Making (anti-) sense out of huntingtin levels in Huntington disease

Patient-derived fibroblasts and human brain samples

Fibroblasts derived from HD patients and controls were purchased from Coriell Cell
Repositories, Camden, USA (Table 1). Fibroblasts were cultured at 37°C and 5% CO₂ in Minimal Essential Medium (Gibco Invitrogen, Carlsbad, USA) with 15% heat inactivated
Fetal Bovine Serum (Clontech, Palo Alto USA), 1% Glutamax (Gibco) and 100 U/ml penicillin/streptomycin
(Gibco).

Table 1. Patient-derived fibroblasts

Post-mortem human brain tissue was obtained from the Neurological Foundation of New
Zealand Human Brain Bank in the Centre for Brain Research, University of Auckland,
and the brain bank from the department of Neurology, Leiden University Medical Center.
Tissue was obtained with the families full consent and with the ethical approval of
the various institutional Ethics Committees. For a complete list of samples and corresponding
clinical information, see Table 2.

Table 2. Post-mortem human brain tissue

CAG repeat sizing

Genomic DNA samples were isolated from patient-derived fibroblasts and human brain
using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA) according to
manufacturer’s instructions and diluted to 50 ?g/ml. The number of CAG repeats in
the HTT gene was determined by PCR using primers “HD-1” and “HD-3” as described previously
24], followed by fragment analysis on an ABI 3130 Automated Capillary DNA Sequencer (Applied
Biosystems, Life Technologies Corporation, Carlsbad, USA). The exact PCR conditions
are available on request. The 3? CAA and following CAG are not counted. For the polyQ
repeat the CAA and CAG triplet are counted and the polyQ repeat is therefore 2 units
longer than the CAG repeat size.

RNA and genomic DNA analysis

Post-mortem brain tissue was homogenized using ceramic MagNA Lyser beads (Roche, Mannheim,
Germany) by grinding in a Bullet Blender (Next Advance, Averill Park, USA) according
to manufacturer’s instructions. Total RNA was isolated from fibroblast cells and brain
tissue using the Aurum Total RNA Mini Kit (BioRad, Hercules, USA), with an on-column
DNase treatment for 30 min. RNA was eluted in 40 ?l elution buffer and cDNA was synthesized
from 1 ?g total RNA using the Transcriptor First Strand cDNA Synthesis Kit with oligo
(dT) primers at 55°C for 90 min (Roche).

PCR was performed using 1 ?l cDNA or genomic DNA, 10x Expand High Fidelity buffer
with 15 mM MgCl₂ (Roche), 200 ?M dNTPs (Roche), 1 M Betaine (Sigma-Aldrich, St. Louis, USA), 15 pmol
of both forward primer HttCAGFw: 5?-ATG GCG ACC CTG GAA AAG CTG AT-3? and reverse
primer HttCAGRev: 5?-TGA GGC AGC AGC GGC TG-3? (Eurogentec, Liege, Belgium), 3 U Expand
High Fidelity enzyme mix (Roche), and PCR grade water to a final volume of 30 ?l.
The PCR program started with a 2 min initial denaturation at 94°C, followed by 35 cycles
of 15 sec denaturation at 94°C, 30 sec annealing at 60°C, 1 min elongation at 72°C,
and final elongation step at 72°C for 7 min.

PCR products were loaded on a 2% agarose gel diluted in Tris/Borate/EDTA (TBE) buffer.
DNA gel electrophoresis was performed for 1 hour at 100 V. Intensities of DNA bands
were quantified using ImageJ software. Intensity of the HTT mRNA band was divided
by the corresponding genomic DNA band to normalize for differences in PCR efficiency
due to CAG repeat length.

SNP genotyping and SNP linkage by circularization (SLiC)

The procedure for SNPs rs362273 genotyping and SNP linkage by circularization on human
brain tissue was adapted from Liu et al. 11]. One ?g of DNase-treated total RNA, together with oligo (dT) primers, was used for
cDNA synthesis using SuperScript III First-Strand Synthesis System (Invitrogen). To
improve reverse transcription of long cDNA templates, 2 M betaine and 0.6 M trehalose
(both Sigma-Aldrich) were added to the reaction mixture 25]. cDNA synthesis was performed at 42°C for 2.5 hours, followed by RNase H treatment
at 37°C for 20 min. Next, 5 ?l cDNA was used as template for long-range PCR and SLiC.

Taqman SNP assay

Quantitative PCR was performed using the LightCycler 480 II (Roche), according to
manufacturer’s instructions, using a mixture containing 45 ng cDNA, 1xTaqMan Universal
PCR Master Mix, no AmpErase UNG (Applied Biosystems), 1xTaqMan SNP Genotyping Assay
(Applied Biosystems), and nuclease-free water (Ambion) in a 20 ?l reaction volume.
ACTB (Applied Biosystems, cat#Hs99999903_m1) was included as reference gene. A standard
curve was generated using pooled equal amounts of cDNA from all samples. Quantification
of the dual-color hydrolysis of both allele-specific fluorescent reporter dyes, FAM
(“G” allele) and VIC (“A” allele), was performed with the LightCycler 480 SW 1.5.1
software (Roche) using the 2^nd derivative method, according to manufacturer’s instructions.

