 cells per dish 24 h before transfection. On the day of transfection, media was renewed without the addition of antibiotics. Transfection was performed using 4000 ng plasmid DNA supplemented with 20 μl Lipofectamine LTX transfection reagent. Cells were lysed and assessed by co-immunoprecipitation, 24 h post transfection.
HEK293 cell lines were transfected by using polyethylenimine (PEI) according to an in house optimized protocol. Briefly, cells were seeded out in a 24 well plate at 7x104 cells per well, 24 h before transfection. On the day of transfection, media was renewed without the addition of antibiotics and containing a reduced serum concentration (2%). 500 ng plasmid DNA was diluted in 36 μl Opti-MEM (Life-Technologies) and in parallel, 2.5 μl PEI was diluted in 36 μl Opti-MEM. The diluted PEI was added to the DNA and mixed by gently vortexing. After 15 min of incubation at room temperature, the solution was added to the cells.
Western blotting
Cells were lysed in lysis buffer (0.5% Nonidet P-40, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 4 mM Sodium orthovanadate, 20 mM Glycerol-2-Phosphate, 10 mM Sodium Fluoride, 1 mM Sodium Pyrophosphate, together with complete protease and Phospho-STOP inhibitor mixtures - Roche Applied Science) for 30 min on ice and cleared by centrifugation for 10 min at 14,000 rpm. After protein concentration was determined, by using the Bradford protein assay (Bio-Rad), cell lysates were boiled for 5 min at 95 °C in reducing Laemmli buffer (Life Technologies) supplemented with 100 mM 1.4-Dithiothreitol (DTT). Proteins were separated on NuPAGE gels (Life Technologies) and transferred to a nitro-cellulose-membrane (Hybond™-P, GE Healthcare). Blocking of the membrane was performed using 5% milk powder diluted in PBS, supplemented with 0.1% Tween 20. Afterwards, membranes were incubated with a primary antibody over night at 4 °C and one hour with a secondary horseradish peroxidase conjugated antibody. Blots were developed by using the Enhanced Chemiluminescence ECL Plus™ detection system (Thermoscientific) and imaged with an ImageQuant imager (GE Healthcare). The following antibodies were used: anti-HSPB1 (Enzo Life Sciences), anti-V5 (Life Technologies), anti-VSV (Sigma-Aldrich) and anti-PCBP1 (Santa Cruz Biotechnology and Novus Biologicals).
Band intensities were determined by quantifying the mean pixel grey values using the ImageJ software [44]. Mean pixel grey values were measured in a rectangular region of interest.
Co-immunoprecipitation
Cells were lysed in lysis buffer (0.5% Nonidet P-40, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 4 mM Sodium orthovanadate, 20 mM Glycerol-2-Phosphate, 10 mM Sodium Fluoride, 1 mM Sodium Pyrophosphate, together with complete protease and Phospho-STOP inhibitor mixtures - Roche Applied Science) for 30 min on ice and cleared by centrifugation for 10 min at 14,000 rpm. Protein concentration was determined, by using the Bradford protein assay (Bio-Rad), in order to equalize the protein concentration from different cell lines. Sepharose 6B (Sigma-Aldrich) and protein G beads (GE Healthcare) or anti-V5 beads (Sigma-Aldrich) were used in a 3:1 ratio. Cell lysates with an equal protein concentration were incubated together with the bead mixture and the desired antibody, when needed, and left on a rotating device overnight at 4 °C. Afterwards the beads were collected by centrifugation and washed repeatedly with the same buffer as used for lysis of the cells. After the last washing step beads were resuspended in reducing Laemmli buffer (Life Technologies) supplemented with 100 mM DTT and boiled for 5 min at 95 °C prior to be loaded on NuPAGE gels (Life Technologies).
Polysome Gradient Analysis
Polysomal and non-polysomal fractions were separated according to a standardized protocol. Briefly, cells were collected by centrifugation, lysed in lysis buffer (30 mM Tris-HCl; pH7.4, 100 mM NaCl, 10 mM MgCl2 and 0.1% Triton X-100) and clarified by centrifugation for 10 min at 10.000 × g. The supernatant was transferred onto a 5–50% sucrose gradient (30 mM Tris-HCl; pH7.4, 100 mM NaCl, 10 mM MgCl2 and 5–50% sucrose) and centrifuged at 37,000 rpm for 3.5 h at 4 °C in a Beckman-Coulter LE-80 K. Fractions of each gradient were collected by monitoring at 260 nm and precipitated with 50% ethanol, 25% methanol and 25% acetone. After precipitation, pellets were washed in 0.3 M Guanidinium-HCl in 96% ethanol. The purified pellet was dissolved in Laemmli buffer (Life Technologies) supplemented with 100 mM DTT and boiled for 5 min at 95 °C prior to be loaded on NuPAGE gels (Life Technologies).
Luciferase assay
HEK293 cells were co-transfected with a luciferase reporter, PCBP1 or EGFP and HSPB1 wild type or mutants by using PEI as described before. Luciferase activity was measured with the Dual-luciferase reporter assay system (Promega) and following the manufacturer’s protocol. The luciferase activity was normalized by the renilla activity in order to correct for inter-well variations.
Immunocytochemistry and immunofluorescence microscopy
Immunostainings were performed according to a standardized protocol. Briefly, cells were seeded out on glass coverslips the day before fixation. Fixation was performed by incubating the cells with 4% paraformaldehyde for 20 min. Afterwards cells were permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS). Blocking was performed with 5% BSA diluted in PBS for 1 h, primary and secondary antibodies were incubated for 1 h diluted in 1% BSA in PBS supplemented with 0.1% Tween 20. The following antibodies were used; anti-HSPB1 (Enzo Life Sciences), anti-V5 (Life Technologies), anti-NFH (Covance), anti-PCBP1 (Santa Cruz Biotechnology), anti-mouse IgG Alexa Fluor 488 (Life Technologies), anti-goat IgG Alexa Fluor 488 (Life Technologies), anti-mouse IgG Alexa Fluor 594 (Life Technologies) and anti-goat IgG Alexa Fluor 594 (Life Technologies). Nuclear staining was performed with DAPI (Life Technologies), afterwards cells were mounted with fluorescent mounting medium (Dako). Images were taken on a LSM700 confocal fluorescence microscope using a 63×/1.40 plan-apochromatic objective. Possible cross-talk of the fluorescence channels was excluded by employing frame-by-frame scanning.
Neurite outgrowth of neuroblastoma cells
The human neuroblastoma cell line SH-SY5Y stably expressing different HSPB1 constructs was cultured in the presence of 20 μM retinoic acid (Sigma Aldrich) to induce neuronal differentiation after seeding. Phase contrast images were acquired with a Carl Zeiss Axiovert 200 M using a 10× objective (Plan NeoFluar Ph1 0.3 NA) and Zeiss AxioCamMR3 camera. Images were acquired of the same microscopic fields of cells on consecutive days, making use of the automated positioning function of the motorized microscope stage. To determine neurite outgrowth, a specific image analysis procedure was developed in ImageJ [44] to extract from each image the neuritic structures from the background and cell bodies. The pipeline consists of a combination of noise reduction filters, bandpass filtering in the Fourier space, local and global intensity thresholding, and combining resulting masks of different segmentation algorithms with Boolean operations. The final neurite mask was derived after a skeletonization step and its total size was used as a measure for the neurite length. The analysis was run in batch on all images using an ImageJ macro script.
RNA isolation from mouse tissues
To measure the abundance of mRNA in various mouse tissues, RNA isolation was performed. Animals were euthanized by CO2 inhalation and the brain, heart, liver, lung, muscle, sciatic nerve and spinal cord were dissected out and snap frozen in liquid nitrogen. The frozen tissues were homogenized in Trizol (Qiagen) and total RNA was isolated by using the RNeasy Lipid Tissue Mini kit (Qiagen) according to manufacturer’s protocol. All mouse experiments were approved by the Ethical Committee for Laboratory Animals (University of Antwerp). The mice were housed and maintained at the Animal Facility Interfaculty Unit, which is accredited by the Association for Assessment and Accreditation of Laboratory Animals.