HTT antisense determination

RNA isolation as described above. PCR was performed using 1.5 ?l cDNA, 10x PCR buffer
with 20 mM MgCl₂ (Roche), 200 ?M dNTPs (Roche), 6 pmol HTTAS_v1 primer, forward: 5?-CAC CGG GGC AAT
GAA TGG-3?, reverse: 5?-GTG CGG ATG GCA AGG ACA G-3?, 2 U FastStart Taq DNA Polymerase
(Roche), 1 M ethylene glycol (Sigma-Aldrich), and PCR grade water to a final volume
of 30 ?l. The PCR program started with a 3 min initial denaturation at 95°C, followed
by 40 cycles of 10 sec denaturation at 95°C, 10 sec annealing at 60°C, 10 sec elongation
at 72°C, after which a final elongation step was performed at 72°C for 7 min.

PCR products were loaded on a 3% TBE agarose gel and bands were extracted using the
NucleoSpin Gel PCR Clean-up kit (Machery Nagel, Düren, Germany). To confirm the
sequence of HTTAS, PCR products were cloned into a pGEM-T Easy vector (Promega) and
analyzed by Sanger sequencing using a T7-specific forward primer.

Protein isolation

Fibroblasts were detached from the culture surface with a 0.5% Trypsin/EDTA solution.
After washing twice with HBSS, cells were resuspended in 200 ?l ice cold lysis buffer,
containing 50 mM HEPES, 50 mM NaCl, 10 mM EDTA, 10 mM DTT, 0.1% CHAPS, and 1 tablet
Complete mini protease inhibitor EDTA free (Roche) per 10 ml buffer 26]. Next, samples were sonicated 3 times 5 sec using ultrasound with amplitude 60 at
4°C. After 1 hour head-over-head incubation at 4°C, extracts were centrifuged for
15 min at 10,000 × g and 4°C and supernatant was isolated.

For brain homogenates, slices from frozen unfixed human brain tissue were collected
using a sliding microtome (Leica SM 2010R). Tissue was homogenized using ceramic MagNA
Lyser beads (Roche) by grinding in a Bullet Blender (Next Advance) for 3 min at strength
8 in lysis buffer with pH 7.2, containing 1% CHAPS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2PO4, 1.4 mM KH2PO4, 5 mM EDTA, 5 mM EGTA, and 1 tablet Complete mini protease inhibitor
EDTA free (Roche) per 10 ml buffer 27]. Homogenates were incubated for 1 hour in a head-over-head rotator at 4°C, and centrifuged
for 15 min at 10,000 × g at 4°C.

Protein concentrations were determined with the bicinchoninic acid kit (BCA) (Thermo
Fisher Scientific, Waltham, USA) using Bovine Serum Albumin (BSA) as a standard. After
addition of 5% glycerol, samples were aliquotted, snap frozen and stored at ?80°C.

Western blotting

SDS-PAGE separation of proteins was performed according to the “shorter CAG repeats”
protocol as described previously 27]. Proteins were transferred to a 0.2 ?m nitrocellulose membrane (Bio-Rad, #170-4159)
using the Trans-blot Turbo (BioRad) at 2.5A (constant)/25 V for 10 min. Membranes
were blocked for 15 min in tris buffered saline containing 5% non-fat milk (Nutricia,
Schiphol, the Netherlands). Next, membranes were incubated with primary rabbit antibody
EPR5526 (Abcam, Cambridge, UK) that recognizes the N-terminus of the htt protein,
diluted 1:5000 in blocking buffer, followed by secondary incubation with rabbit IRDye800
(LI-COR, Lincoln, USA) diluted 1:5000 in blocking buffer. Blots were analyzed on an
Odyssey reader (LI-COR). Protein bands corresponding to were quantified using the
Odyssey software version 3.0 (LI-COR). Background correction was performed by sampling
an empty area of the blot of the same size as the area that contained the positive
protein band. Quantification of wild-type and mutant htt protein relied on densitometry
measurement of both western blot bands separately. In case separation was small, we
could magnify our scanned blots using the software’s “zoom” function to aid in a proper
alignment of the densitometry calculation boxes over both separate bands. Wild-type
and mutant htt protein expression levels relative to total htt protein expression
were calculated by dividing wild-type and mutant htt band intensities with total htt
band intensity (wild type?+?mutant).

Statistical analyses

Experiments were performed at least six times per subject (3 biological and 2 technical
replicates). PCR linearity was evaluated by GraphPad Prism version 6.02 by determining
the individual linear regression coefficients (r2) of the band intensities of wild-type and mutant HTT expression versus the number
of PCR cycles.

GraphPad Prism version 6.02 was used to create whisker boxplots (whiskers?=?min to
max), showing all mean values per sample. IBM SPSS Statistics Version 20.0.0 was used
for statistical analysis. All datasets were tested for a Gaussian distribution by
visual inspection after plotting the residuals. Significance of the pairwise differences
was determined using a linear mixed model.