RNA-immunoprecipitation (RNA-IP)
Total mouse brain was lysed in lysis buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT together with complete protease inhibitor mixture - Roche Applied Science), put on ice for 30 min and cleared by centrifugation for 10 min at 14,000 rpm. 40 Units/ml RNase OUT (Life Technologies) was added to the cleared lysate. After determining the protein concentration by using the Bradford protein assay (Bio-Rad), the lysate was diluted to a final concentration of 1 mg/ml in immunoprecipitation buffer (50 mM Tris-HCl; pH 7.4, 200 mM NaCl, 0.1% Triton X-100 and 2 mg/ml Heparin) and prepared for preclearing. Preclearing was performed on the diluted lysate by adding 50 μl Dynabeads (Life Technologies) without the presence of an antibody. After leaving the beads on a rotating device for 60 min at 4 °C, supernatant was taken for immunoprecipitation by adding 50 μl Dynabeads supplemented with 7 μg PCBP1 antibody (Santa Cruz Biotechnology). In parallel, a negative control was created by using 50 μl Dynabeads and 7 μg normal goat IgG (Santa Cruz Biotechnology) in order to check for background mRNAs binding to the Dynabeads. As for the preclearing, the samples for immunoprecipitation were left on a rotating device for 60 min at 4 °C. Afterwards beads were washed four times with washing buffer (50 mM Tris-HCl; pH 7.4, 200 mM NaCl, 0.1% Triton X-100 and 1 mM MgCl2). After the final wash beads were resuspended in elution buffer (200 mM NaOAc, 1 mM EDTA and 0.5% SDS) and heated up to 75 °C for 5 min. RNA was isolated on this fraction by phenol/chloroform extraction.
RNA sequencing
Purified RNA originating from the RNA-IP was sequenced on an Illumina HiSeq platform by paired-end sequencing. Library prepping was performed by using the TruSeq RNA Library Prep Kit from Illumina. The sequencing data were mapped to the mouse mm10 reference genome (Release date: December 2011) and thoroughly analyzed. Differential gene expression analysis was performed with the DESeq algorithm (Bioconductor 2.13).
RT-qPCR
Isolated RNA was assessed on concentration and purity by Nanodrop (Thermoscientific) measurement. DNase treatment was performed by using the TURBO™ DNAse treatment kit (Life Technologies) and cDNA conversion was performed by using the M-MLV Reverse Transcriptase system (Life Technologies) according to manufacturer’s protocol. To determine the amount of a transcript being present in the RNA-IP, relative expression levels were determined by qPCR experiments. The reactions were performed using Power SYBR® Master Mix (Life Technologies) according to manufacturer’s instructions and run on a ViiA 7 Real-Time PCR system (Applied Biosystems). All the reactions were preformed in triplicate and normalized to IgG as negative control. Primers were designed making use of Primerbank (http://pga.mgh.harvard.edu/primerbank). Primers were validated for specificity and amplification efficiency.
Calculation motif frequencies
Biomart and Perl API from Ensembl was used to extract the genomic coordinates and the sequences for the exons, 5′UTRs, 3′UTRs and introns belonging to the coding transcripts in mouse database v82. Only the transcripts having complete annotation and sequences available for all 4 features: exons, 5′UTR, 3′UTR and introns were used. The motif frequency was calculated in each of the 4 features of each transcript using the count of the exact match for the motif CTCCTCCTCCTCC (and reverse complement; GAGGAGGAGGAGG) in the feature and normalized to the transcript length.
Statistical analysis
For all experiments, results are shown as average with standard deviation. GraphPad Prism software was used for all statistical analysis. One way ANOVA with Holm-Sidak multiple comparison Test was used to analyze the quantification of the ratio PCBP1/HSPB1 in transiently transfected HeLa cell lines. To analyze the median 3′- and 5′-UTR length of the top 50 PCBP1-regulated targets a One Sample Wilcoxon signed-rank test was performed. Multiple t-test with Holm-Sidak as a correction for multiple comparisons was performed as statistical test for the qPCR validation graphs. The p-values were calculated on at least three independent experiments. A two-sample one-sided Kolmogorov-Smirnov test in R package was performed to test statistical significance in the motif frequencies analyzed in 3′-UTR, 5′-UTR, introns and exons of enriched target genes.